Aptamer-Based Detection and Characterization of Ovarian Cancer

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Title:
Aptamer-Based Detection and Characterization of Ovarian Cancer
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1 online resource (143 p.)
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english
Creator:
Lopez-Colon,Dalia
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University of Florida
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Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
Tan, Weihong
Committee Members:
Horenstein, Nicole A
Fanucci, Gail E
Smith, Benjamin W
Narayan, Satya

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Subjects / Keywords:
aptamer -- ctc -- ovarian -- selex
Chemistry -- Dissertations, Academic -- UF
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Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract:
Whereas ovarian cancer presents only 3% of the new cases of cancer in women, it is the 5th deadliest malignancy overall in the United States. Due to a lack of effective methods to screen for ovarian cancer, 75% to 80% of ovarian cancers are diagnose in stage III or stage IV. At these stages, the 5-year survival is only 15-20% as compared to an 80-90% survival rate for ovarian cancer diagnosed at early stages of the disease. Therefore, detection of the ovarian cancer tumor at an early stage will improve the outcome for ovarian cancer patients. For this purpose, we selected a panel of aptamers for the detection and characterization of ovarian cancer. Aptamers are single-stranded nucleic acids (DNA or RNA) capable of recognizing a specific target with high affinity and specificity. Aptamers are selected from a random pool containing about 1015 distinct sequences. During the selection process, unbound sequences are washed away, while sequences binding to the target are eluted, PCR amplified and used for subsequent rounds of selection. This cycle is repeated until the pool has been fully enriched for the target. The enriched pool is then sequenced and potential aptamer sequences are identified by looking at sequence homology. In this work, we have identified six aptamer sequences capable of binding to the epithelial ovarian adenocarcinoma cell line CAOV3. From these sequences, DOV-2a, DOV-3, DOV-4 and DOV-6a were further characterized. These aptamers showed binding affinities between 30-130 nM. Furthermore, the aptamers specificity was tested with other cancer cell lines. DOV-4 showed no binding to normal immortalized lung epithelial cells, while DOV-2a showed no binding to the ovarian benign cysts cell line. The selected ovarian cancer aptamers were further characterized to identify the target in the cell membrane. The aptamer binding was assayed after trypsin, proteinase K and glycosidase treatment. Finally, the aptamers were used to develop an ovarian cancer detection assay using whole blood. The assay developed allows the detection of 500 ovarian cancer cells spiked in 1 mL of whole blood with little pretreatment of the blood sample.
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In the series University of Florida Digital Collections.
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Includes vita.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Dalia Lopez-Colon.
Thesis:
Thesis (Ph.D.)--University of Florida, 2011.
Local:
Adviser: Tan, Weihong.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-02-29

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1 APTAMER BASED DETECTION AND CHARACTERIZATION OF OVARIAN CANCER By DALIA L"PEZ COL"N A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Dalia Lpez Coln

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3 To my daughter, Dalianis, my inspiration To my mother, my pillar of strength

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4 ACKNOWLEDGMENTS I would like to thank my advisor, Dr Weihong Tan of the Chemistry Depar tment, University of Florida for his guidance and constructive comments throughout my PhD career. I would also like to especially thank Dr Rebecca Sutphen of the College of Medicine, University of South Florida. She has been of tremendous help in my resea rch work, as she has provided me with guidance and advice on our collaborated efforts. I am also grateful to my entire Ph.D research committee members, including Dr. Ben Smith, Dr. Satya Narayan, Dr. Gail Fanucci and Dr. Nicole Horenstein for their suppor t and constructive comments that have helped me to refine this dissertation. I thank the support staff at the Department of Chemistry, University of Florida, for their silent contribution during my research work. My special thanks go to Ms. Lori Clark, Dr. Kathryn Williams and the staff at the editorial office and ETD office for their continuing help with numerous tasks during the preparation and submission of this dissertation. During all this years, the Tan group has been like a family away from home. Bot h old and new members have contributed greatly to my academic advancement. I would like to thank Dr. Karen Millians and Dr. MariCarmen Estevez for their unconditional help, their friendship and support during the early stages of my graduate studies. I am a lso grateful to Elizabeth Jimenez, Dimitri Van Simaeys, Basri Gulbakan and Meghan to the most extraordinary member of the Tan group, Dr. Kwame Sefah, as he has not only tr ained and guided me throughout my PhD career, but has also been a true friend through out all these years. I am equally thankful to the entire Tan group members for their friendship, support and encouragement throughout the years.

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5 I thank my friends outside the Tan lab for their friendship and support. To my puertorrican friends, Emelyn Cordones, Sheila Sanchez, Jose Valle, Mariselis Luna, Evelyn Lopez and Rafael Ruiz, thank you so much for your honest friendship and support. To my Zumba friends, Madel Sot omayor, Sandy Highsmith, Michelle Lucas, Mimi Zarate, Marisol, Cecilia Mejias, Nicole Alibrandi, Eric Ocane and Enrico Eastmond, for their unconditional friendship. You all made my life brighter and my problems insignificant. I feel very blessed to have yo u in my life. I would like to give special thanks to my family for their unconditional support throughout my PhD career. I thank my parents for, without them, I would not be here. Thank you for believing in me, and for supporting me. I thank my cousin, Eve lyn Navarro Colon, for her support and friendship during one of the hardest parts of my life. I thank my cousin, Evelyn Lebr n, for her friendship, and her advice during many difficult moments. I also thank my aunts, Magali Estrada and Gloria Lopez, for yo ur unconditional support. Finally, I thank the rest of my family, as, one way or another, each one has helped me throughout my Ph D career. Last but not least, I thank God for putting on my path so many wonderful persons. I have been blessed by having so many great friends and a supportive family. I thank God for giving me the strength to continue on my path even when I encountered moments of adversity.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 14 ABSTRACT ................................ ................................ ................................ ................... 16 CHAPTER 1 BACKGROUND AND SIGNIFICANCE ................................ ................................ ... 18 Ovarian Cancer ................................ ................................ ................................ ....... 18 Causes of Ovarian Cancer ................................ ................................ ............... 19 Risks of Ovarian Cancer ................................ ................................ ................... 20 Hereditary Breast Ovarian Cance r syndrome (HBOC): Breast Cancer gene (BRCA) mutations ................................ ................................ .......... 21 Lynch syndrome or Hereditary Nonpolyposis Colon Cancer (HNPCC) ...... 22 Ovar ian Cancer Subtypes ................................ ................................ ................ 22 Staging and Treatment ................................ ................................ ..................... 23 Symptoms of Ovarian Cancer ................................ ................................ .......... 25 Current Detection Methods ................................ ................................ ............... 26 Early Diagnosis ................................ ................................ ................................ 27 Aptamers ................................ ................................ ................................ ................ 28 Aptamers Characteristics ................................ ................................ ................. 29 Systematic Evolution of Ligands by EXponential Enrichment (SELEX) ............ 29 Aptamer Target Intera ctions ................................ ................................ ............. 30 Cell SELEX ................................ ................................ ................................ ...... 31 Rationale ................................ ................................ ................................ .... 32 Generation of a panel of ap tamers ................................ ............................. 32 Aptamers in Cancer Research ................................ ................................ ......... 33 Overview of Dissertation ................................ ................................ ......................... 37 2 SELECTION OF OVARIAN CANCER APTAMERS BY CELL SELEX ................... 42 Introductory Remarks ................................ ................................ .............................. 42 Materials and Methods ................................ ................................ ............................ 43 Instrumentation and Reagents ................................ ................................ ......... 43 Design of the DNA Library ................................ ................................ ................ 43 Chemical Synthesis of the Random Library by 3400 DNA Synthesizer ............ 44

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7 DNA Deprotection and Preparation for High Performance Liquid Chromatography (HPLC) Purification ................................ ............................ 46 HPLC ................................ ................................ ................................ ................ 47 Polymerase Chain Reaction (PCR) Optimization of the Library and Primer Set ................................ ................................ ................................ ................. 49 Gel Electrophoresis ................................ ................................ .......................... 50 Cell Culture ................................ ................................ ................................ ....... 51 Cell Based SELEX ................................ ................................ ........................... 51 PCR Amplification of the Firs t Selected Pool ................................ .................... 53 Preparation of Single Stranded DNA ................................ ................................ 53 Flow Cytometry ................................ ................................ ................................ 54 Results ................................ ................................ ................................ .................... 55 Design and Optimization of the Selection Library ................................ ............. 55 Monitoring of the Selection Progress ................................ ................................ 57 Concluding Remarks ................................ ................................ .............................. 59 3 BIOCHEMICAL CHARACTERIZATION OF THE SELECTED ATPAMERS ........... 63 Introductory Remarks ................................ ................................ .............................. 63 Materials and Methods ................................ ................................ ............................ 63 454 Sequencing ................................ ................................ ............................... 63 Preparation of the DNA library ................................ ................................ ... 64 Purification of the PCR product ................................ ................................ .. 64 Verification of the DNA product ................................ ................................ .. 65 Sequence Alignment and Selection of Aptamer Candidates ............................ 65 Screening of the Aptamer Candidates ................................ .............................. 65 Confocal Microscopy ................................ ................................ ........................ 66 Determining Aptamer Affinity (binding constant, apparent Kd) ......................... 66 Determining Aptamer Specif icity ................................ ................................ ...... 67 Effect of Temperature on the Aptamer Binding ................................ ................ 67 Competition Assay ................................ ................................ ........................... 67 Internalization Studies ................................ ................................ ...................... 68 Results ................................ ................................ ................................ .................... 68 Analysis of the Sequencing Data ................................ ................................ ...... 68 Binding Affinity ................................ ................................ ................................ .. 69 Selectivity Assay ................................ ................................ .............................. 70 Competition Studies ................................ ................................ ......................... 70 Temperature Effect in Aptamer Binding ................................ ............................ 71 Internalization Studies ................................ ................................ ...................... 71 Concluding Remarks ................................ ................................ .............................. 72 4 APTAMER BASED DETECTION OF OVARIAN CANCER CELLS IN BLOOD ...... 80 Introductory Remarks ................................ ................................ .............................. 80 Materials and Methods ................................ ................................ ............................ 81 Binding in Complex Media ................................ ................................ ................ 81 Detection of spiked ovarian cancer in whole blood ................................ ........... 81

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8 Direct incubation in blood ................................ ................................ ........... 82 Detection of spiked cells after ficoll ................................ ............................ 82 Dual de tection ................................ ................................ ............................ 83 Results ................................ ................................ ................................ .................... 83 Aptamer Binding in Complex Media ................................ ................................ 83 Detect ion of Spiked Ovarian Cancer Cells in Whole Blood .............................. 84 Concluding Remarks ................................ ................................ .............................. 85 5 ELUCIDATION OF THE APTAMER TARGET ON THE CELL MEMBR ANE .......... 90 Introductory Remarks ................................ ................................ .............................. 90 Materials and Methods ................................ ................................ ............................ 90 Protease Treatment ................................ ................................ .......................... 90 Trypsin ................................ ................................ ................................ ....... 91 Proteinase K ................................ ................................ .............................. 91 Inhibitors of Protein G lycosylation ................................ ................................ .... 91 Monensin ................................ ................................ ................................ ... 91 Tunicamycin ................................ ................................ ............................... 92 Swainsonine ................................ ................................ ............................... 92 Benzyl 2 acetamido 2 deoxy D galactopyranoside ............................... 93 Glycosidase Treatment ................................ ................................ .................... 93 PNGase F ................................ ................................ ................................ .. 93 Neuraminidase ................................ ................................ ........................... 94 Fucosidase ................................ ................................ ............................. 94 Treatment of Cells with Azide Labeled Sugars ................................ ................. 94 Results ................................ ................................ ................................ .................... 96 Protease Treatment ................................ ................................ .......................... 96 Inhibitors of Glycosylation ................................ ................................ ................. 97 Glycosidase Treatment ................................ ................................ .................... 97 Treatment of Cells with Azide Labeled Sugars ................................ ................ 98 Concluding Remarks ................................ ................................ .............................. 99 6 CONCLUSION ................................ ................................ ................................ ...... 109 Summary of Dissertation ................................ ................................ ....................... 109 Future Work ................................ ................................ ................................ .......... 110 APPENDIX A SELECTION OF MYCOPLASMA POSITIVE CELLS ................................ ........... 112 Introduction ................................ ................................ ................................ ........... 112 Materials and Methods ................................ ................................ .......................... 113 Instrumentation and reagents ................................ ................................ ......... 113 Cell culture and buffers ................................ ................................ ................... 114 SELEX library and primers ................................ ................................ ............. 114 In Vitro cell SELEX ................................ ................................ ......................... 114

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9 Cloning using TOPO T/A ................................ ................................ ................ 115 Aptamer binding studies by flow cytometry ................................ .................... 116 Results ................................ ................................ ................................ .................. 117 Concluding re marks ................................ ................................ .............................. 118 B CHEMICAL APTAMER CONJUGATION TO THE AAV CAPSID ......................... 121 Introduction ................................ ................................ ................................ ........... 121 Materials and Methods ................................ ................................ .......................... 124 Chemical Conjugation of the Aptamer Capsid ................................ ................ 124 Construction of AAV BAP for biotinylati on of AAV in cells .............................. 124 Aptamer Mediated AAV Infection of CEM Cells ................................ ............. 125 Results ................................ ................................ ................................ .................. 125 Chemical Conjugation of the Aptamer Capsid ................................ ................ 125 Construction of AAV BAP for Biotinylation of AAV in Cells ............................. 125 Aptamer Mediated AAV Infection of CEM Cells ................................ ............. 125 Concluding Remarks ................................ ................................ ............................ 126 LIST OF REFERENCES ................................ ................................ ............................. 131 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 143

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10 LIST OF TABLES Table page 1 1 List of aptamers selected for cancer cell lines ................................ .................... 41 2 1 Summary of the selection progress. ................................ ................................ ... 62 3 1 List of aptamers selected and their affinity ................................ ......................... 79 3 2 Summary of the aptamer selectivity assay. ................................ ........................ 79

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11 LIST OF FIGURES Figure page 1 1 Ten leading cancer types for the estimated new can cer cases and deaths for women, 2010 ................................ ................................ ................................ ...... 38 1 2 The major histologic subtypes of ovarian carcinoma ................................ .......... 38 1 3 Stages of ovarian cancer ................................ ................................ .................... 3 9 1 4 Schematic of cell based aptamer selection (cell SELEX) ................................ ... 40 2 1 Representative structure of a phosphora midite: cytosine ................................ ... 60 2 2 Representative structure of a newly synthesized oligomer ................................ 60 2 3 Gel electrophoresis images ................................ ................................ ................ 61 2 4 Monitoring of the selection progress ................................ ................................ ... 61 3 1 Representation of the 454 primers ................................ ................................ ..... 74 3 2 Representativ e sample of sequence alignment: DOV 1 ................................ ..... 74 3 3 Representation of aptamer candidate binding to CAOV3 cells. .......................... 75 3 4 Confocal microsc opy of CAOV3 cells with TMR labeled DOV 3, DOV 4 and DOV 6a ................................ ................................ ................................ .............. 76 3 5 Aptamer binding to the target cell line at 4 o C and 37 o C. ................................ ..... 76 3 6 Competition studies using excess FITC labeled DOV 2a competing against PE Cy5.5 labeled DOV 2a, DOV 3 and DOV 4. ................................ ................. 77 3 7 Internalization studies by flow cytometry ................................ ............................ 77 4 1 Aptamer binding to CAOV3 cells in complex media. ................................ .......... 87 4 2 Direct incubation of the aptamer DOV 4 for the detection of CAOV3 cells spiked on whole blo od ................................ ................................ ........................ 87 4 3 Detection of CAOV3 cells spiked in whole blood by FACS. ................................ 88 4 4 Dual labeling of CAOV3 cells using EPCAM FITC and DOV 4 PE Cy5.5 ......... 88 4 5 Dual detection of CAOV3 cells spiked in whole blood by FACS ......................... 89 5 1 Effect of protease treatment on the aptamer binding ................................ ........ 101

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12 5 2 DOV 3 binding after treatment of CAOV3 cells with 0.1 mg/mL and 0.5 mg/mL proteinase K. ................................ ................................ ........................ 101 5 3 DOV 4 binding after treatment of CAOV3 cells with 0.1 mg/mL and 0.5 mg/mL proteinase K. ................................ ................................ ........................ 102 5 4 Effect of the treatment with inhibitors of glycosylation on the aptamer binding 102 5 5 PNGase F treatment: Optimization of enzyme concentration ........................... 103 5 6 PNGase F Treatment: Optimization of incubation time ................................ ..... 104 5 7 PNGase F treatment, followed by treatment with proteinase K for 20 min. ...... 104 5 8 Neuraminidase treatment: optimization of e nzyme concentration .................... 105 5 9 Neuraminidase treatment: Optimization of the incubation time ........................ 105 5 10 Neuraminidase treatment, follo wed by treatment with proteinase K for 20 min 106 5 11 Fucosidase treatment, followed by treatment with proteinase K for 20 min. ..... 106 5 12 Structures of azide sugars and biotin alkyne ................................ .................... 106 5 13 Schematic representation of the labeling of carbohydrates with neutravid in via azide alkyne chemistry and biotin neutravidin interaction. .......................... 107 5 14 Aptamer binding after the metabolic incorporation of azide labeled sugars. Aptamer binding was confirmed for untreate d cells, cells labeled with all three sugars, azide glucose, azide mannose or azide galactose. ............................. 107 5 15 Optimization of the alkyne incubation time. Black: untreated cells. Blue: cells treated with alkyne solution for 3 hours. Red: cells treated with alkyne solution for 2 hours. ................................ ................................ .......................... 107 5 16 Effect of proteinase K treatment on the aptamer binding ................................ .. 108 A 1 Monitoring of the selection progress ................................ ................................ 119 A 2 Aptamer binding to mycoplasma negative CAOV3 cells. ................................ .. 119 A 3 Candidate aptamer binding to mycoplasma negative CAOV3 cells .................. 120 B 1 Chemical biotinylation of the AAV capsid ................................ ......................... 127 B 2 Construction of AAV BAP for biotinylation of AAV in cells ................................ 127

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13 B 3 Metabolic biotinylation of the AAV capsid. Biotinylation of AAV particle was confirmed by Western blot analysis ................................ ................................ .. 128 B 4 Transfection of CEM cells using aptamer labeled AAV. Chemical biotinylation of the AAV capsid proteins. ................................ ................................ .............. 129 B 5 Transfection of CEM cells using aptamer labeled AAV. Metabolic biotinylation of the AAV capsid proteins. ................................ .......................... 130

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14 LIST OF ABBREVIATION S 2D PAGE two dimensional polyacrylami de gel electrophoresis CA125 carbohydrate antigen 125 BRCA breast cancer gene CD cluster of differentiation CTC circulating tumor cell Cy5 cyanine derivative 5 Cy5.5 cyanine derivative 5.5 DNA deoxyribonucleic acid dNTP deox yribonucleotide triphosp hate DPBS Dulbecco's phosphate buffered saline EPCAM epithelial cell adhesion molecule FBS fetal bovine serum FITC fluorescein isothiocyanate FL1 channel one on the flow cytometer, emission of 525nm (FITC) FL4 channel three on the flow cytometer, e mission >610 (PE) FSC Forward Scatter HBOC Hereditary Breast Ovarian cancer syndrome HNPCC Hereditary nonpolyposis colon cancer HPLC high performance liquid chromatography IC50 half maximal inhibitory concentration IDT Integrated DNA Technologies MRI magnetic resonance imaging mRNA messenger ribonucleic acid MS mass spectrometry

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15 NP Nanoparticle Nucleotide nt PAGE polyacrylamide gel electrophoresis PBS Phosphate buffer solution PCR polymerase chain reaction QD quantum dot RNA ribonucleic acid SELDI TOF surface enhanced laser desorption/ionization time of flight SELEX systematic evolution of ligands by exponential enrichment siRNA small interfering RNA SSC Side Scatter ssDNA single stranded deoxyribonucleic acid TVS Transvaginal ultrasound

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16 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy APTAMER BASED DETECTION AND CHARACTERIZATION OF OVARIAN C ANCER By Dalia Lopez Colon August 2011 Chair: Weihong Tan Major: C hemistry Whereas ovarian cancer presents only 3% of the new cases of cancer in women it is the 5 th deadliest malignancy overall in the United States. Due to a lack of effective methods t o screen for ovarian cancer, 75% to 80% of ovarian cancers are diagnose in stage III or stage IV. At these stages, the 5 year survival is only 15 20% as compared to an 80 90% survival rate for ovarian cancer diagnosed at early stages of the disease. Theref ore, detection of the ovarian cancer tumor at an early stage will improve the outcome for ovarian cancer patients. For this purpose, we selected a panel of aptamers for the detection and characterization of ovarian cancer. Aptamers are single stranded nucl eic acids (DNA or RNA) capable of recognizing a specific target with high affinity and specificity Aptamers are selected from a random pool containing about 10 15 distinct sequences. During the selection process, unbound sequences are washed away, while se quences binding to the target are eluted, PCR amplified and used for subsequent rounds of selection. This cycle is repeated until the pool has been fully enriched for the target. The enriched pool is then sequenced and potential aptamer sequences are ident ified by looking at sequence homology.

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17 In this work, we have identified six aptamer sequences capable of binding to the epithelial ovarian adenocarcinoma cell line CAOV3. From these sequences, DOV 2a, DOV 3, DOV 4 and DOV 6a were further characterized. The se aptamers showed binding affinities between 30 130 nM. Furthermore, the aptamers specificity was tested with other cancer cell lines. DOV 4 showed no binding to normal immortalized lung epithelial cells, while DOV 2a showed no binding to the ovarian beni gn cysts cell line. The selected ovarian cancer aptamers were further characterized to identify the target in the cell membrane. The aptamer binding was assayed after trypsin, proteinase K and glycosidase treatment. Finally, the aptamers were used to devel op a n ovarian cancer detection assay using whole blood. The assay developed allows the detection of 500 ovarian cancer cells spiked in 1 mL of whole blood with little pretreatment of the blood sample.

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18 CHAPTER 1 BACKGROUND AND SIGNIFICANCE Ovarian Cancer Ovarian cancer is a malignancy in which normal ovarian cells begin to grow in an uncontrolled, abnormal manner and produce tumors in one or both ovaries. It is considered a disease of postmenopausal women, as the peak age of development of ovarian cancer i s between the ages of 75 79. Tumors can develop from germ cells, stromal cells or the connective tissue within the ovaries that generate reproductive hormones, as well as from the epithelia ovarian lining. Epithelial ovarian cancers are the most common typ e of ovarian malignancies, accounting for more than 80% of the ovarian cancer ( 1 ) Ovarian cancer is the fifth most common cause of cancer related death in women, and it is the most letha l gynecologic malignancy ( 2 ) This disease is characterized by few early symptoms, typical presentation at an advanced stage and poor survival statistics. According to the American Cancer Society, there were an estimated 21,880 newly diagnosed cases of ovarian cancer and 13,850 deaths in 2010 (Figure 1 1) ( 3 ) This dismal outcome is due, in part, to the inability to detec t ovarian cancer at an early, curable stage. More than two thirds of patients with ovarian cancer have widespread metastatic disease at initial diagnosis, with a 5 year survival rate of 15 20% Conversely, at early stages, the long term survival rate is a pproximately 90% ( 4 7 ) These statistics provide a compelling basis for development of an effe ctive early detection strategy. There is currently no effective screeni ng strategy for ovarian cancer. The only currently used clinical biomarkers, CA125 ( 8 10 ) and the recently marketed Ova1 test

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19 ( 11 12 ) are not used for general population screening, but only as adjunct tests at the time of clinical symptoms or diagnostic work up of a pelvic mass. Unfortunatel y, not only is it currently im possible to reliably detect ovarian cancer early, it is not even poss ible to accurate ly diagnose it. Even among women presenting with clinical symptoms suggesting the possibility of ovarian cancer, using all available biomarker and imaging tools, it is currently not possible to accurately distinguish women with ovarian cancer from those wh o do not. Thus, a staggering 80% of women who undergo surgery for suspected ovarian cancer do not, in fact, have cancer, but benign disease that could be managed far less invasively, avoiding significant anxiety, distress, morbidity and associated costs ( 11 12 ) This is an issue of major public health impact, given that the American College of Obstetrics and Gynecology estimates that 5 10% of all women in the U.S. will undergo such surgery during their lifetime ( 13 ) Causes of O varian C ancer The molecular events responsible for the initiation and propagation of ovarian cancer are still unknown. Nonetheless, several theories have been postulated as an attempt to explain t he physiological processes that give rise to the malignant transformations leading to ovarian cancer. The most accepted theory is the Incessant Ovulation Theory ( 14 ) This theory suggests that the ovarian surface epithelium undergoes malignant transformations due to continuous ovulation and repair. During her ovulations break the ovarian surface to release an ovule. As the ovarian surface is ruptured, ovarian epithelial cells undergo rapid proliferation to repair the damage. With each, menstrual cycle, the possibility of having mutations accumulate due to errors during the repair process increases, thus leading to an increased risk of ovarian cancer

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20 with age (i.e. the risk of ovarian cancer is a function of the total number of ovulatory cycles) This theory is supported by the observation that reduction of ovulation cycles by pregnancy, lactation and use of birth control reduces the risk of ovarian cancer. ( 15 ) The second theory postulated to explain the initiation of ovarian cancer is the Pituitary Gonadotropin Hypothesis. This theory postulates that the ovarian epithelium is stimulated by high levels of follicle stimulating hormone (FSH) or leuteniz ing hormone (LH) to proliferate in excess, leading to accumulation of deleterious mutations and to eventual malignant transformation. Both hormones are known to result in the production of estrogen, which stimulates the ovarian epit helium to form inclusion cysts. Furthermore, FSH is known to activate many oncogenes. This theory is also supported by the observation that reduction of ovulation cycles by pregnancy, lactation and use of birth control reduces the risk of ovarian cancer, as well as the observatio n that infertility and polycystic ovarian syndrome increase the risk of ovarian cancer, while it decreases with the use of progesterone only birth control ( 4 ) Risks of O varian C ancer As mentioned above, the molecular events that give rise to the malignant transformation of the ovarian surface epithelial cells are unknown. Yet, several risk factors have been associated wi th epithelial ovarian cancer ( 15 ) Some of the risk factors are : Nulliparity Early menarche Late menopause Increasing age Family history Personal history of breast cancer Ethnic background

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21 Women having a larger number of menstrual cycles (ovulation) seem to be at higher risk of ovarian cancer. Thereof, nulliparity (not having conceived), early menarche, late menopause and increasing age are considered risk factors of ova rian cancer ( 16 ) Woman who have not given birth are at double risk of developing ovarian cancer. The protective effect of pregnancy increases with the number of births. This effect is hypothesized to be caused by a reduction in the number of ovulations. It has also been h ypothesized that malignant cells are shed during pregnancy. Ethnic background and race also play a role in ovarian cancer. Although the reason why different races present different risk of ovarian cancer are unknown, it has been observed that white women h ave the highest incidence of ovarian cancer, while black and Hispanic women have the lowest risk. Inherited germline mutations genetic predisposition and personal history of breast cancer also increase the risk of ovarian cancer. Inherited germline mutati ons are the most important and best understood risk factor for ovarian cancer, as risk of ovarian cancer increases from 1.4% in the normal population to 15 60% on woman having a family history of ovarian cancer. These factors will be discussed individually in the following sections ( 5 17 ) Hereditary Breast Ovarian C ancer syndrome (HBOC) : Breast Cancer gene ( BRCA ) mutations Hereditary genetic dis orders account for about 10% of all ovarian cancer cases. The most common genetic disorder in ovarian cancer is the Hereditary Bre a st Ovarian cancer syndrome, which is characterized by BRCA1 and BRCA2 mutations, accounting for 90% of all cases of hereditar y ovarian cancer. Mutations on the BRCA genes increase the risk of ovarian cancer to 60 70% ( 18 ) BRCA1 and BRCA2 are involved in signaling of DNA damage, activation of DNA repair, induc tion of apoptosis and cell cycle

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22 checkpoints ( 19 ) Thus, c ells having aberrant or non functional BRCA have increased aneuploidy (abnormal chromosome number), centrosome amplification and chromosomal aberrations, making them susceptible to further mutations. The HBOC syndrome is particularly common in woman of Ashkenazi ancestry, as 35 40% carry the BRCA1 or BRCA2 mutation, as compared to a 10% occurrence on the general population ( 4 5 ) Lynch syndrome or Hereditary N onpolyposis C olon C ancer (HNPCC) Patients with Lynch syndrome account for 10% of all cases of hereditary ovarian ca ncer (1 2% of all cases). This syndrome is characterized by defects on the mismatch repair mechanism (mutations on repair genes such as MSH2 MSH6 and MLH1) thus resulting in genetic instability and susceptibility to accumulate multiple mutations which c an lead to malignant transformation of the cell ( 4 ) Ovarian C ancer S ubtypes Ovarian cancer is a very heterogeneous disease. In general, ovarian cancer tumors can originate from three dif ferent cell types: Germ cells (5 10%), stromal cells or the connective tissue within the ovaries that generate reproductive hormones (10 15%) and the epithelial ovarian lining (>80%). Most ovarian cancer patients present cancer of epithelial origin; there fore, this introduction will focus on epithelial ovarian cancers and its subtypes Epithelial ovarian cancer is divided into several subtypes: serous, mucinous, endometrioid, clear cell carcinoma, squamous cells mixed epithelial tumors Brenner tumors tr ansitional cell undifferentiated carcinomas and unclassified tumors ( 1 4 5 ) Figure 1 2 shows the f our major histologic subtypes for epithelial ovarian carcinoma : serous, endometrioid, mucinous and clear cell ( 7 ) Although, these subtypes derive from the same cell type, poorly develope d differentiated mesothelial cells forming

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23 the ovarian surface epithelium, they resemble tissues from the female reproductive track in morphology. For instance, serous carcinomas resemble cells from the fallopian tube epithelium, while endometrioid carcino mas resemble endometrial glands. Each tumor subtype is further classified as benign, malignant or borderline; and described as low or high grade malignancy. Research based on genetic and biomarker profiling of ovarian cancer, as well as mRNA expression, h as shown that each subtype has a unique molecular signature ( 20 21 ) This indicates that each ovarian cancer subtype represents a distinct dis ease and should be treated as such in the context of detection, treatment and prognosis. Therefore, proper histological classification is highly important as each subtype has a different behavior and response to therapy. Staging and T reatment Ovarian canc er staging involves the determination of the degree of disease spread on the patient Most ovarian cancers are staged at the time of surgery (Laparotomy), when tissue samples are obtained from different parts of the pelvis and abdomen for diagnosis and sta ging ( 5 22 ) histology and staging, as well as complete or partial tumor removal (cytoredu ction). Also, during the exploratory laparotomy, biopsies of at risk tissues are taken (systematic sampling) to ensure metastasis has not occurred. Staging is imperative as ovarian cancers have a different prognosis at different stages and require a differ ent treatment. The accuracy of the staging may determine the survival of the patient ( 4 ) As shown on Figure 1 3 ovarian cancer is divided into four stages depending on how far has the c ancer spread beyond the ovary. During the first stage of disease, the cancer is limited to one or both ovaries ( F igure 1 3 A). Patients with stage 1 ovarian cancer undergo total abdominal hysterectomy (removal of the uterus) and bilateral

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24 salpingo oopho r ec tomy (removal of both ovaries and the fallopian tubes), omentectomy (removal of the omenetum or abdominal lining), with tumor removal as initial therapy. A biopsy of lymph nodes and other tissues in the pelvis and abdomen is performed to ensure the disease has not spread When ovarian cancer presents in young women, hysterectomy is avoided. Also, if the disease is confined to one ovary the patient undergoes unilateral salpingo oophorectomy (removal of the affected ovary and fallopian tube) leaving the hea lthy ovary intact In all cases, the tumor is classified into the corresponding histological subtype, and classified as low grade or high grade. Pat ients with low grade ovarian cancer do not undergo any further treatment, but remain under observation. I f t he tumor is high grade the patient may receive combination chemotherapy (i.e. intravenous chemotherapy with carboplatin plus taxane) During stage two, the cancer has spread into the pelvic region (fallopian tubes, uterus), but has not yet reached the ab dominal organs (Figure 1 3 B). Patients diagnosed with stage 2 ovarian cancer undergo hysterectomy and BSO with removal of as muc h of the tumor as possible. L ymph nodes and other tissues in the pelvis and abdomen that are suspected of harboring cancer are b iopsied as well After the surgical procedure, treatment may be one of the following: 1) combination chemotherapy with or without radiation therapy or 2) combination chemotherapy. Stage 3 ovarian cancer patients present metastasis into the abdominal organs (Figure 1 3 C). Treatment is the same as for Stage II ovarian cancer. Following the surgical procedure, the patient may either receive combination chemotherapy possibly followed by additional surgery to find and remove any remaining cancer.

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25 Finally, stage four ovarian cancer presents metastasis to distant organs, including the lungs, liver and lymph nodes (Figure 1 3 D). Experts agree that aggressive or radical surgery should be performed to remove as much of the tumor as possible, reaching optimum cytoreduc tion (no visible residual disease). After surgery, the patient is treated with combination chemotherapy (intravenous chemotherapy with carboplatin plus a taxane) ( 7 ) Symptoms of O varian C ancer Ovarian cancer has long been known as the silent killer, as most woman report not having symptoms during early stage ovarian cancer. Yet, o varian cancer does present a series of nonspecific symptoms. These symptoms are typically attributed to other diseases, leading to a delay on ovarian cancer diagnosis. Ovarian cancer symptoms include ( 4 23 ) : Bloating Difficulty eating or feeling full q uickly Pelvic or abdominal pain Abnormal menstrual cycles Constipation Increased gas Indigestion Lack of appetite Nausea and vomiting Sense of pelvic heaviness Swollen abdomen or belly Unexplained back pain that worsens over time Vaginal bleeding Vague low er abdominal discomfort Weight gain or loss Excessive hair growth Increased urinary frequency or urgency

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26 Current D etection M ethods Epithelial ovarian cancer usually presents no symptoms until the disease has spread outside the ovaries. This is largely due to the anatomical position of the ovaries deep inside the pelvis, offering little interference with nearby structures until the tumor has grown significantly or metastasis has ensued ( 4 ) Therefore, the need for a screening method for the early detection of ovarian cancer is imperative. To this end, many ovarian cancer biomarkers have been identified ( 5 24 ) Unfortunately, the diagnostic and prognostic tumor biomarkers in use today are not adequate in distinguish benign from malignant ovarian neoplasma. They also fail to differentiate among the various histological and clinicall y aggressive forms of ovarian cancer ( 25 26 ) To date, there are no screening tests for epithelial ovarian cancer available to the general pop ulation. However, women with a high risk of developing epithelial ovarian cancer are tested in two possible ways: transv aginal sonography and serum biomarker testing ( 27 ) Transvaginal s onography (TVS) is a type of sonogram used to non invasively image the ovary size and shape in detail, facilitating the detection of ovarian cancer tumors. Although the technique has a reported specificity of about 98%, it does not provide adequate sensiti vity as an early detection tool as it is incapable of distinguishing cancerous from benign masses. Therefore, tumors detected using this technique need to be surgically evaluated to make the final diagnosis ( 28 31 ) Furthermore, TVS can only detect tumors which cause an increase in the size of the ovary. Therefore, this technique cannot be used to identify early stage serous ovarian cancer, as this histology is characterized by rapid spread of the disease to other pelvic sites prior to ovarian enlargement. Transvaginal sonography cannot absolutely diagnose cancer.

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27 Early detection of disease based on serum biomarker testing is ideal as it presents a non invasive, cost effective, easily administered and non subjective method. The most commonly used biomarker for clinical screening and prognosis in patients with ovarian canc er is ovar ian cancer antigen 125 (CA 125) ( 7 ) CA125 is a transmembrane glycoprotein of the mucin family which is typically elevated in epithelial ovarian cancer patients. CA 125 serum l evels directly correlate with the protein production in the tumor, indicating that the tumor is undergoing active growth. Although present in the fallopian tubes, endometrium and endocervix, it is not expressed my normal ovarian epithelia. CA 125 is detect ed at low levels (<35 U/mL) in the serum of normal individuals, but its value is elevated in about 80% of women with advanced stage epithelial ovarian cancer. However, it is only elevated in 50% to 60% of women with stage I disease ( 10 32 33 ) Forty percent of patients with early stage ovarian cancer will have a negative CA 125 test result. At the same time, CA 125 is elevated by other gynecological conditions, including pelvic inflammation, endometriosis and ovarian cysts. The high rate of false positive results with CA 125 screening may lead to unnecessary follow up testing and invasive procedures. Th erefore, the only role f or CA 125 testing is in monitoring disease progression and recurrence and in assessing the effects of treatment for ovarian cancer. Similarly, TVS although sometimes used for screening or early detection, is not a reliable too l f or diagnosing ovarian cancer ( 7 ) Therefore, it is imperative to create a new and reliable screening method for the early detection of ovarian cancer. Early D iagnosis The potential benefi ts of earlier diagnosis of ovarian cancer are evident in current 5 year survival statistics: 80% to 90% for stage I tumors, 65% to 70% survival with

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28 stage II disease, 30% to 60% for tumors diagnosed in stage III and 20% or less when diagnosed with stage IV ovarian cancer. Unfortunately, at present, 75% to 80% of ovarian cancers are detected in stage III or stage IV, and the prognosis is poor ( 7 ) Experts agree that development of a targete d screening strategy for effective early detection of ovarian cancer is : 1) likely to require complementary approaches and 2) that an efficient plan for development is to first focus on methodology to accomplish accurate diagnosis (e.g., distinguish, amon g women with clinically suspected ovarian cancer, between those who have it and those who do not) before applying the methods to screening (i.e., can the methods for identification of ovarian cancers associated with clinical symptoms also detect cancers be fore clinical symptoms develop?). This suggests development of methods that are based on : 1) inherited characteristics of disease risk that are present before development of disease and 2) characteristics of cancer cells present at the earliest stages of disease. Aptamers Aptamers are probes capable of specifically binding to biomarkers expressed by targeted tumor cells, and represent a feasible alternative for ovarian cancer early detection and diagnosis. Aptamers are short single stranded oligonucleoti des (<100 bases, RNA or DNA) selected from large combinatorial pools of sequences by SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for their capacity to bind to many types of different targets, ranging from small molecules to proteins o r nucleic acid structures. ( 34 39 ) They have a definite tertiary structure that confers them the selectivity towards their target ( 40 42 )

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29 Aptamers C haracteristics Aptamers present the same high specificity and affinity for their targets as antibodies. With respect to antibodies, aptamers present several advantages for in vivo applications. First, aptamers do not appear to trigger an immune response, which is one of the major limitations of antibodies for in vivo use ( 43 47 ) Second, their smaller size could promote a better tissue penetration ( 48 51 ) Third, aptamers may be chemically synthesized for relatively low prices, allowing better batch to batch reproducibility and easier incorpora tion of chemical modifications, therefore conferring plasmatic resistance to their degradation or improved pharmacokinetic ( 52 55 ) Finally, aptamers are easily developed in a short time as compared to antibodies. Also, multiple aptamers against the different targets in the cell membrane may be developed at the same time This is a very important advantage as using multiple aptamers for the detection of a certain type of cell may decrease the false positive and false negative results. Many aptamers are now being developed against biomedical relevant extracellular targets, such as membrane receptor proteins, hormones, neuropeptides and coagulation factors ( 56 57 ) Furthermore, one aptamer has recently been appro ved by the FDA and several more are now in clinical trials ( 58 62 ) Systematic Evolution of Ligands by EXponential E nrichment (SELEX) Aptamers are selected from a random library of about 10 15 different sequences by the Systematic Evolution of Ligands by EXponential enrichment (SELEX ). Each sequence on the random library contains a stretch of 30 45 random bases (N) flanked by to primer sequences. Because there are four different bases, the pool has a theoretical number of 4 N different sequences. For a library containing 30 random base s, the pool can contain 4 30 or 1.1 x 10 18 different sequences. Nonetheless, preparing a

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30 DNA library with a larger number of random bases improves the library variety. Another fact that needs to be considered is the library concentration used for selection. In order to ensure the pool used for selection indeed contains 10 15 different sequences, a minimum concentration of 10 15 molecules/6.02x10 23 molecules/mole or 1.66 n moles must be used Once the library is prepared, the selection process is started. For t he first round of selection, between 2 20 nmoles of the synthetic library are incubated with the target. A few of the sequences will bind to the target, while most of the sequences will not. Unbound sequences are then removed from the pool. Meanwhile, the bound sequences are eluted and PCR amplified to be used as pools for subsequent rounds. This process is repeated between 10 20 times until the pool is fully enriched for the target. Since some molecules can have similar motifs, a negative selection step ca n be incorporated into the selection process to ensure that the pool will recognize only the target. For this purpose, a counter selection step is added to the selection process. The counter selection step can be performed before or after incubating the ta rget with the pool. During the counter selection step, the pool is incubated with a negative target and the unbound sequences are recovered. The rest of the selection process is continued as described above ( 63 ) Aptamer T arget I nteractions Aptamer target recognition is mainly governed by the three dimensional structure adopted by the aptamer sequence This interaction, which in volves the formation of many wea k and specific interactions w ith the target, confer s and selectivity. In solution, the aptamer adopts the most stable conformation at that temperature, salt concentration and pH. Once the aptamer encounters its target,

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31 hydrogen bonding Van der Waals stac king (for flat ligands) and electrostatic interactions are formed between both species ( 64 ) Electrostatic interactions are especially important when the aptamer target carries a positiv e charge, as charge complementarity creates the perfect environment for the target to bind. Furthermore, the aptamer three conformation whi ch further improves the interact ion between the aptamer and it s target ( 41 65 66 ) the aptamer structure folds to form a binding pocket for the target On the other hand, when the aptamer is binding to a bigger molecule, such as a protein, the aptamer itself adopts a structure which will target typically falls in the nanomolar to sub pico molar range. Cell SELEX The SELEX method has been applied for the selection of aptamers against whole cells. The process of selecting aptamers for whole cells has been te rmed cell SELEX and is shown in figure 2 4 Cell SELEX exploits differences on the membrane of different cells, for example between diseased and normal cells, to generate a panel of aptamers that is capable of differentiating between the two cells. During the selection process, the target cells are incubated with the random unselected library (around 10 15 sequences). Some sequences on this library will bind to the cell, while most will not. Unbound sequences removed by washing the cells with washing buffer. Meanwhile, bound sequences are recovered by heating the cells at 95 o C, followed by centrifugation. These sequences can then be incubated with a counter selection cell line. During counter selection, sequences binding to common markers in the cell membran e are removed, leaving only those sequences selective for the target cell line.

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32 After recovering of the unbound sequences, the selected pool is PCR amplified and used as starting library for subsequent rounds. The selection process is then repeated until t he pool has been fully enriched for the target cell line, while showing no binding to the counter selection cell line. Rationale Disease cells differ from normal cells in many degrees. First, disease cells, such as cancer ous cells undergo mutations in the ir genome, which can add, remove or change one or more bases on the DNA strand These mutations are then carried over to mRNA expression resulting in changes on the mRNA sequence Changes in mRNA sequence can result in changes in the protein sequence duri ng translation, or can result in early termination of protein synthesis C hanges in protein sequence may, in turn, cause changes in protein conformation and/or loss of protein activity. The protein itself may undergo changes in post translational modificat ions, such as glycosylation, methylation, phosphorylation, etc. Furthermore, the protein expression level may also be affected by the disease. Therefore, many changes at the genomic, and protein level exist which can be exploited to select aptamer probes c apable of differentiating between normal and diseased cells. Generation of a panel of aptamers Since the selection process is performed against the whole cell, multiple aptamers binding to different markers in the cell membrane can be selected at the same time. Moreover, because these molecular markers are unknown at the time of selection, the selected aptamers can be used as tools for the identification of new disease related membrane markers for that particular disease. Therefore, the result of the selec tion is a panel of aptamers that can selectively bind to the target cell and not to normal cells.

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33 This panel of aptamers can then be employed for the detection diagnosis and profiling of the disease. The most significant feature of using a panel of marker s for disease detection, diagnosis and profiling is the reduction of false positive and false negative results Because of disease heterogeneity, every patient can present a different expression level and even expression pattern of membrane markers. If onl y one or few of these markers are used for monitoring of disease, the probability of that particular marker having a different expression pattern between two patients is high. On the other hand, if a larger number of markers is used during diagnosis, the o verall error in diagnosis is greatly decreased as the probability of having every single marker changing between different patients is much smaller. Therefore, using cell SELEX to generate a panel of aptamers for the early detection of ovarian cancer is a feasible and valuable alternative for disease diagnosis. Aptamers in C ancer R esearch Aptamers have been used for the detection, imaging and targeted therapy of cancer. Table 1 lists some of the cancer types used for the in vitro selection of aptamers. Seve ral functional aptamers have recently been selected, and their function has been monitored by fluorescence microscopy. Xiao et al. demonstrated that sgc8, an aptamer selected against T cell leukemia (CEM), is internalized into the endosome by observing its colocalization with transferring (a serum glycoprotein known to be internalized via receptor mediated endocytosis) ( 67 ) Li et al used a Cy5 labeled aptamer against angiogenin to monit or the internalization of angiogenin in real time by human umbilical vein endothelial cells (HUVEC) and human breast cancer cells (MCF 7). By monitoring the fluorescent signal from the aptamer by confocal microscopy, the

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34 authors demonstrated that the aptam er angiogenin conjugate was internalized into intracellular organelles ( 68 ) Similarly, an aptamer selected against the mouse transferring receptor wa s used to deliver streptavidin C y5 o L iduronidase (lysosomal enzyme which is deficient in lysosomal storage disease or LSD) into enzyme deficient mouse fibroblasts. The aptamer successfully delivered the streptavidin cy5 into the cell, as observed by confocal microscopy. The aptamer was L iduronidase, as the deficient cell line showed no glycosaminoglycan accumulation after treatment ( 69 ) In clinical settings, aptamer probes can be used for disease diagnosis, in particular for the detection of leukemia cells in blood by fluorescence microscopy and flow cytometry. Aptamers selected against T (Ramos), non cute Myeloid leukemia (HL60) ( 70 73 ) have been shown to recognize leukemia cells in patient samples, showing great promise for their use in profiling for these diseases ( 74 ) Furthermore, leukemia cells (Toledo, Ramos and CEM) were sorted, enriched and detected from a mixture of cells by using a PDMS microfluidic device design containing aptamers for each cell type immobilized in different regions of the device. These cells where shown to be 96% pure, viable, and capable of growing at the same speed as cultured cells. Once immobilized in the PDMS device, each cell line can be individually recovered for further analysis or can be analyzed in situ ( 73 ) Their use as imaging probes has al so been explored. Recently, the Missailidis group published two papers on the use of a 99m Tc labeled anti MUC1 (cell surface mucin glycoprotein) aptamer (AptA) as an in vivo imaging and radiopharmaceutical

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35 probe. In the first report, a new 99m Tc cyclene ba sed chelator was synthesized to improve the stability of the ligand metal complex. Fu r thermore, commercially available chelators were used to create a multi aptamer complex, which showed increased tumor retention and circulation time, while maintaining rap id penetration and low immunogenicity ( 75 ) In the second report, two anti MUC1 aptamers (AptA and AptB) where conjugated to MAG2 and labeled with 99m Tc, and their in vivo biodistributio n was end with an inverted thymidine to provide resistance to nuclease degradation. Both aptamer conjugates showed accumulation in the tumor site and rapid blood clearance ( 76 ) Poly(D,L lactic co glycolic acid) block poly(ethylene glycol) (PLGA b PEG) nanoparticles conjugated to the A10 RNA aptamer, which targets the prostate specific membrane antigen (PSMA), have been developed to target PSMA positive cells in vivo but their potential as imaging probes was not explored ( 77 78 ) A ptamers have also been used as optica l imaging probes in vivo ( 79 80 ) Cy5 labeled TD05, an aptamer selected against the IgM heavy chain expressed in Ramos cells (B cell lymphoma) was administered by tail vein injection into tumor bearing BALB/c nude mice, and whole body fluorescence imaging was used to determine the temporal biodistribution of the Ramos cells. The aptamer probe was shown to circulate through the animal, producing signals in all tissues min after injection. After 4 hours post injection, the non specific signal observed in non target tissues faded, while the tumor signal remained even after 6 hours post injection. To demonstrate the specificity of the aptamer probe, a control aptamer, Cy5 Sgc8a, and free Cy5 dye were also administered, and they cleared from the system, showing no signal in the tumor site afte r 3.5 hours post injection To further

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36 demonstrate the specificity of the TDO 5 aptamer, the aptamer binding to tumors produced from two cell lines, Ramos cells (target cell line) and CEM (non target cell line, T cell leukemia), was compared. The Cy5 labeled TDO5 aptamer accumulated only in Ramos tumors, with minimum non specific accumulation in CEM tumors. Therefo re, these results demonstrate that dye labeled aptamers selected by in vitro cell SELEX are useful probes for in vivo optical imaging, with good specificity, sensitivity and biostability (71) Furthermore, these probes can be further explored as reporting probes for monitoring in vivo cell function and distribution. However, more research is needed to further demonstrate the potential of dye labeled aptam ers for in vivo studies. Nanoparticle based contrast agents have shown great promise, because they can be easily modified with targeting moieties, such as aptamers, as well as with drugs or other reporting molecules, such as organic dyes, for the developme nt of multimodal nanoparticles. For example, Hwang et al developed magnetic fluorescent nanoparticles (cobalt ferrite nanoparticles coated with a silica shell containing rhodamine) conjugated to gallium 67 ( 67 Ga) and the nucleolin aptamer AS1411. The apta mer nanoparticle conjugates were administered by intravenous injection into tumor bearing nude mice, and their biodistribution was analyzed. The conjugates showed rapid blood clearance and accumulation in the tumor site as observed by scintigraphic images and MRI. Furthermore, accumulation of the conjugate was corroborated by fluorescence imaging of the tumor after organ extraction. However, the conjugate was also shown to accumulate in the liver and intestine ( 79 ) Aptamers have been successfully employed for the in vitro and in vivo imaging of live cells, thus providing many potential applications in biomedicine. These probes are

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37 easily synthesized and conjugated to numerous nanomaterial s, such as silica, gold and magnetic nanoparticles and quantum dots, providing them with many characteristics attractive for their use as imaging and drug delivery probes. However, their application is mainly restricted by the limited number of apta mer pro bes currently available. Therefore, effort is needed on the development of new aptamers capable of detecting other diseases, as well as probes that will allow the monitoring of disease progression. Furthermore, incorporation of nuclease resistant nucleotid es into the aptamer design to increase their lifetime in blood could greatly extend their use for in vivo studies. In conclusion, aptamers show great promise as detection, characterization and targeted therapy probes in both academic and clinical settings, and these applications will increase in number as new nanomaterials capable of conjugating to aptamers become available. Overview of D issertation The scope of the research work presented here involves the development of aptamer probes capable of binding a nd recognizing ovarian cancer. The project was initiated by applying the cell SELEX technology to select a panel of aptamers against ovarian cancer cells. The selected aptamers were further characterized to determine their binding affinity and selectivity against other cancer cells. Furthermore, preliminary studies were performed to determine the type of molecule in the cell membrane the aptamers recognize. Once characterized, the best aptamer was used to develop a FACS based cancer detection method from wh ole blood.

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38 Figure 1 1: Ten leading cancer types for the estimated new cancer cases and deaths for women, 2010 Figure 1 2 : The major histologic subtypes of ovarian carcinoma. Serous carcinomas resemble fallopian tube epithelium, endometrioid carcinom as resemble endometrial glands, and mucinous carcinomas resemble endocervical epithelium. Photographs show representative tumor sections stained with hematoxylin and eosin. The shaded circle represents the general anatomical location from which ovarian car cinomas are thought to arise. The pink and blue entities within the cross sected ovary represent maturing ovarian follicles (Adapted from Karst, A. M., and Drapkin, R. (2010) Ovarian cancer pathogenesis: a model in evolution, Journal of oncology 2010 9323 71) 1 1 Copyright 2010 Alison M. Karst and Ronny Drapkin. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, dis tribution, and reproduction in any medium, provided the original work is properly cited.

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39 A B C D Figure 1 3: Stages of ovarian cancer: A) Stage 1 ovarian cancer B) Stage 2 ovarian cancer C) Stage 3 ovarian cancer D) Stage 4 ovarian cancer (Adapted from www.ovarian cancer facts.com )

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40 Figure 1 4 : Schematic of cell based aptamer sele ction (cell SELEX). Briefly, ssDNA pool is incubated with target cells. After washing, the bound DNAs are eluted by heating in binding buffer. The eluted DNAs are then incubated with control cells (negative cells) for counter selection. After centrifugatio n, the unbounded ssDNAs in supernatant are collected, and then amplified by PCR. The amplified DNAs are used for the next round of selection. The selection process is monitored using fluorescent analysis by flow cytometry. The final pool is cloned and the n sequenced (Adapted from Shangguan, D., Li, Y., Tang, Z., Cao, Z. C., Chen, H. W., Mallikaratchy, P., Sefah, K., Yang, C. J., and Tan, W. (2006) Aptamers evolved from live cells as effective molecular probes for cancer study, Proceedings of the National A cademy of Sciences of the United States of America 103 11838 43) 2 2 Copyright by the National Academy of Sciences

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41 Table 1 1: List of aptamers selected for cancer cell lines Cancer type Aptamer Applications Ovarian cancer (CAOV3 and TOV 21G) Multiple: TOV 1 to TOV 10, DOV 1 to DOV 6a Imaging by flow and confocal microscopy ( 81 ) Lung cancer (H69 SCLC and A549 NSCLC ) Multiple Imaging by flow, confocal microscopy and histology ( 82 ) Colon cancer (DLD 1, HCT116, ) Multiple Imagi ng by flow, confocal microscopy ( 83 84 ) (Ramos) Multiple: TDO5 Imaging by flow and confocal microscopy, in vivo imaging, targeted therapy ( 85 ) T cell leukemia (CEM) Multiple: SGC8 Imaging by flow and confocal microscopy, in vivo imaging, targe ted therapy, drug delivery ( 86 87 ) Glioblastoma (U251) Multiple Imaging by flow and confocal microscopy Liver Cancer ( IMEA ) Multiple : TLS11 a Imaging by flow and confocal microscopy ( 37 47 74 88 90 ) Prostate cancer Multiple: PSMA Imaging by flow, confocal microscopy, in vivo imaging, targeted therapy, drug delivery ( 46 91 )

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42 CHAPTER 2 SELECTION OF OVARIAN CANCER APTAMERS BY CELL SELEX Introductory R emarks DNA aptamers that target cell surface markers on the ovarian cancer cell membrane with high affinit y and specificity will expand the repertoire of molecular probes and offer opportunity for molecular targeting. Furthermore, they offer the opportunity for extensive research on the molecular aspect of the disease. Molecular markers that can be used to pr edict therapy effectiveness, residual disease detection and long term prognosis will benefit clinical therapy in maximizing survival and minimizing toxicity. Aptamer probes selected against these molecular markers can be used as a panel to profile ovarian cancer cells and to construct molecular profiles of ovarian cancer. These profiles can be used to classify ovarian cancer into subgroups with favorable or unfavorable prognosis within genetically defined subclasses. These molecular differences possess grea t significance in aiding the understanding of the biological processes and mechanisms of the disease ( 92 97 ) Cell based selection of high affinity aptamers for ovarian cancer involved several rounds of incubation of the live target cell with a randomized pool of DNA sequences (library). After the incubation, washing buffer was used to remove the unbound sequences. Following this step, the bound sequences were eluted, amplified by PCR and used as the enriched library pool for subsequent selectio n. The entire process involved (i) cell based SELEX, (ii) flow cytometric analysis, and (iii) sequencing and alignment of the selected pool ( 46 47 72 74 )

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43 Materials and M ethods Instrumentation and R eagents DNA primers and libraries were synthesized using a n ABI 3400 DNA/RNA synthesizer (Applied Biosystems, Fo ster City, CA) All the reagents needed for DNA synthesis were purchased from Glen Research. Varian Prostar reverse phase HPLC system was used to purify all DNA sequences. PCR was performed on Biorad Thermocycler and all reagents were purchased from Fisher Scientific. The enrichment of the selected pool monitoring, characterization of the selected aptamers and identification of the target protein assays were performed with flow cytometric analysis using a FACScan cytometer (BD Immunocytometry Systems). Des ign of the DNA L ibrary For all aptamer selection s, the process is started by designing a proper DNA library of about 10 15 random sequences. Each library used for aptamer selection is designed to contain the sequence of the forward primer followed by 30 45 synthesizer will chose random bases to place on each N, thus creating the random library) and ends with the complementary sequence of the reverse primer. The primer sequences are constant for all the random sequences contained on the pool and are used for PCR amplification of the pools during selection. The primary or forward primer end for the preparation of ssDNA after PCR amplification. Preparation of the ssDNA will be explained below. The primer and library sequences used for this project were designed using the program Oligo Analyzer 3.0 available online through the Integrated

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44 webpage. When designing the DNA library, several details have to be considered. Because the library has to be denatured at 95 o C during PCR amplification, it cannot form a stable hairpin. To avoid having a library that is too stable, the s elected sequence cannot have a melting temperature above 10 o C. Because the library contains a fluorescein isothiocyanide (FITC) label end, the design avoids having guanine end as it can quench the fluorophore. Each DNA sequence contains two primer sites between 18 20 bases long When designing each primer sequence, care must be taken to not choose a sequence with forms self dimers (binds to itself with three or more consecutive bonds and o ). It is also i mportant that the primers do not form heterodimers (primer 1 binds to primer 2 with three or more consecutive bonds and o ). If the primers form dimers, the dimers will compete with the full sequence for PCR amplification. Furthermore, PCR p refers the amplification of shorter DNA sequences. The primers must anneal into the library at a temperature between 55 60 o C. To obtain such an annealing temperature, the primer must have a GC content of 50% or more. Since we can only choose one annealing temperature on the PCR machine, both primers must have the same annealing temperature (1 o C). As with the library, the primers cannot form stable structures. Therefore, the selected sequences must form hairpins with a melting temperature lower than 10 o C All these requirements were met when designing the library used for the selection. Chemical S ynthe sis of the R andom L ibrary by 3400 DNA S ynthesizer Synthetic nucleic acids (DNA or RNA) are chemically synthesized by solid phase using phosphoramidite chemi stry. In nature, DNA synthesis occurs when the DNA

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45 energy stored on the tri phosphate bond to catalyze the formation of a new phosphodiester bond between the bases. Chemical sy nthesis of synthetic nucleic acids does not involve the use of tri phosphate nucleotides, but rather uses phosphoramidite takes place on a solid support know as controlled pore glass (CPG). This solid support is a non swellable glass bead with high surface area and narrow pore size distribution. The end is covalently bound to the solid support and remains attached until the synthesis is compl eted. The subsequent bases are then introduced as phosphoramidites. In order to avoid the incorporation of the base in the wrong position, all active groups on the phosphoramidite are blocked by protecting groups (Figure 2 1). As seen on figure 2 1 the ox a dimethoxytrityl (DMT) group. This group is also present in the solid support. On the phosphate group on the phosphoramidite is protected with a di i sopropyl amine and 2 cyanoethyl group. Finally the base is protected by the incorporation of a benzoyl group. To initiate the DNA synthesis the DMT protecting group on the solid support needs to be removed prior to the addition of the following base by introducing dichloroacetic acid or trichloroace tic acid. In order to incorporate the next base, the di phosphate group is removed by simultaneously delivering the base and tetrazole (activator). The tetrazole replaces the di isoprolyl amine becoming a good leavi ng group The OH in the previous base (attached to the CPG) can now do a nucleophilic attach to the phosphate group, displacing the di isopropyl amine Since no reaction is 100% efficient, some sequences will fail to

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46 incorporate the base leaving a free OH In order to avoid building oligo mer s with different base composition and length unreacted OH groups are capped using acetic anhydride. These sequences are truncated during this step. Finally, an oxidation step is carried out to oxidize the phosphite linkage formed to a more stable phosphate linkage. This cycle is repeated until the desired oligo mer is synthesized. Because the DMT group has an absorbance at 412 nm the DNA synt hesizer measures DMT absorbance during each base incorporation an d uses this value to calculat e the DNA synthesis efficiency for each base incorporation. As mentioned on the previous chapter, the longer the library is, the more diversity it contains. Therefore, in theory, the library should be as long as possible. Unfor tunately, the DNA synthesis yield decreases with each base incorporation. To have an idea of the overall synthesis yield, assuming every base incorporation has an average yield of 99%, the overall yield for an 80 base long oligo mer would be (99%) 79 (the fi rst base is already present in the solid support) or 45%. Therefore, the length of the DNA library used during selection is limited by the DNA synthesis efficiency. To obtain a good synthesis yield, a lengt h of 70 80 bases is preferred. DNA D eprotection an d P reparation for High Performance Liquid Chromatography ( HPLC ) P urification After DNA synthesis is complete, the newly synthesized oligo mer s remain covalently at end. T he free oxygen in the phosphate group is protected by a 2 cy anoethyl group, the bases remain protected with a benzoyl group and the 3 end is protected by the DMT group (Figure 2 2) Also, the synthesis produc t consists of a mixture of full length oligo mer s and truncated sequences, thus requiring HPLC purification. Before the oligo mer can be used, the oligo mer needs to be cleaved

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47 from the CPG and the protecting groups (except for DMT) need to be removed. This was accomplished by incubating the DNA synthesis pr oduct in an ammonium hydroxide: methylamine (1:1) solution at 65 o C for 30 min The DMT group needs to be retained as it is required for HPLC purification. To prepare the synthesis product for HPLC purification, deprotected DNA was precipitated by incubating in cold ethanol and 3 .0 M sodium chloride (NaCl) for 1 h our at 20 o C. The precipitate was then collected by centrifugation at 4,000 rpm for 30 min at 4 o C. The supernatant was discarded and the DNA was resuspended in 0. 1 0 M triethylamine acetat e ( TEAA ) This solution was centrif uged one more time at 14,000 rpm for 1 min to remove any remaining CPG that may have been transferred during deprotection and precipitation. This step is critical as any CPG remaining in the solution can block the HPLC column. HPLC As mentioned above, the DNA synthesis product consists o f a mixture of full length oligo and truncated sequences. Therefore, all truncated sequences need to be removed and purified full length oligo mer collected. This was accomplished by using reverse phase ion pairing HPLC. A C 18 column (Econosil, 5 4.6 mm) from Alltech (Deerfield, IL) was used as stationary phase along with acetonitrile (ACN) and 0.1 M triethylamine acetate (TEAA) in water as mobile phases on a ProStar HPLC station (Varian, CA) As the stationary phase is non polar, polar compounds e lute faster than non polar compounds. Because DNA is ionic, it does not interact well with the mobile phase by itself. Therefore, the anions on the DNA molecule are paired with triethylene amine cations, making the DNA molecule neutral and more non polar.

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48 The DNA product was introduced into the column and allowed to interact with the stationary phase. As mentioned in the previous section, full length DNA product contains a DMT group on it end. On the other hand, truncated sequences contain an acetate g roup, which is smaller and more polar than the DMT group. The presence of this group aids in the separation of DNA product from the truncated sequences, allowing the full length DNA to interact longer with the stationary phase. Therefo re, the truncated seq uences eluted from the column first. The elution was achieved by using a gradient elution with acetonitrile and 0.1 0 M triethylamine acetate i n water. The chromatography began at 100% ACN, which is non polar. As the chromatography progressed the percentag e of TEAA was gradually increased until the sample was eluted. The gradient used is dependent on the length of the oligo being purified. F or example, to purify an 80 base pair long library, a gradient of 10% TEAA to 60% TEAA in 30 min is typically used. Fo r smaller sequences, a faster gradient is more suited (e.g. 10% TEAA to 85% TEAA in 30 min). The 80 base pair long library was purified using a gradient of 10% TEAA to 60% TEAA in 30 min. Once the DNA oligo was purified, the sample was dried. Because the o ligo mer still contained the DMT group end a final incubation with 80% acetic acid for 30 min was performed to remove the final protecting group. After removal of the DMT group, the acetic acid was evaporated and the DNA was resuspended in water. The DNA concentration in the stock solution was then calculated using the Beer Lambert the DNA oligo, b is the light path of the cuvette and c is the sample concentra tion.

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49 Polymerase Chain Reaction ( PCR ) O ptimization of the L ibrary and P rimer S et Polymerase chain reaction (PCR) is a technique used to exponentially expand DNA. PCR is divided into three stages: denaturation stage, annealing stage and elongation stage PC R uses Taq HS DNA Polymerase. This Polymerase contains a mixture of Taq Polymerase and a monoclonal antibody to Taq Polymerase, which binds to the polymerase until the temperature is elevated. The binding of this antibody prevents nonspecific amplification due to mispriming and/or formation of primer dimers during reaction assembly. The antibody is then denatured in the initial PCR DNA denaturation step, releasing the polymerase and allowing DNA synthesis to proceed. To perform PCR, a 10 stock solution o n both primers (forward and reverse primers were mixed at a 10 concentration each) and a 10 12 M library stock solution (template) were prepared. Following this step, optimization of the number of cycles was performed to obtain the best amplification of the library pool without observing any nonspecific amplification. The PCR samples were prepared using 1x PCR buffer, 125 U/ Hot stat Taq polymerase, primers and library. Four samples were prepared as well as three corresponding control samp les, which contain everything except the library. Each sample and corresponding control was removed from the PCR machine after each desired number of cycles had completed. Once the PCR was completed, the PCR products were analyzed by agarose gel electroph oresis (3% agarose) using ethidium bromide as a dye to observe the DNA bands. The optimized number of cycles is the cycle that gives the brightest band and displays no nonspecific amplification. Once the number of cycles was successfully optimized, the nex t step was to optimize the annealing temperature. The PCR machine is capable of providing with

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50 eight (8) different temperatures for simultaneous PCR amplification. In order to optimize the annealing temperature, a temperature gradient was used with the low est primer melting temperature (57 o C ) 3 o C as the range. The PCR selected the eight temperatures depending on the temperature range. Once the PCR was completed, the PCR products were analyzed by agarose gel electrophoresis using ethidium bromide as a d ye to observe the DNA bands. The optimized annealing temperature is the temperature at which the brightest band is observed without any nonspecific amplification. The optimized annealing temperature was subsequently used for the amplification of the select ed library pool. Gel E lectrophoresis The PCR product was analyzed by resolving the amplified DNA products on a 3% agarose gel by electrophoresis. The DNA was visualized using 0.5 mg/ml ethidium bromide staining, which intercalates with DNA and fluoresces upon exposure to UV light. The 3% agarose gel was prepared by adding 1.2 g agarose to 40 ml of 1 X Tris/Borate/EDTA (TBE) buffer (Fisher Scientific Inc., Pittsburg, PA ) and melted using a microwave for 1 minute. The solution was poured on a gel casting sys tem using a n 8 well comb and allowed to cool down prior to sample analysis. Eight microliters of the PCR product or control (contains PCR buffer, dNTPs and primes, but does not contain template) were mixed with 2 of loading buffer (bromophenol blue and glycerol) and loaded into the gel. A 25 bp DNA ladder was used to estimate the size of DNA as shown in Figure 2 3. The gel was run at a constant voltage (100 V) for 30 min to move the negatively charged DNA throug h the gel towards the positive electrode. The bands were visualized

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51 Cell C ulture Adherent CAOV3 (human ovarian epithelial adenocarcinoma cells) and HeLa (Human cervical epithelial adenocarcinoma cells) were obtained from ATCC (A merican Type Culture Collect ion ). CAOV3 cells were cultured in Medium 199: MCDB 105 mixed in a 1:1 ratio (Sigma Aldrich Co., St. Louis, MO ) HeLa cells were cultured in RPMI 1640 (ATCC Manassas, VA ). Other cells tested for binding to the selected aptamers were cultured as described by ATCC. All media were supplemented with 10% Fetal Bovine Serum (FBS) (Heat Inactivated from GI BCO) and 100 IU/ml penicillin streptomycin. Cell Based SELEX Ovarian cancer cells are adherent cells. This means that they grow attached to each other and to the bottom of the flask. To perform SELEX, the cells need to be suspended in the media by breaking the adhesion interactions between them. This is achieved by incubating the cells with trypsin for 45 sec s. Following trypsin treatment, the cells are placed in the incubator for two hours to regenerate the proteins that may have been digested by the enzyme. Once the cells are suspended in the media, the cell concentration is determined using the heamocytometer. The selection process is then initialized. To perfor m SELEX, the ssDNA library pool (20 nmole s ) is dissolved in binding buffer (4.5 g/L glucose, 5 .0 mM MgCl 2 0.1 mg/mL tRNA and 1 .0 mg/mL BSA, all in DNA is denatured by heat ing at 95 o C for 15 min and is rapidly cooled on ice for 10 min before incubation with the cells. This forces the random library sequences to fold into the most favorable structures. Target cell culture containing approximately 2 3 x 10 6 cells is centrifug ed at 950 rpm for 5 min at 4 o C to pellet the cells. The pellet is washed

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52 twice with washing buffer (5 .0 mM MgCl 2 4.5 g/L glucose, all in 1 X PBS). The cell pellet is then incubated with the DNA library by resuspending the cells in the ssDNA library pool. The incubation mixture is placed on ice in an orbital shaker for 30 min. The cells are then washed three times with washing buffer to remove unbound DNA sequences. The bound sequences are eluted in 500 L binding buffer by heating at 95 o C for 15 min an d centrifuged at 14,000 g for 1 min. The supernatant containing the DNA sequences is then incubat ed with a negative cell line (5 fold excess of positive cell line) to perform a subtraction of the sequences that bind. These sequences are removed since they are not selective binders for the target cell line. The remaining sequences are amplified by PCR using FITC and biotin labeled primers. The selected sense ssDNA is separated from the biotinylated antisense ssDNA by strep tavidin coated Sepharose beads ( 63 ) For the first round selection, the counter selection was eliminated because of the possibly low amount of bound sequences. The entire selection process was then repeated, starting wit h the target positive cells To acquire aptamers with high affinity and specificity, the washing strength was enhanced gradually by extending the wash time (2 5 min), increasing the volume of washing buffer (3 5 mL) and the number of washes (3 5 washes ) T he enrichment of specific sequences was assayed using flow cytometry as expl ained below. Once the library wa s enriched for the target, it was sequenced and subject to a sequence alignment using Clustal X Potential aptamers were identified by selecting se quences from families showing high sequence homology. This is explained in the following chapter.

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53 PCR Amplification of the First Selected P ool During the first round of selection, the pool contains, in theory, only one copy of every sequence. Therefore, if only a portion of this pool is used for amplification and further selection rounds, only a fraction of the sequences present will be carried over to the next round. In order to avoid loosing potential binding sequences, the whole eluted pool on the first round was subject to an initial amplification. The PCR mix was prepared as described above in a final volume of 1 000 and PCR amplified for 10 cycles. After the first PCR amplification, all the PCR products in each tube were pooled together and used as the template for the next PCR procedure. Preparation of S ingle S tranded DNA After PCR amplification, the selected pool was turned into dsDNA, where the sense strand is FITC labeled and the antisense strand is biotin labeled. Since SELEX requires the use o f a n ssDNA po ol, the PCR amplified pool needed to be converted back into ssDNA. For this purpose, streptavidin coated high performance Sepharose beads (GE Healthcare, NJ) were used to make a small affinity column. The dsDN A product was passed through the c olumn five times allowing the binding of biontiylated dsDNA to the streptavidin beads The beads were then washed with 2.5 mL of PBS to remove any remaining forward primer. After washing, the dsDNA was de hybridized with alkaline 0.2 M NaOH solution, rele asing the fluorophore labeled strand (sense strand) In order to remove the sodium salts, a size exclusion NAP was used to desalt the pool The ssDNA was loaded on the column, allowed to interact with it and eluted with water. The larger DNA mole cules were eluted first leaving the salts in the column. The desalted ssDNA was quantified by a UV spectrophotometer and

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54 vacuum dried. The PCR products were re suspended in binding buffer just before use in next round of selection Flow C ytometry Flow cyto metry uses the principles of light scattering, light excitation, and emission of fluorochrome molecules to generate specific multi parameter data from particles and cells in the size range of 0.5 m diameter. Cells are hydro dynamically focused in a sheath of PBS before intercepting an o ptimally focused light source (F igure 8). The sample is injected into the center of a sheath flow. The combined flow is reduced in diameter, forcing the c ell into the center of the stream, one cell at a time. As the cells or particles of interest intercept the light source they scatter light and fluorochromes are excited to a higher energy state. This energy is released as a photon of light with specific sp ectral properties unique to different fluorochromes. Scattered and emitted light from cells and particles are converted to electrical pulses by optical detectors. Forward SCatter (FSC) gives information on relative Size and Side SCatter (SSC) give data on relative granularity or internal complexity. Flow cytometers can detect chromosomes, proteins, or molecules such as nucleic acids if attached to a particle such as a microsphere ( 63 70 84 85 98 ) To monitor the enrichment of aptamer candidates after selection, the F ITC labeled ssDNA pool was incubated with 1 x 10 6 positive or negative cells in 200 L binding buffer and incubated on ice for 2 0 min. Cells were washed twice with binding buffer and resuspended in 250 L buffer. The fluorescence was determined with a FACS can cytometer (BD Immunocytometry Systems) by counting 30,000 events. The FITC labeled unselected ssDNA library was used as a negative control.

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55 Results Design and O ptimization of the S election L ibrary In order to initiate the selection process, a DNA libra ry was designed using the parameters described in the Materials and Methods section. A n 80 nt long ssDNA library with a randomized core of 42 nt flanked on both, the 3' and 5' ends by a 19 mer fixed primer binding sites was designed, synthesized and HPLC pu rified FITC labeled library was synthesized using an ABI 3400 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA) at the 1 state phosphoramidite chemistry as described above The sequence of the library was as follows, 5 ACT CAA CGA ACG CTG TGG A N 42 TGA GGA CCA GGA GAG CAG T The DNA was cleaved off the solid support CPG and ethanol precipitated. Reverse phase HPLC (RP HPLC) was employed to purify the product with the protecting DMT group. An HPLC program with a 0.1 M TEAA gradient of 10% 65% and run time of 30 min was used to purify the library. The HPLC purified product was then deprotected in mild acid ic conditions using acetic acid, vacuum dried and re suspended in water. A Cary Bio 300 UV spectrometer (Varian, Walnut Creek, CA) was used to quantify the DNA. A square quartz cuvette with 1 cm path length was used for the absorption measurements. DNA was stored at 20 C before use. The primers were acquired from IDT technologies. The forward primer was FITC label and the reverse primer was labeled with biotin. The primers were diluted in water to a final concentration of 500 The fo r ward primer was a 19 mer sequence FITC ACT CAA CGA ACG CTG TGG A wi th a melting temperature of 56.8 C and GC content of 52.6 %, an extinction coefficient of 208560 L/mole.cm and a molecular weight of 6359.3 g/mole The reverse primer was a 19 mer

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56 seque nce Biotin ACT GCT CTC CTG GTC CTC A with a melting temperature 57.7 C and GC content of 57.9 % with a molecular weight of 6084.1 g/mole, an extinction coefficient of 163700 L/mole.cm. PCR efficiency is critical in cell based SELEX because eluted DNA se quences for each round of selection have to be amplified by PCR. In order to achieve a reproducible and efficient PCR which yields adequate products, the reaction conditions must be optimized. The PCR was optimized for the annealing temperature, number of cycles as well as the concentrations of all reagents including primers and library. All PCR mixtures are prepared by combining 50 mM KCl, 10 mM Tris HCl (pH 8.3), 1.5 mM MgCl 2 dNTPs (each at 2.5 mM), 0.5 M each primer, and Hot start Taq DNA polymerase ( 5 U /L). Amplifications were carried out in a Biorad 1 Cycler at 95 C for 30 sec (denaturation), 57 C for 30 sec (annealing), and 72 C for 30 sec (extension), followed by a final extension step for 3 min at 72 o C. Once the DNA library and primer set wa s synthesized, the PCR conditions were optimized to determine the annealing temperature for the primer set. In order to determine the annealing temperature of the primer set, the number of cycles was optimized. Seven tubes were prepared, four samples and t hree controls. Tubes were picked after 14, 16, 18 and 20 cycles (F igure 2 3 A) After the PCR, a 3% agarose gel was run and stained with ethidium bromide. Sixteen cycles of amplification was selected because it allowed the amplification of the DNA sequence s with enough product but without any nonspecific amplification, which usually appears as small bands slightly greater or smaller than the required sequences.

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57 Once the number of cycles was optimized, the annealing temperature was optimized using the best number of cycles. To optimize the annealing temperature, a temperature gradient was performed. The temperature gradient was set from 55.3 o C to 60.0 o C. The machine selected the gradient to run as follows: 55.3 o C, 55.7 o C, 56.3 o C 57.0 o C 58.2 o C 59. 1 o C 59.7 o C, and 60.0 o C. After the PCR, a 3% agarose gel was run and stained with ethidium bromide. The best band was observed at 55.3 o C (Figure 2 3 B) Since at low temperatures nonspecific amplification is promoted, a higher annealing temperature wa s used. The annealing temperature used during selection for the PCR amplification of the selected sequences was 57 o C. Due to an increase of non specific amplification during the selection process, the annealing temperature was increased to 60 o C to reduce the non specific bands and improve the PCR yield. Monitoring of the S election P rogress In order to yield aptamers that bind and recognize ovarian cancer cells, the CAOV3 cell line was used as positive cell line and HeLa cells were used as a negative cell line. As we desire to select aptamers selective for ovarian cancer and not normal ovarian cells, the best negative cell line would be a one derived from normal epithelial human ovarian cells. Unfortunately, normal ovarian cell lines are not commercially a vailable. We, therefore, chose HeLa cells as negative control. Selection was started by dissolving 20 nmol of library into 500 L of binding buffer (4.5 g/L glucose, 5 .0 mM MgCl 2 0.1 mg/mL tRNA and 1 mg/mL BSA). Before incubation with approximately 4 x 1 0 6 CAOV3 cells, the DNA pool was denatured at 95 o C for 15 min and quickly cooled on ice. During the first selection round, the unselected pool was incubated with the CAOV3 cells for 60 min at 4 o C with constant shaking. After washing 3 times with 3 mL of washing buffer, the selected pool was eluted in water by heating at

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58 95 o C and PCR amplified. For the first round of selection, the whole pool was PCR amplified using 10 cycles to ensure multiple copies of each sequence are present. This is a critical step, as only part of the pool is PCR amplified and used for the following selection round. After the initial amplification, the number of PCR cycles was optimized and the pool was amplified for an additional 5 cycles of PCR. The amplified pool was used for the following round. This process was repeated for the following rounds, except the initial amplification of the pool is omitted (it is unnecessary as the pool contains multiple copies of each sequence at this point). The washing strength was increase with su bsequent rounds. Also, the incubation time of the pool with the target cell line, as well as the number of target cells used was reduced during subsequent rounds. Negative selection was incorporated during the 15 th round of selection. Negative selection wa s performed for 1 hour at 4 o C prior to positive selection. The selection process is summarized on Table 2 1. The enrichment of the library through successive selection rounds was monitored by flow cytometry. As the selection progresses, the number of seq uences binding to the target cell line increases. Therefore, the enriched pool shows an increase in mean fluorescence intensity. Once the pool shows no increase in fluorescence intensity, the pool has been fully enriched and the selection has been complete d. The selection showed enrichment after 9 rounds of SELEX. (Figure 2 4 A). The pool also showed binding to the negative cell line (Figure 2 4 B). This enrichment was removed by negative selection. After 19 rounds of selection, the pool showed no further i ncrease in fluorescence intensity (Figure 2 4 A). As the selection reached its end, the pool was

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59 sent for sequencing. The sequencing, alignment and aptamer characterization are discussed in the following chapter. C oncluding R emarks An 80 base long library was designed using the IDT Oligoanalyzer 3.1 software. The library was synthesized and purified in our laboratory, while the primers were ordered from IDT. The PCR conditions for the amplification of the library were optimized. After optimization, the libr ary was used for selection. Cell SELEX was successfully applied for the enrichment of a random library for binding to the CAOV3 cell line. Enrichment was observed after 9 rounds of SELEX, with binding to the negative cell line (HeLa). After incorporation o f negative selection, the enrichment for HeLa cells was reduced. The selection reached its end after 19 rounds of SELEX.

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60 Figure 2 1 : Representative structure of a phosphoramidite: cytosine Figure 2 2 : Representative structure of a newly synthesized oligo mer

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61 A B Figure 2 3 : Gel electrophoresis images (A) showing PCR cycles used to determine the best amplification cycle and (B) showing PCR cycles used to determine the best annealing temperature. C = control. A B Figure 2 4: Monitoring of the selec tion progress. (A) Binding of the enriched pools to CAOV3 cells. The red histogram corresponds to cells only. The green histogram corresponds to unselected library. Dark blue corresponds to the 9 th round. Pink corresponds to the 15 th rounds and light blue corresponds to the 19 th rounds. (B) Binding of the enriched pools to HeLa cells. The red histogram corresponds to cells only. The green histogram corresponds to unselected library. The light blue histogram corresponds to the 9 th round. The pink histogram corresponds to the 15 th round. The Yellow histogram corresponds to the 19 th round

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62 Table 2 1: Summary of the selection progress. Round CAOV3 cells Incubation time PCR cycles DNA eluted Wash HeLa 1 8 60 10 + 5 381.5 3 mL 3x N/A 2 7 55 14 150.3 3 mL 3x N/ A 3 6 50 18 934 3 mL 3x N/A 4 5 45 18 265 3 mL 3x N/A 5 5 40 18 252.2 3 mL 3x N/A 6 4 35 10 161.2 3.5 mL 3x N/A 7 3 35 14 266.9 3.5 mL 3x N/A 8 3 35 14 137.8 3.5 mL 3x N/A 9 4 35 16 117.9 3.5 mL 3x N/A 10 3 35 14 503 3.5 mL 3x N/A 11 3 35 16 346.3 3.5 mL 3x N/A 12 3 35 16 153.2 4 mL 3x N/A 13 3 35 18 369.2 4 mL 3x N/A 14 3 35 18 81.7 4 mL 3x N/A 15 3 35 18 213.8 4 mL 3x 10 16 3 35 18 145.3 4 mL 3x 10 17 3 35 18 152.2 4 mL 3x 10 18 3 35 18 134 4 mL 3x 10 19 2 35 20 597 4 mL 3x 10

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63 CHAPTER 3 BIOCHEMICAL CHARACTERIZATION OF THE SELECTED ATPAMERS Introductory R emarks During the selection process, the pool is enriched for sequences that have an affinity for the target cell. Nonetheless, some non binder sequences remain in the pool. It is, the refore, ne cessary to screen the pool for potential aptamer sequences. Those aptamer candidates are selected based on sequence homology; one or two sequence members of the same homologous family are selected, synthesized and tested for binding to the target and control cell line. Once the aptamer sequences have been identified, each aptamer is further characterized to determine binding affinity and selectivity. In this chapter, the biochemical characterization of the aptamers selected against CAOV3 cells is described. Materials and M ethods 454 S equencing 454 sequencing is a next generation sequencing based on sequencing by synthesis. During 454 sequencing, each sequence on the pool is immobilized into a 454 bead. Each bead bound library is then emulsified wit h amplification rea gents in a water in oil mixture. Each unique library sequence is PCR amplified within the bead, resulting in several million copies of each library sequence per bead. Each bead is then removed from the emulsion and loaded onto a Pico Tit er Plate device for sequencing. Each bead is then incubated with sequencing enzymes. The sequencing system flows each individual nucleotide in a set order. For every nucleotide complementary to the template added, a chemiluminescent signal is emitted and r ecorded by the CCD camera. The

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64 signal strength is proportional to the number of nucleotides incorporated in a single nucleotide flow. Each signal is then converted into the sequence for each bead ( 99 ) Preparation of the DNA l ibrary Once the selection was completed and full pool enrichment was achieved, the selected pool s were sequenced to identify the potential aptamer candidates. To sequence the pool s a primer containing the 454 prime rs, a barcode sequence (MID) was synthesized (Figure 3 1) The barcode is used to identify different pools which are sequenced together. These primers were used to PCR amplify the selected pool using FastStart High Fideli ty PCR System (Roche). To PCR amplify the selected pool using 454 primers, a PCR mix containing 1X FastStart High Fidelity Reaction buffer, 200 dNTP solution, 0.4 primer solution, 100 pg 10 ng selected pool and 2.5 U of FastStart High Fidelity Enzyme in a final volume of 50 was prepared. The mix was then PCR amplified as described on the previous chapter. Purification of the PCR p roduct T he PCR product was purified using the QIAquick PCR purification kit The PCR purification kit has a maximum binding capacity of 10 ug The maximum elution volume is 30 After PCR amplification, the enriched pool (now double stranded DNA) was diluted in 5 volumes of buffer PB (supplied by the purification kit) to 1 volume of the PCR sample. The QiAquick spin column was placed in the provided 2 mL collection tube and the sample was added and centrifuged at 13,000 rpm for 30 60 s ec The flow through was dis carded and the column was centrifuge d at 13,000 r p m for 30 60 s ec After centrifugation, the column was washed with 750 Buffer PE and centrifuge d at 13,000 rpm for 30 60 s The flow through is discarded and the column is centrifuged for an additional 1 min. The QIAquick column was then transferred to a clean 1.5 mL

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65 centrifuge tube and the DNA was eluted by adding 50 B uffer EB (10 mM Tris CL, pH 8.5) or water (pH 7.0 8.5) to the center of the QIAquick membrane and centrifuge d the column for 1 min. Verification of the DNA product After purification, the DNA pool was analyzed on a 3% agarose gel to ensure the PCR produc t was the right length and purified. To analyze the PCR product, 10 of the PCR product were mixed with 2 of loading dye, loaded on a 3% agarose gel and separated using a constant voltage of 100 V. The DNA concentration was quantified by measuring absorbance at 260 nm using the following equation: u g/mL = A 260 X 50 u g/mL Once quantified, the PCR product was diluted with tris buffer to a final concentration of 5 ug/mL and submitted for sequencing to the ICBR sequencing core. Sequence A lignment and S election of A ptamer C andidates After sequencing, the primer regions of each sequence were removed and the random p ortions of the library aligned using Clustal X to group the sequences into families wi th sequence homology (Figure 3 2 ). Sequence candidates were selected from each homologous family, synthesized with a biotin la bel and HPLC purified. Other sequences from smaller families were selected as well for screening. Each aptamer candidate was screened for binding to the target cells as described in the following section. Screening of the A ptamer C andidates To determine if the aptamer candidates are indeed binding to the target cell line, 1 x 10 6 target cells were incubated with biotin labeled aptamer or biotin labeled library in 200l binding buffer and incubated at 4 o C for 20 min. After washing twice with washing buffer, cells are incubated with streptavidin PE Cy5.5 for 20min on ice. After the

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66 incubation, cells were washed twice with binding buffer and resuspended in 250l buffer. The fluorescence was determined with a FACScan cytometer (BD Immunocytometry Systems) by co unting 30,000 events. Confocal M icroscopy TMR. The cells were seeded in a glass bottom petri dish and allowed to grow to confluency. After washing twice with 2 mL of washing buffer, cells were incubated with 250 nM aptamer or library solution at 4 o C for 20 min. After washing three times with washing buffer, cells were imaged by confocal microscopy using an Olympus FV500 IX81 confocal microscope (Olympus America Inc., Melville, NY). A 5 mW 543 nM He Ne laser is the excitation source for TAMRA throughout the experiments. The objective used for imaging is a PLAPO60XO3PH 60x oil immersion objective with a numerical aper ture of 1.40 from Olympus (Melville, NY). Determining A ptamer A ffinity (binding constant, apparent Kd) The binding affinity of the aptamers was determined by flow cytometer as explained above. The biotin labeled unselected library was used as a negative control to determine nonspecific binding. All binding assays were performed in triplicates. The mean fluorescence intensity of the unselected library was subtracted from that of the aptamer with the target cells to determine the specific binding of the lab eled aptamer. The equilibrium dissociation constant (Kd) of the aptamer cell interaction was obtained by fitting the dependence of intensity of specific binding on the concentration of the aptamer s to the equation Y = B max X/(K d + X), using Sigma Plot (Ja ndel, San Rafael, CA).

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67 Determining A ptamer S pecificity The selected aptamers were screened against different cell lines to verify selectivity. For this purpose, the selected aptamers were incubated with 1 x 10 6 cells of each cancer cell lines (Table 3 2). The unselected library was used as the negative control. After washing, the binding patter n for each aptamer was determined by flow cytometry as described above. Effect of T emperature on the A ptamer B inding Since the selection is performed at 4 o C, it is important to verify if the aptamer retains its binding to the target cell line at 37 o C. Those aptamers able to bind to the target cell at 37 o C can be used to further develop in vivo studies. Binding assays were performed as described above. To determine binding at 37 o C 1 x 10 6 target cells were incubated with biotin labeled aptamer or biotin labeled library in 200 l binding buffer and incubated at 4 o C or 37 o C for 20 min. After washing twice with washing buffer, cells are incubated with streptavidin P E Cy5.5 for 20 min on ice. After the incubation, cells were washed twice with binding buffer and resuspended in 250 L buffer. The fluorescence was determined with a FACScan cytometer (BD Immunocytometry Systems) by counting 30,000 events. Competition A ss ay As some of the sequences posses some sequence homology, competition studies were performed to the determine if the aptamers bind to the same target. For this purpose, 1 x 10 6 target cells were incubated with 3 (1000X excess) FITC labeled aptamer in 2 00 L binding buffer at 4 o C for 20 min. Biotin labeled aptamer was then directly added to the solution to a final concentration of 250 nM. After washing twice with washing buffer, cells are incubated with streptavidin PE Cy5.5 for 20 min on ice.

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68 After the incubation, cells were washed twice with binding buffer and resuspended in 250 l buffer. The fluorescence was determined with a FACScan cytometer (BD Immunocytometry Systems) by counting 30,000 events. Internalization S tudies There are several examples e xists on the literature showing the internalization of aptamers ( 99 ) Therefore, the selected aptamers were tested for cell internalization. In order to determine if the aptamers selecte d against CAOV3 cells can be internalized, about 2 x 10 6 target cells were wa s h ed twice with washing buffer and incubated with 250 nM biotin labeled aptamer or library solution at 37 o C or 4 o C for one hour. After washing, the cells were incubated with str eptavidin PE Cy5.5. After washing, the cells were analyzed by flow as described above. The internalization was further confirmed using a Zeiss confocal microscopy. Cells were seeded on a glass bottom petri dish (MatTek Corporation, Ashland, MA) to conflue nce. Cells were incubated with 250 nM aptamer PE Cy5.5 or library PE Cy5.5 for 2 hours at 37 o C. Half an hour prior to the end of the incubation, Hoechst 33342 (MP, Solon, OH) was added to a final concen imaged by confocal microscopy. Results Analysis of the S equencing D ata In order to align the sequences, the constant primer regions were removed. This is important because the alignment software will align the pri mer regions rather than the random stretch. Because the 454 sequencing yields a large number of sequences (about 3,000 per pool), a program written in perl (program was written by Dimitri Van

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69 Simaeys ) was used. Once the primer sequences were removed, the c onstant regions were aligned using Clustal X. Three large homologous families and three smaller families were identified. A representative sequence from each family was chosen, synthesized in house with a end and screened for binding to the target and control cell line. From the 15 different aptamer ca ndidates tested for binding, seven sequences showed binding to the target cell line (Figure 3 3 and Table 3 2) Although the enriched pools showed minimum binding to HeLa cells, all the selected sequences showed binding to these cells Since DOV 3, DOV 4 and DOV 6a showed the highest mean fluorescence intensity, their binding was verified by confocal microscopy as described above As shown on F igure 3 4 b oth DOV 3 and DOV 4 showed higher fluorescence intensity than the library. On the other hand, the fluorescence signal obtained with DOV 6a was the same as that observed for DOV 6a. Therefore, both DOV 3 and DOV 4 have the potential of being used as probes for tissue imaging. Binding A ffin ity We determined the apparent dissociation constant for three of the selected aptamers, and all were in the nanomolar range (Table 3 1). These aptamers were select ed because they showed the highest fluorescence intensity. As mentioned on the introduction, aptamers have been shown to have high affinities for their target, ranging from to pM. Therefore, the affinities observed for the aptamers selected are comparable to other aptamers selected against whole cells and are comparable to antibody affinity.

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70 S electivity A ssay Once the aptamer candidates are screened for binding and characterized for binding affinity towards the target, the aptamers need to be screened for selectivity. Theoretically, these aptamers should be selective to only the target cell. Ho wever, it has been reported that one aptamer from a group of aptamers selected with the counter selection strategy could still bind to the negative cell line ( 100 ) This shows that the counter selection may not be efficiently enough to eliminate all nonspecific sequences. In order to determine if the aptamers selected are indeed selective for CAOV3 cells, a ll the selected aptamers were tested for binding to other cancer cell lines. As s hown on T able 3 2, all the aptamers showed binding to several of the cancer cell lines tested. Interestingly, none of the aptamers bind to leukemia cell (CEM and Ramos) or the liver cancer cell line LH 60. Furthermore, all the aptamers, except DOV 2, bind to the counter selection cell line, HeLa. These findings suggest that the aptamers bind to a common marker for epithelial cancer. Competition S tudies Although the aptamers selected are members of different families, we wished to determine if they bound to the same target. To do so, competition studies were performed between DOV 2a, DOV 3, DOV 4 and DOV 6 The binding of the competitor aptamer present in excess was verified by observing the fluorescence intensity of the FITC label on channel one as a means of demonstrating its presence in the cell membrane. Meanwhile, the aptamer binding was monitored on channel three (monitoring the signal from PE Cy5.5). None of the aptamers seem to compete with each other Figure 3 5 shows the results obtained for DOV 2a The signal from excess FITC labeled DOV 2a (pink histograms on the top) remains the same for all the

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71 samples. Meanwhile, when incubating excess FITC labeled DOV 2a with PE Cy5.5 labeled DOV 2a, a reduction in the PE Cy5.5 is observed. This is expected, a s DOV 2a should compete with itself. On the other hand, the signal from PE Cy5.5 labeled DOV 3 and DOV 4 are not affected by the presence of excess DOV 2a. The same procedure was repeated using FITC labeled DOV 3 and DOV 4 (data not shown); similar results were obtained for all the aptamers. Therefore, each aptamer seems to be binding to different targets on the cell membrane. Temperature E ffect in A ptamer B inding The stability of aptamer binding to target cells was also assayed at different temperatures. A s aptamers were selected at 4 o C, a study was done to verify the suitability of using these aptamers at physiological temperature. For this purpose, the aptamers were incubated with the target cells at 4 o C and 37 o C. As shown in Figure 3 6 there was no s ignificant change in fluorescence intensities among the two temperatures tested for DOV 3, DOV 4 or DOV 6a. However, DOV 2a showed a significant decrease in binding intensity when incubated with the target cell line at 37 o C. Nonetheless, all the aptamers retain binding to CAOV3 cells at physiological temperature. This observation is important as it is an indication that all the aptamers can be successfully used in vivo studies. Internalization S tudies Aptamer internalization was assayed by flow cytometry u sing biotin labeled aptamers. Cells were incubated with biotin labeled aptamers at either 4 o C or 37 o C. After washing, the cells were further incubated with streptavidin PE Cy5.5 to labeled the biotin moiety on the aptamer. If the aptamer is internalized, the streptavidin PE Cy5.5 wont be able to bind to the biotin group on the aptamer. If the aptamer is not

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72 internalized, the biotin will remain available for binding to the streptavidin PE Cy5.5. The aptamer incubated at 4 o C serves as the positive control, as internalization is inhibited at this temperature. As shown in F igure 3 7 the aptamers incubated at 37 o C showed a reduced fluorescence signal as compared to the same aptamer incubated at 4 o C. Therefore, the aptamers seem to be internalized. Aptamer i nternalization was further verified by confocal microscopy As shown in Figure 3 8, cells incubated with DOV 3 showed higher signal intensity than the library, thus indicating that the aptamer is binding to the cells. The fluorescence signal from the aptam er was observed at the same plane as the signal coming from Hoesch, the nuclear dye. Furthermore, most of the aptamer fluorescence signal was observed from within the cells inside what seems to be endosomes. Therefore, the DOV 3 is internalized after bindi ng to its target on the cell membrane. Concluding R emarks Seven aptamer sequences have been successfully selected against the CAOV3 cell line. Three of this aptamers were characterized for their bin ding affinity showing apparent K observed for other aptamers selected against whole cells The aptamers were also tested for binding to other cancer cell lines. All the aptamers showed binding to some of the cancer cell lines tested. Alth ough having an aptamer that is selective for ovarian cancer only is ideal, the fact that the aptamers bind to other cancer cells does not mean the aptamers are not useful for disease detection. Indeed, the aptamers have the potential of being used as gener al probes for the detection of cancer. More importantly, since the aptamers show a different binding pattern f or each cell line, this pattern along with the binding patter of other aptamers previously selected for other cancer cell lines

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73 can be used for the profiling of cancer. Furthermore, since the aptamers were shown to be internalized by flow cytometry and confocal imaging they can be used for drug delivery studies.

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74 Forward primer (Primer A Key): TCAG {MID} {Forward primer s equence} Reverse primer (Primer B Key): TCAG {MID} {Reverse primer sequence} Figure 3 1: Representation of the 454 primers Figure 3 2 : Representative sample of sequence alignment: DOV 1

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75 Figure 3 3: Representation o f aptamer candidate binding to CAOV3 cells.

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76 Figure 3 4 : Confocal microscopy of CAOV3 cells with TMR labeled DOV 3, DOV 4 and DOV 6a Figure 3 5 : Aptamer binding to the target cell line at 4 o C and 37 o C.

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77 Figure 3 6 : Competition studies using excess FITC labeled DOV 2a competing against PE Cy5.5 labeled DOV 2a, DOV 3 and DOV 4. Figure 3 7: Internalization studies by flow cytometry

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78 Figure 3 8: Internalization studies of DOV 3 on CAOV3 cells by confocal microscopy.

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79 Table 3 1: List of aptamers selected and their affinity Table 3 2: Summary of the aptamer selectivity assay. Aptamer CAOV3 TOV H 23 LH 60 HCT 116 HeLa Ramos CEM DLD 1 A172 HT29 DOV 1 + ++ ++ + ++ + DOV 2 + + + ++ ++ DOV 3 ++++ ++++ ++++ ++++ +++ ++++ ++++ +++ DOV 4 ++++ ++++ ++++ ++++ +++ ++++ ++++ +++ DOV 5 ++ ++ + + + + DOV 6 a ++ + + ++ + Note: Cy5.5 labeled library was chosen so that 99% of the cells have cy5.5 labeled aptamer was incubated with the cells, the percentage .1 <10%, + 10 30%, ++ 30 60%, +++ 60 80%, and ++++ >80% Aptamer Sequence Kd (nM) DOV 1 ACT CAA CGA ACG CTG TGG ACG TTG GAT GTT AAT TAC ACA TGT ATT TGG GTA TGG TGA GGA CCA GGA GAG CA ND DOV 2 ACT CAA CGA ACG CTG TGG ATA GCG GAC GAA CTT TTA GCA GAA CAC AGC CTC GGC TGA GGA CCA GGA GA G CA ND DOV 2a ACT CAA CGA ACG CTG TGG ATG TGA AAG AAG GAG GAT CTT TAG GCT TGG ACT GTA TGA GGA CCA GGA GAG CA ND DOV 3 ACT CAA CGA ACG CTG TGG ATG CAG AGG CTA GGA TCT ATA GGT TCG GAC GTC GAT GAG GAC CAG GAG AGC A 13030 DOV 4 ACT CAA CGA ACG CTG TGG AGG GCA TCA GAT TAG GAT CTA TAG GTT CGG ACA TCG TGA GGA CCA GGA GAG CA 4020 DOV 5 ACT CAA CGA ACG CTG TGG ATT GGT GTG GAC GGT AGT TGT ACA GGG GGT AAC CAA TGA GGA CCA GGA GAG CA ND DOV 6a ACT CAA CGA ACG CTG TGG AAT GTT GGG GTA GGT AGA AGG TGA AGG GGT TTC A GT TGA GGA CCA GGA GAG CA 4020

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80 CHAPTER 4 APTAMER BASED DETECTION OF OVARIAN CANCER CELLS IN BLOOD Introductory R emarks There is currently no recommended blood screening for ovarian cancer in the general population, due to the l ack of an effective blood test. The only clinically avail able blood tests for ovarian cancer are used in the context of suspected ovarian cancer at the t ime of clinical presentation CA125 and the recently marketed Ova1 test are both used as adjunct tests to aid i n diagnosis of ovarian cancer ( 67 68 ) Unfortunately, neither of these tests is sufficiently accurate for clinical decision making. For example, CA125 is elevated in only approximately 50% o f patie nts with early stage disease ( 32 ) and is elevated in many benign cond itions. As a result, there currently is no effective way to reliably diagnose ovarian cancer short of surgery and more than 50,000 women annually undergo surgery for suspected ovarian cancer when they actually have benign conditions that could be managed much less invasively. Although CA125 blood testing is recommended for cancer screening in women with major pre disposition to ovarian cancer due to inheritance of germline susceptibility mutations (i.e., BRCA 1 or 2 mutations, etc.), this recommendation comes with a clear warning that there is no known effective strategy for early detection ( 67 68 ) Development of a blood based strategy that could reliably diagnose ovarian cancer would be a major improvement in management of this disease. Additionally, it could serve as a foundation for additional investigation of its utility in screening and early detection. A promising strategy for development of such a test might focus on a marker that is found across malignant phenotypes of ovarian cancer, but not in normal or benign conditions such a marker has been elusive. Recent findings regarding exfoliated

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81 tumor cells present in the circulation, and specifically those demonstrating features of stem cells, suggests that these cells migh t represent a fruitful t arget. Such Ci rculating tumor cells (CTCs) have been demonstrated across all malignant cancer types, including ovarian cancer ( 101 102 ) The fact that CTCs can be found in patients before the primary tumor is detected and are not present in the blood of healthy individuals or individu als with nonmalignant disease makes them a very promising target for our efforts to develop an effective blood test for accurate d iagnosis and early detection. Materials and M ethods Binding in C omplex M edia Since the aptamer selection is performed in binding buffer (PBS), it is imperative to determine if their binding will be retained when used in complex media. To asse s s if the aptamer binding is retained in more complex media, aptamer binding studies where performed in cell culture media, 100% FBS and human plasma as described in the previous chapter. In short, about 2 x 10 6 cells were resuspended in cell culture media, FBS or human plasma con taining biotin labeled aptamer or biotin labeled library to a final concentration of 250 nM, and incubated for 20 min at 4 o C. After washing twice with washing buffer, cells were incubated with streptavidin Pe Cy5.5 for 20 min at 4 o C. After washing, cells were analyzed by flow cytometry. Detection of spiked ovarian cancer in whole blood Since DOV 4 shows the best signal intensity by flow cytometry, this aptamer was chosen for further applications. In order to determine if the aptamer DOV 4 can be used for the detection of CAOV3 cells spiked in whole blood, different CAOV3 cell concentrations were spiked in 1 mL of whole b lood ranging from 1million to 1 0,000 cells.

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82 Further treatment of the blood was applied as a means of improvi ng the limit of detection. Direct incubation in b lood In order to determine the limit of detection, 1 mL of whole blood was spiked with different CAOV3 concentrations ranging from 1 million to 10,000. After spiking the blood, biotin labeled DOV 4 or librar y was added to a final concentration of 250 nM and incubated for 20 min at 4 o C without any pretreatment. After washing, the samples were incubated with streptavidin PE Cy5.5 for 20 min at 4 o C. After washing, the samples were analyzed by flow cytometry. A total of 10 million events were counted per sample. Detection of spiked cells after ficoll In order to further improve the detection limit, spiked blood was submitted to a Ficoll gradient to remove red blood cells. For Ficoll gradient, blood was layered o n top of the F icoll layer avoiding mixing of both layers. The samples were then submi tted to centrifugation at 800 rpm for 30 min with the centrifuge break off. Because the red blood cells contain iron, they are heavier than the rest of the blood cells and therefore, pellet at the bottom of the tube. Once the centrifugation was completed, the sample contained thre e layers. The top layer consisted of blood plasma, the bottom la yer contained the F icoll along with red blood cells and the interface contained t he rest of the blood cells along with the spiked CAOV3 cells. In order to recover the blood cells on the interface, the plasma was collected and discarded without removing any of the cells in the interface. The interface was then collected and further wash ed by centrifugation. The bottom layer, containing the Ficoll and red blood cells, was discarded. After washing to remove any remaining Ficoll, the blood cells were incubated with 250 nM biotin labeled aptamer or library for 20 min at 4 o C. After washing, the samples

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83 were incubated with streptavidin PE Cy5.5 as described above. After washing, the samples were analyzed by flow cytometry. A total of 5 million cells were count ed for every sample Dual detection As the detection of CAOV3 cells with the DOV 4 ap tamer had a high detection limit, we explored the use of a dual labeling to further lower the assays sensitivity. Since the CAOV3 cells are EPCAM positive, a dual detection method was developed using EPCAM FITC and DOV 4 PE Cy5.5. For this purpose differen t CAOV3 cell concentrations, ranging from 5,000 to 500 cells, were spiked in 1 mL of whole blood pretreated for the removal of red blood cells by Ficoll gradient and incubated with EPCAM FITC and 250 nM DOV 4 PE Cy5.5 or Isotype FITC and 250 nM Library. A fter washing, the samples were incubated with streptavidin PE Cy5.5 as described above. The samples were washed twice and analyzed by flow. A total of 5 million cells were counted for every sample. Results Aptamer B inding in C omplex M edia Since the aptamer selection is performed in an artificial buffer, the aptamer binding has to be confirmed when used in more complex and biologically relevant media. Therefore, the aptamer binding was confirmed for the incubation with the cells in full cell culture media, 1 00% FBS and human plasma. As observed on Figure 4 1, the aptamers tested retain their binding in the tested medium. Therefore, these aptamers have the potential of being used for the development of a cancer detection method from biological fluids. This pot ential aptamer use was explored in the following sections.

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84 Detection of S piked O varian C ancer C ells in W hole B lood The aptamer DOV 4 was selected to used for the development of a FACS based detection method for ovarian cancer circulating tumor cells. In or der to assess the detection limit of the method, several COAV3 cell concentrations were spiked in 1 mL of whole blood and assayed with DOV 4 for the detection by flow cytometry. The dot plot was used to analyze the data. In order to determine the amount of detected cells, a threshold was set using the l ibrary treated blood sample such that 99% of the cells were within this threshold The number of cells counted plotted against the number of cells spiked showed a linear relationship with an R 2 of 0.97 (Figur e 4 2). As shown in Figure s 4 2 and 4 3 as few as 10,000 CAOV3 cells spiked in 1 mL of whole blood could be detected by Flow without any pretreatment. By using the linear regression obtained by excel, the theoretical limit of detection (LOD) was calculate d by dividing the standard deviation of the line (also known as standard error of the predicted y value for each x in the regression) by the slope and multiplying by three The theoretical LOD was determined to be 380 cells per 1 mL of blood. Yet, this LOD cannot be achieved with the method as described above as the control resembles the sample. Indeed, the sample and control are identical when less than 10,000 cells are spiked in 1 mL of whole blood. In order to further improve the detection limit, the sam ples were subject to a Ficoll gradient to remove red blood cells As red blood cells are the major group of cells present in blood, their removal reduces the sample background. Therefore, a lower detection limit was expected after pretreatment of the sampl es. Unfortunately, the detection limit was not significantly improved (data not shown). This limitation was mainly due to the fact that target cells were not fully separated from the other blood cells in the dot plot. Therefore, a dual labeling using EPCAM FITC and DOV 4 PE Cy5.5 was

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85 used to further separate the target cells fro m the blood cells. As shown on F igure 4 4, the dual labeled CAOV3 cells can be fully separated from the blood cells. By using this method, as few as 500 cells was achieved (Figure 4 5). The number of cells counted plotted against the number of cells spiked showed a linear relationship with an R 2 of 0.99 (Figure 4 5). The theoretical LOD for this method was calculated as described above and was determined to be 91 cells. Nonetheless, t he limit of detection could not be further impr oved because of limitations of f low cytometry. As can be see on Figure 4 5, the analysis of samples spiked with 500 cells yielded the detection of less than 100 cells. The detection of less than 100 cells by f low cytometry is unreliable as the samples resemble the control. C oncluding R emarks We have shown the potential use of DOV 4 in tandem with EPCAM labeling for the detection of circulating ovarian cancer cells in whole blood. The method developed is easy t o perform and requires minimum sample preparation. Nonetheless, the limit of detection is limited to 500 cells per 1 mL of whole blood. In order to consider a circulating tumor cell detection method clinically relevant, a limit of detection of 1 5 tumor ce lls per 1 mL of whole blood must be achieved. Unfortunately, this limit of dete ction cannot be achieved using f low cytometry, as the cell background is too high and the method cannot differentiate between controls and samples The circulating tumor cells are far outnumbered by normal cells in the blood (1 CTC per 10 6 10 7 normal blood cells) especially during the ea rly development of the disease ( 103 ) Therefore, a successful molecular platform must be able to enrich them in addition to detecting them. To accomplish this, we propose utilizing the aptamer selected and conjugated them to magnetic nanoparticles for the specific recognition o f

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86 CTCs/CSCs in ovarian cancer. The work presented here serves as a proof of the feasibility of using the selected aptamers for the detection of ovarian cancer. Therefore, we plan to utilize these ovarian cancer specif ic molecular probes and improve the methodo lo gy for enrichment of CTCs/CSCs and depletion of other blood cells, for the capture and detection of ovarian cancer cells in blood. known. Therefore, the development of a new method showing high capture efficiency will be use therefore, to develop a blood based detection method for ovarian cancer.

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87 Figure 4 1: Aptamer binding to CAOV3 cells in complex media. Figure 4 2: Direct incubation of the apt amer DOV 4 for the detection of CAOV3 cells spiked on whole blood

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88 Figure 4 3: Detection of CAOV3 cells spiked in whole blood by FACS. Figure 4 4: Dual labeling of CAOV3 cells using EPCAM FITC and DOV 4 PE Cy5.5

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89 Figure 4 5: Dual detection of CAOV3 cells spiked in whole blood by FACS

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90 CHAPTER 5 ELUCIDATION OF THE APTAMER TARGET ON THE CELL MEMBRANE Introductory R emarks Since cell based SELEX is performed without prior knowledge of the identity of the target on the cell membrane, it is important to f ind out what type of molecule the aptamer binds to. The most obvious possibility is that the aptamer binds to a protein. This can be confirmed by protease digestion. If the target molecule is a protein, protease digestion often destroys the ability of the aptamer to bind the cells. Using this approach, the targets of several aptamer selected in the Tan group have been identified as membrane proteins (Sgc8 aptamer selected against CCRF CEM cells binds to PTK 7; TD05 aptamer selected against Ramos cells binds to surface membrane protein IgM) ( 37 86 ) These pieces of evidence suggest surface membrane proteins are common candidates for aptamer target s. On the other hand, other biomolecules can be targeted during the selection process. For example, carbohydrate moieties on the cell membrane, as well as lipids can also serve as targets during the selection process. Thereof, these biomolecules should als o be considered during the process of aptamer target identification. Materials and M ethods Protease T reatment As observed by other cell SELEX, the principal aptamer targets are membrane proteins. In order to determine if the aptamers selected bind to membr ane proteins, the cells were treated with proteinase K and trypsin prior to performing aptamer binding assays. Trypsin is a serine protease with substrate selectivity for the carboxyl side of lysine and arginine. Meanwhile, proteinase K is a broad spectrum serine protease.

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91 Trypsin For trypsin digestion, approximately 5 x 10 6 CAOV3 cells were dissociated with non enzymatic buffer, washed with PBS and then incubated with 1 mL of 0.05% trypsin/0.53 mM EDTA in HBSS for 0, 5, 15 and 20 min at 37 o C. After incub ation, t he activity of the proteinases was quenched with FBS. After washing with binding buffer, the treated cells were used for aptamer binding assay using flow cytometer as described above. Proteinase K For proteinase K, approximately 5 x 10 6 positive c ells were dissoci ated with 1 mL of 0.05% trypsin/ 0.53 mM EDTA in HBSS washed with PBS containing 10% FBS and then incubated with 1 mL 0.1 mg/mL or 0.5 mg/mL proteinase K in PBS at 37 o C for 0, 5 15 and 2 0 min. The activity of the proteinases was quenche d with FBS. After washing with binding buffer, the treated cells were used for aptamer binding assay using flow cytometer as described above. Inhibitors of P rotein G lycosylation In order to determine if the aptamer target is a glycoprotein, the cells were treated with four different inhibitors of glycosylation. Two inhibitors of O linked glycosylation and two inhibitors of N linked glycosylation were incubated with CAOV3 cells for 24 hours. After incubation, the cells were assayed for aptamer binding as de scribed above. If the target is a glyc oprotein, a reduction in the ap t a observed Monensin Monensin transports ions, such as Li + Na + K + Rb + and Tl + across the cell membrane, blocking protein transport. CAOV3 cel ls were seeded on a petri dish and

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92 incubated with 1 2 and 5 monensin for 24 hours. After the incubation, the cells were tested for binding to the aptamer as described above. Untreated cells were used as positive control. If the aptamer binds to a glycoprotein, a reduction in the mean fluorescence intensity should be observed. Tunicamycin Tunicamycin inhibits GlcNac phosphotransferase (GPT), blocking all N linked glycosylation and inhibiting the synthesis of all N linked glycoproteins. CAOV3 cells were seeded on a petri dish and incubated with 1 tunicamycin for 24 hours. After the incubation, the cells were tested for binding to the aptamer as described above. Untreated cells were used as positive control. If the aptamer binds to a N linked carbohydrate moiety from a glycoprotein, a reduction i n the mean fluorescence intensity of the aptamer, as compared to untreated cells, will be observed. Swainsonine Swainsonine is a known inhibitor of N linked glycosylation. It inhibits glycoside hydrolases and disrupts Golgi alpha manosidase II This inhibi tor has been previously used in the literature to determine if the target of interest is a N linked glycoprotein (104, 105) CAOV3 cells were seeded on a petri dish and incubated with 5 and 10 swainsonine for 24 hours. After the incubation, the cells were tested for binding to the aptamer as described above. Untreated cells were used as positive control. If the aptamer binds to a N linked carbohydrate moiety from a glycoprotein, a reduction in the mean fluorescence intensity of the aptamer, as compared to untreated cells, will be observed.

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93 Benzyl 2 acetamido 2 deoxy D galactopyranoside Benzyl 2 acetamido 2 deoxy D galactopyranoside is a know inhibitor of O linked glycosylation. Also inhibits 2,3(O) sialyltransferase and disrupts glycoprotein targeting (104) CAOV3 cells were seeded on a petri dish and incubated with 1mg/mL b enzyl 2 acetamido 2 deoxy D galactopyranoside for 24 hours. After the incubation, the cells were tested for binding to the aptamer as described above. Untreated cells were used as positive control. If the aptamer binds to a n O linked carbohydrate moiety from a glycoprotein, a reduction in the mean fluorescence intensity of the aptamer, as compared to untreated cells, will be observed. Glycosidase T reatment C arbohydrate moieties on the surface of membrane proteins (glycoproteins), as well as lipids (glycol ipids) present possible candidates for the aptamer target. To address if the aptamer recognizes a carbohydrate moiety on the surface of the protein, a series of glycosidases (Sigma Aldrich Co., St. Louis, Mo ) were used to digest all carbohydrate moieties o n the ce ll membrane. PNGase F PNGase F is a glycosidase capable of removing most N linked carbohydrate moieties. This enzyme removes the full length N linked carbohydrate. For the PNGase F digestion, approximately 2 x 10 6 positive cells were resuspended w ith non enzymatic buffer, washed with PBS and then incubated with 5,000 to 50,000 U of PNGase F in reaction buffer (provided with the kit) at 37 o C for 1, 2 and 3 hours. The concentration of the glycosidases and the incubation time were optimized to obtain maximum carbohydrate digestion without affecting the cell viability. After washing with binding

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94 buffer, the treated cells were used for aptamer binding assay using flow cytometry as described above. Neuraminidase Neuraminidase is a n O linked glycosidase. Although PNGase F is capable of removing the full length N linked sugars, no such O linked glycosidase exists. Since most O linked carbohydrates contain a sialic acid moiety at the end of the chain, cells were treated with Neuraminidase. Neuraminidase is a glycosidase that cleaves the glycosidic linkage of neuraminic acids (or sialic acid). For the Neuraminidase digestion, approximately 2 x 10 6 positive cells were resuspended with non enzymatic buffer, washed with PBS and then incubated with 50 and 150 U o f Neuraminidase in reaction buffer (provided with the kit) at 37 o C for 1, 2 and 3 hours. The concentration of the glycosidases and the incubation time were optimized to obtain maximum carbohydrate digestion without affecting the cell viability. After wash ing with binding buffer, the treated cells were used for aptamer binding assay using flow cytometry as described above. Fucosidase A lpha 1,2 Fucosidase is a glycosidase which releases alpha 1,2 fucose from the non reducing end of complex carbohydrates For the Fucosidase digestion, approximately 2 x 10 6 positive cells were resuspended with non enzymatic buffer, washed with PBS and then incubated with Fucosidase in reaction buffer (provided with the kit) at 37 o C for 1 hour After washing with binding buf fer, the treated cells were used for aptamer binding assay using flow cytometry as described above. Treatment of C ells with A zide L abeled S ugars Click chemistry by azide alkyne recognition is a very selective reaction. The metabolic incorporation of azide labeled sugars (Figure 5 12A)

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95 carbohydrates has been previously explored ( 106 107 ) Their incorporation into the c ell does not affect cellular functions, thus allowing the use of these artificial sugars in vitro as well as in vivo ( 106 ) Many alkyne reagents exist that can be chemical conjugated to these azide sugars, such as alkyne biotin (Figure 5 12B) thus allowing the modification of these sugars for multiple applications To determine if the aptamer target is carbohydrates were labeled with biotin groups v ia click chemistry and further conjugated to neutravidin (Figure 5 13) If the target of the aptamer is indeed a carbohydrate, the neutravidin molecule will block access to the target, and a reduction in the mean fluorescence intensity will be observed. Th e cell labeling was accomplished by metabolically labeling all the carbohydrates on the cell with azide labeled glucose, galactose and mannose (Fisher, Rockford, Il) In order to incorporate the azide label to the cells, t azide labeled sugars (Figure 5 12A) for 7 passages to allow complete incorporation of the non natural carbohydrate moieties. After incubation, the aptamer binding was verified by incubating 5 x 10 5 cells with 250 nM biotin label ed aptamer or library solution for 20 min at 4 C. After washing, the cells were incubated with streptavidin PE Cy5.5 for 20 min at 4 C. After washing, the cells were analyzed by flow cytometry as described above. A total of 20,000 events were counted. On ce the cells were labeled with the azide sugars, all sugars were labeled with a biotin group using click chemistry by incubating 3 x 10 6 alkyne reagent ( Fisher, Rockford, Il, Figure 5 12B) for 2 hour or 3 hours at room temperature. After washing, the biotin labeled sugars were block ed by incubating

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96 with 5 mg/mL n e u travidin solution dissolved in BB. In order to monitor the incorporation of the azide sugars into the cell, streptavidin Alexa 488 was added to the neutra vidin After washing, streptavidin was blocked with 1 mM biotin solution prior to aptamer incubation. After washing, the cells were incubated with 250 nM biotin labeled aptamer or library solution for 20 min at 4 C. After washing, the cells were incubated with streptavidin PE Cy5.5 for 20 min at 4 C. After washing, the cells were analyzed by flow cytometry as described above. A total of 20,000 events were counted. To further determine if the decrease in aptamer bindi ng was due to the due to steric interference, the cells were treated with 0.1 mg/mL proteinase K for 20 min oteins prior to treatment of the cells with the alkyne biotin reagent and introduction of the neutravidin group as described above. Results Protease T reatment Trypsin and Proteinase K have previously been used to determine if the aptamer target on the cell membrane is a p rotein ( 37 86 ) In order to determine if the aptamers selected for CAOV3 bind to a membrane protein, cells were incubated with trypsin or proteinase K and tested for binding to DOV 2a, DOV 3, DOV 4 and DOV 6a. As can be seen on Figure 5 1, the aptamer binding is conserved after treatment with both proteases. Furthermore, the mean fluorescence intensity increases after proteinase K treatment. In order to determine if the protease concentration used was insufficient, cells were also treated with a higher proteinase K concentration (0.5 mg/mL). As shown

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97 in Figures 5 2 and 5 3, the aptamer binding for DOV 3 and DOV 4 (respectively) is retained and no difference is observed between the treatment with 0.1 mg/mL or 0.5 mg/mL. These results suggest that the aptamer target is either not a cell membrane protein, or the protein is not accessible to proteases Inhibitors of G lycosylation Since protease cleavage might be hindered by heavy glycosylation the cells were further treated with inhibitors of glycosylation. The inhibitors were selected to have different effects on the protein glycosylation and transport. Monensin was selected as an inh ibitor of prot ein transport. Tunicamycin and s wainsonine were selected as both inhibit N linked glycosylation. Finally, Benzyl 2 acetamido 2 deoxy D galactopyranoside was selected as an inhibitor of O linked glycosylation. After treatment with four inhi bitors of glycosylation, the aptamer binding is maintained (Figure 5 4) Because the inhibitors used reduce protein glycosylation and export to the cell membrane, these results suggest the aptamer target is unlikely to be a glycoprotein. Glycosidase T reatm ent Since the aptamer target seems unlikely to be a protein or glycoprotein, carbohydrate moieties on the cell membrane were explored as potential aptamer targets. To do so, cells were treated with different glycosidases : PNGase F, Neuraminidase and Fucosi dase PNGase F is a known N linked glycosidase and was chosen to determine if the aptamer target is a N linked carbohydrate moiety. Three different PNGase F co ncentrations were tested, 5,000, 20,000 and 50,000 U. As shown on Figure 5 5, no ch anges in the a ptamer binding were observed. In order to determine if the glycosidase incubation time was insufficient, the enzyme was incubated with the cells for 1, 2 and 3 hours. As can be observed in Figure 5 6, no change was observed.

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98 As a final assessment of the ef fect of PNGase F on the aptamer binding, cells were treated with 0.1 mg/mL Protei nase K for 20 min at 37 C prior to treat ment of the cells with 50,000 U PNGase F for 3 hours. After treating the cells w ith bo t h the protease and glycosidase, the aptamer bi nding was not affected by the glycosidase treatment (Figure 5 7). As the aptamer binding is retained after treatment with a N linked glycosidase, the possibility of having a n O linked glycosidase as target was explored. Since there is no known O linked gly cosida se capable of removing the full length sugar, cells were treated with Neuraminidase. Two glycosidase concentrations were tested, 50 and 150 U. As shown on Figure 5 8, the aptame r binding improves with the treatment of the cells with Neuraminidase. Th e cells were further treated with 150 U Neuraminidase for 1, 2 and 3 hours, but no change in the aptamer binding was observ ed (Figure 5 9). The cells were also treated with 0.1 mg/mL proteinase K for 20 min at 37 C followed by treatment with 150 U of Neur aminidase for 3 hours. As observed in Figure 5 10, the mean fluorescence intensity further increases after the sequential treatment of the cells with proteinase K and Neuraminidase. Treatment with F ucosidase yielded similar results (Figure 5 11). These res ults suggest the aptamer target is not a N linked carbohydrate moiety, yet the possibility of having the aptamer binding to the core of a n O linked protein remains Treatment of Cells with Azide Labeled Sugars In order to verify if the incorporation of t he azide group on the sugar moieties after metabolically labeling CAOV3 cells with azide labeled glucose, mannose or galactose, as well as cells labeled all three suga rs. As shown on Figure 5 13, the

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99 incorporation of these non recognizing its target on the cell membrane. The incorporation of the azide group into the cell was corroborated by incubating the cells w ith streptavidin Alexa 488 (data not shown). In order to ensure full labeling of the azide sugars with the alkyne biotin moiety, t he alkyne biotin concentration was optimized to 250 nM. Furthermore, the alkyne incubation time was optimized to 3 hours (Fig ure 5 14). As shown on Figure 5 14, treatment of the azide labeled cells with 250 nM alkyne solution for 3 hours, followed by incubation with 5 mg/mL neutravidin solution resulted in a decrease in the aptamer binding to the target cells. To further determ ine if the decrease in aptamer binding was due to the or if the effect is merely due to steric interference the cells were treated with 0.1 mg/mL proteinase K for 20 min at 37 C to remove cells with the alkyne biotin reagent and introduction of the neutravidin group. As shown on Figure 5 15, treatment of the cells with proteinase K prior to the introduction of the neutravi din group results in lower mean fluorescence intensity, thus a reduction in the aptamer binding is observed. Since previous experiments showed that treatment of the cells with proteinase K result in an increase in the aptamer binding, this reduction in apt amer binding must be due to the blocking of the aptamer target by the neutravidin group. Concluding R emarks Initial efforts to identify the aptamer target have been explored. Cell treatment with proteases showed no effect in the aptamer target, suggesting the aptamer target is unlikely to be a membrane protein. Furthermore, treatment with inhibitors of

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100 glycosylation further disproved glycoproteins as potential aptamer targets. Although the cells were treated with glycosidases to determine if the aptamer tar get is a carbohydrate, the results are inconclusive. Treatment of the cells with PNGase F showed no change in the aptamer binding. This can be due to the fact that PNGase F is not as efficient when used in native proteins. Therefore, other N linked glycosi dases capable of cleaving N linked sugars from native proteins need to be explored. Furthermore, treatment with Neuraminidase further improves the aptamer binding to the cells. These results may suggest the aptamer might be binding to the core of an O link ed carbohydrate. On the other hand, since sialic acid has a negative charge and DNA is a poly anion, the removal of sialic acid residues with Neuraminidase may be improving the aptamer binding by reducing the repulsion between equal charges. The results ob tained by blocking the carbohydrate moieties with neutravidin suggest the aptamer target may be oligosaccharide. Yet, f urther treatment with other glycosidases and the use of a cocktail of glycosidases such as N DEGLY and E DEGLY (S igma Aldrich Co., St. L ouis, Mo ), will be explored to further prove or disprove carbohydrates as the aptamer target.

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101 A B Figure 5 1 : Effect of protease treatment on the aptamer binding. (A) Treatment of CAOV3 cells with trypsin for 20 min at 37 o C (B) Treatment of CAOV3 cells with Proteinase K for 20 min at 37 o C Figure 5 2: DOV 3 binding after treatment of CAOV3 cells with 0.1 mg/mL and 0.5 mg/mL proteinase K.

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102 Figure 5 3: DOV 4 binding after treatment of CAOV3 cells with 0.1 mg/mL and 0.5 mg/mL proteinase K. A B Figur e 5 4 : Effect of the treatment with inhibitors of glycosylation on the aptamer with 1 mg/mL Benzyl 2 acetamido 2 deoxy D galactopyranoside (C) treatment with Swainsonine

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103 C Figure 5 4 : Con tinued Figure 5 5 : PNGase F treatment: Optimization of enzyme concentration

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104 Figure 5 6 : PNGase F Treatment: Optimization of incubation time Figure 5 7 : PNGase F treatme nt, followed by treatment with p roteinase K for 20 min.

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105 Figure 5 8 : Neuramin idase treatment: optimization of enzyme concentration Figure 5 9 : Neuraminidase treatment: Optimization of the incubation time

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106 Figure 5 10 : Neuraminidase treatme nt, followed by treatment with p roteinase K for 20 min Figure 5 11: Fucosidase treatme nt, followed by treatment with proteinase K for 20 min. A B Figure 5 12: Structures of azide sugars and biotin alkyne: (A) Azide sugars used. (B) biotin alkyne reagent used.

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107 Figure 5 13: Schematic representation of the labeling of carbohydrates with n eutravidin via azide alkyne chemistry and biotin neutravidin interaction. Figure 5 14: Aptamer binding after the metabolic incorporation of azide labeled sugars Aptamer binding was confirmed for untreated cells, cells labeled with all three sugars, azi de glucose, azide mannose or azide galactose. Figure 5 15: Optimization of the alkyne incubation time. Black: untreated cells. Blue: cells treated with alkyne solution for 3 hours. Red: cells treated with alkyne solution for 2 hours.

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108 A B Figure 5 16: Effect of proteinase K treatment on the aptamer binding. (A) Treatment with proteinase K followed by treatment with PNGase F. (B) Treatment with proteinase K followed b y treatment with Neuraminidase.

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109 CHAPTER 6 CONCLUSION Summary of D issertation A panel o f six aptamers binding to CAOV3 cells was selected by cell SELEX. Each aptamer was characterized to determine binding affinity and selectivity. The aptamer affinities lie between 40 and 13 0 nM. The aptamers showed cross reactivity to other cancer cell line s, including the negative cell line (HeLa). Although the aptamers bind to other cell line, they show no binding to normal blood cells. Furthermore, one of the selected aptamers showed no binding to a ovarian benign cyst cell line, while a second aptamer sh owed no binding to normal epithelial lung cancer. Therefore, their use as probes for the enrichment and detection of circulating tumor cells in blood remains a potential application for disease detection. This application was explored and a proof of concep t method for the detection of ovarian cancer cells spiked in whole blood was developed using flow cytometry. After testing the aptamer DOV 4 and EPCAM as dual labeling for the detection of circulating tumor cells by Flow cytometry, a limit of detection of 500 cells per milliliter of whole blood was observed. Thereof, this aptamer shows great potential for the detection of circulating tumor cells. Nonetheless, their utility needs to be validated using real samples. On the other hand, the aptamer target on th e cell membrane needs to be identified. Our initial efforts of identifying the aptamer target have shown that the target is unlikely to be a membrane protein as the aptamer binding is retained after treatment with trypsin and proteinase K Treatment of th e cells with inhibitors of glycosylation, as well as treatment with several glycosidases showed no effect in the aptamer binding. Nonetheless, f urther testing of the cells with different glycosidases needs to be performed.

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110 Future W ork As the aptamer target remains elusive, further efforts will be done to identify it. Other glycosidases will be used for the cleavage of carbohydrate moieties on the cell membrane. Furthermore, the glycosidases will be used as a cocktail to ensure full removal of the carbohydra te moieties. If the aptamer bi n ding remains after further glycosidase treatment, the target is more likely to be a lipid. As an attempt to identify the target lipid, purified commercially available lipids will be used to form micelles. The micelles will be tested for binding to the aptamers. Furt her aptamer applications will also be explored. A mice protocol was written and approved by the IACUC (protocol # 200801491). Here, we propose to grow CAOV3 solid tumors in nude mice. The tumors will then be imaged in vivo using DOV 4 conjugated to a NIR dye or a NIR dye labeled magnetic nanoparticle. The use of aptamers in conju n ction with ultrasound contrast agents will also be explored. Furthermore, the aptamer will be immobilized into a nanoparticle containing ci splatin for the targeted therapy of CAOV3 solid tumors. The aptamers selected will be further used for the profiling of ovarian cancer. Before the aptamers can be used for profiling, they have to be validated for the binding and detection of ovarian cancer patient samples. In order to accomplish this goal, patient samples provided by the H. Lee Moffitt Cancer Center and Research Institute will be assayed with the aptamers. Those aptamers that are capable of recognizing the ovarian cancer patient samples wil l be used to create a profile for ovarian cancer staging. Each aptamer will be assayed for binding to patient samples from all four stages to determine which aptamers bind to each stage. The binding pattern of each aptamer will be analyzed and correlated t o the different stages of ovarian cancer.

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111 In order to accomplish this goal, 50 patient samples will be evaluated. Also, 20 samples from normal epithelial cells will be used as negative controls. To perform the analysis, the tissue samples will be suspende d in solution using non enzymatic cell dissociation solution Cell Strainer (BD Falcon). Once the cells are put into solution, 10 5 10 6 cells from each patient sample and negative control will be washed three times with washing buffer and incubated with 250 nM FITC labeled aptamer in binding buffer on ice for 20 min. After washing the samples three times with washing buffer, the aptamer binding will be determined using flow cytometry as explained above. The binding pattern for each aptamer will be evaluated and correlated with the sta ge of ovarian cancer represented by each patient sample. Once this task is successfully completed, the aptamer panel can be used not only to diagnose, but to stage ovarian cancer. This is i mportant as the treatment of ovarian cancer depends on the cancer stage and classification. Also, the development of multiple aptamers capable of recognizing ovarian cancer will provide with the selectivity and sensitivity needed for the development of an assay for early detection of ovarian cancer. Using this panel of a ptamers for early detection will provide with lower false negative and false positive results.

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112 APPENDIX A SELECTION OF MYCOPLASMA POSITIVE CELLS Introduction Mycoplasma is a genus of bacteria frequently present in cell cultures around the world, and pres ent a serious problem in cell based research ( 108 ) These bacteria contain no cell wall, making them resistant to most antibiotics, such as penicillin. Because of their small size (arou nd 0.3 0.8um), mycoplasma cannot be observed by phase contrast microscopy. Furthermore, unlike other bacterial contamination, mycoplasma species grow very slowly and, therefore, do not cause visible changes to the cell culture media nor destroy the cell cu lture. Because of the lack of visible signs of mycoplasma contamination, it is estimated that 15% of all cultures in the United States are affected ( 109 110 ) Although no visible changes are present, mycoplasma contamination affects nearly every cellular function and parameter, including growth, metabolism, morphology, cell attachment, membrane characteristics and chromosomal changes. Therefor e, p ublications and data gathered using mycoplasma contaminated cells are suspect and subject to scrutiny ( 108 111 112 ) Moreover, many mycoplasma detection methods are commercially available and should be used for routine mycoplasma contamination testing on all mammalian cell cultures ( 110 111 113 115 ) Cell SELEX or cell based Systematic Evolution of Ligands b y EXponential enrichment is a technique used to select aptamer probes capable of binding to cell surface markers. Aptamers are short (around 100nt) single stranded nucleic acids, DNA or RNA, which adopt a well defined three dimensional structure, allowing them to recognize and bind to the target wit h high affinity and specificity ( 37 63 82 116 )

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113 Because this recognition capability depends on the presence of membrane surface markers, the success of cell SELEX can be jeopardized by mycoplasma contamination. In this work, we demonstrate the importance of mycopla sma contamination in Cell SELEX, which was employed to select a pool enriched for mycoplasma positive ovarian cancer cells. The selected aptamers were tested with to mycoplasma negative cells, but no binding was observed. It is, therefore, likely that the aptamer target of the tested sequences was induced by the mycoplasma contamination. Materials and Methods Instrumentation and reagents All oligonucleotides were synthesized by standard phosphoramidite chemistry using a 3400 DNA synthesizer (Applied Biosyst ems) M scale, and were purified by reversed phase HPLC (Varian Prostar) using a C18 column and 0.1 M TEAA in water and acetonitrile as mobile phases. All PCR mixtures contained 50 mM KCl, 10 mM Tris HCl (pH 8.3), 2.0 mM MgCl 2 dNTPs (each at 2.5 mM), 0.5 and Hot start Taq DNA polymerase (5 Thermocycler and all reagents were purchased from Takara. Monitoring of pool enrichment, characterization of the selected aptamers, and identification of the target protein assays were performed by flow cytometric analysis using a FACScan cytometer (BD Immunocytometry Systems). Trypsin and Proteinase K were purchased from Fisher Biotech. The TOPO T/A cloning kit and E. coli were purchased from Fisher. C ells were tested for mycoplasma contamination using the PCR based mycoplasma detection kit from Takara (CAT# 6601). The DNA sequences were determined by the Genome Sequencing Services Laboratory at the University of Florida.

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114 Cell culture and buffers The CA OV 3 and HeLa cell lines where obtained from the American Type Cell Culture (ATCC). The CAOV 3 ovarian cancer cell line was maint ained in culture with MCBD 105: Medium 199 (1:1); the HeLa cell line was cultured in RPMI 1640. All media where supplemented wit h 10% FBS and 100 U /mL Penicillin Streptomycin. All cell lines where incubated at 37 C in a 5% CO 2 atmosphere. During the selection, cells were washed before and after incubation with wash buffer (WB), containing 4.5 g/L glucose and 5 mM MgCl 2 in Dulbecc buffered saline with calcium chloride and magnesium chloride (Sigma). Binding buffer (BB) used for selection was prepared by adding yeast tRNA (0.1 mg/mL) (Sigma) and BSA (1 mg/mL) (Fisher) to the wash buffer to reduce background binding. SEL EX library and primers The HPLC purified library contained a randomized sequence of 40 nucleotides (nt) flanked by two 20 nt primer hybridization sites: ACT ACC AAC GAG CGA CCA CT (N) 40 AGA GTT CAG GAG AGG CAG GT FITC, and the reverse primers were biotin. In Vitro cell SELEX In this study, mycoplasma positive CAOV3 was used as the target cell line and mycoplasma positive HeLa was used for counter selection. For the first round, the cells were incubated with 20 pmol of nave ssDNA library dissolved in BB. For later rounds, 75 pmol of enriched pool dissolved in BB to a final concentration of 250 nM was used for incubation. Before incubation, the ssDNA pool was denatured by heating at 95 C for

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115 5 min and was cooled rapidly on ice for 5 min, allowing each sequence to form the most stable tertiary structure. The cells were washed twice with 3mL of WB and incubated with the DNA pool on ice in an orbital shaker for 30 min. The bound sequences were eluted in 500 L BB by heating at 95 C for 15 min, cooled on ice for 5 min and centrifuged at 14,000 rpm for 2 min. The supernatant containing the DNA sequences was then incubated with a negative cell line (HeLa) for 60 min on ice in an orbital shake r remove sequences binding to general surface markers. The unbound sequences were collected and amplified by PCR using the FITC and biotin labeled primers. Amplifications were carried out at 95 C for 30 s ec 60 C for 30 s ec and 72 C for 30 s ec follow ed by final extension for 3 min at 72 C. The selected sense ssDNA was separated from the biotinylated antisense ssDNA by streptavidin coated sepharose beads (Amersham Bioscience). The ssDNA was eluted from the sepharose beads by melting in a 0.2 M NaOH so lution. As the selection progressed, the cells were washed with increased stringency to remove weakly binding sequences (increased WB volume and number of washes). The enrichment of specific sequences was assayed using flow cytometry as explained below. Wh en the level of enrichment reached a plateau, pools of interest were cloned and submitted for sequencing. Cloning using TOPO T/A The T/A cloning strategy was selected for cloning mainly on the basis of its ease of use pertaining to the cloning of PCR prod ucts. T/A cloning takes advantage of the terminal transferase activity of Taq polymerase, which adds a single 3' A overhang to each end of the PCR product. In order to prepare the PCR product for T/A cloning, the

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116 enriched selected ssDNA pool was subjected to PCR amplification using non labeled primers, as described above. The PC R was performed with a 15 min final extension stage to ensure efficient adenylation of the PCR product. Once the PCR product was fully adenylated, it was then ligated into the TOPO T/A vector by incubating 2 L of fresh PCR product with 1 L of the TOPO vector in ligation buffer in a fin al volume of 10 L for 20 min at room temperature. Then, the plasmid was transfected into E. coli by incubating 2 L of the TOPO cloning reaction mix ture with one shot of chemically competent E. coli provided by the cloning kit at 37 o C for 30 min in an orbital shaker The transformed E. coli was grown in petri dishes containing Luria Bertani (LB) medium supplemented with 15 g/L of agar, 50 g/mL of am picillin and 40 L of 40 mg/mL X gal. Since the plasmid contains a gene that provides resistance to the antibiotic used in the medium, only the bacteria that contain the plasmid will grow on the medium. As the PCR product is inserted inside the galactos idase gene in the plasmid, those bacteria containing a PCR product successfully ligated into the plasmid will form white colonies, while those bacteria containing empty plasmid will form blue colonies. The colonies containing PCR product were collected and submitted for sequencing by the Universit y Aptamer binding studies by f low cytometry To determine the binding of the aptamers, the target cells (510 5 ) were incubated with 250 FITC labeled aptamers on ice for 20 min in 100 then washed twice with 500 FITC labeled random sequence as the negative control. All the experiments for binding assays were repeated at least 2 times.

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117 Results In order to prove that mycoplasma contamination can affect the results obtained during Cell SELEX, two cancer cell lines were chosen to perform a full selection. The ovarian cancer cell line CAOV3 was chosen for the selection of ovarian c ancer aptamers. In order to identify aptamers that specifically bind to ovarian cancer cells, the cervical cancer cell line HeLa was used for counter selection. Both cell lines were mycoplasma positive during the selection process. The SELEX procedure is d escribed briefly below. A detailed description is provided in the experimental section. To start the selection process, 20 pmol of naive library was incubated with mycoplasma positive CAOV3 cells to yield an enriched pool. Sequences showing non specifi c binding to general cell surface markers were removed by incubating the enriched pool with HeLa cells (rounds 6 to 13). The eluted pool for each round of SELEX was amplified through PCR, after which the ssDNA pools of interest were recovered and monitored for enrichment toward CAOV3 by flow cytometry. As the selected pools were enriched with sequences that recognize and bind to the target cell line, an increase in fluorescence signal was observed (Figure 1a). The enriched pools showed minimum enrichment fo r HeLa binding sequences (Figure 1b). After 13 rounds of SELEX, an enriched pool that specifically bound to the CAOV3 cell line, but marginally to HeLa cells, was obtained (Figure 1). Thus, the pool was successfully enriched for sequences binding surface markers expressed by CAOV3 cell line, but not by cervical cancer cells. To demonstrate that the selection was compromised by the presence of mycoplasma in the cell culture, the pools were also tested for binding to mycoplasma negative cells, and little enr ichment was observed (Figure 2).

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118 Following completion of the selection process, two pools were chosen and submitted for cloning and sequencing: the final pool (round 13) and the previous pool (round 12). The sequences were aligned into families according t o homology, and aptamer candidates were chosen from each family. Six sequences showing the best homology throughout the pools were selected as aptamer candidates, synthesized and tested for binding to mycoplasma negative CAOV3 cells, and no binding was o bs erved (Figure 3). Concluding remarks In conclusion, we have selected a pool that is capable of recognizing mycoplasma positive CAOV3 cells but not mycoplasma negative cells. These results are significant, as the effect of mycoplasma contamination was not e xpected to change the cell profile to the extent of selecting aptamers unable to cross react with mycoplasma negative CAOV3 cells. Therefore, this selection shows the importance of performing mycoplasma testing throughout the entire cell SELEX process (at least weekly). Otherwise, the researcher runs the risk of selecting for a cell target that is an artifact produced by the mycoplasma infection. Furthermore, publications involving the selection of aptamers against whole cells should be required to show neg ative results for mycoplasma testing of the target cells to ensure that the data are reliable.

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119 A B Figure A 1: Monitoring of the selection progress: (A) Binding of the enriched pools to CAOV3 cells. The red histogram corresponds to cells only. The pink histogram corresponds to unselected library. Dark blue corresponds to the 10 th round. Yellow corresponds to the 11 th rounds. Light blue corresponds to the 12 th rounds. Purple corresponds to 11 th pool repeat. (B) Binding of the enriched pools to HeLa cells. The red histogram corresponds to cells only. The pink histogram corresponds to unselected library. Dark blue corresponds to the 10 th round. Yellow corresponds to the 11 th rounds. Light blue corresponds to the 12 th rounds. Purple corresponds to 11 th pool r epeat. The green histogram corresponds to the binding of Sgc8 to HeLa cells. Figure A 2: Aptamer binding to mycoplasma negative CAOV3 cells.

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120 Figure A 3: Candidate aptamer binding to mycoplasma negative CAOV3 cells

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121 APPENDIX B CHEMICAL APT AMER CONJUGATION TO THE AAV CAPSID Introduction Gene delivery is one of the most promising therapeutic approaches available nowadays for the treatment of several diseases ( 117 118 ) This method is based on the introduction of genetic material into cells for the production of therapeutic proteins or blocking the expression of harmful proteins. Gene therapy can be done in vivo by directly implan ting or injecting the therapeutic DNA. It can also be done ex vivo by injection of the cells into the patient ( 119 ) An ideal gene transfer vector should be: safe, it must not induce cellular toxicity or provoke an immune reaction, economic, convenient, it must be capable of being delivered by injection, efficiently injecting target cells, and express ing the transgene product at a therapeutic level and under tight regulation for the required amount of time. Viral and non viral vectors systems have been developed for gene therapy. Non viral vectors include liposomes, cell penetrating peptides, polymers, and naked DNA. They involve physical or chemical transfer of genetic material, and are dependent on cellular transport mechanisms for uptake and expression in the host cell. Non viral vectors a re easy to make, accept different sizes of inserted DNA and di splay fewer immunological and safety problems. The principal drawback is the poor transduction efficiency, transient transgene expression and non selective cell targeting ( 117 120 ) Viral vectors are considered appealing delivery vehicles. Several virus families (adeno retro herpes and AAV viruses) have been used as gene transfer vectors for gene therapy. Viral vectors are very efficient as their transfection effic i ency approaches

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122 100% and c an achieve prolonged expression But they have several limitations. There are safety concerns about the use of viral vectors, as random integration into host genome might give rise to insertion muta genesis and they may elicit an immune response. Furthermore, the virus effect is governed by its v iral tropism (target specificity) their a bility to elicit strong and stable transgene expression is limited and i s not possible to produce the vector at high titers ( 121 122 ) AAV type 2 has shown a great promise for gene therapy as it posses several advantages: Non pathogenic to humans d oes not induce a strong immune response can transduce both dividing and quiescent cells v iral particle is stable c an be produced at high titers and has p otential for long term expression Still, there are several limitations that need to be addressed such as s ize limitation of AAV capsid c ontrol of the tropism of the vector and n eutralization by antibodies ( 123 ) AAV is a small, non enveloped, single stranded linear DNA parvovirus. The v irion is icosahedral in shape and measures 20 25nm in diameter. The capsid contains three proteins, VP1, VP2 and VP3, in a 1:1:10 ratio with a total of 60 proteins per capsid. VP1, VP2 and VP3 differ from each other at their N terminus and have apparent mo lecular masses of 87, 72 and 62 kDa, respectively ( 124 125 ) enco des the non structural capsid proteins. The Rep proteins are required in all phases of the viral life cycle, including transcription, replication, encapsidation, integration and rescu e form the latent state. The capsid proteins are crucial for rescue, replication, packaging, and integration of AAV.

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123 Eleven serotypes have been identified, each having different intrinsic properties. The greatest divergence between them lies in the capsid proteins leading to differences in both tropism and serological neutralization. AAV2 is the most widely used serotype and is the best characterized. AAV2 uses heparin sulfate proteoglycan (HSPG) as a primary receptor for cell attachment and it also utilize s co receptors to assist its internalization. The construction of recombinant AAV vectors is based on transient triple transfection protocols of target/producer cells, which requires: a p lasmid with the sequence of the rAAV genome (cap and rep genes are d eleted and replaced with the desired gene), a p lasmid with sequences encoding the two AAV ORFs of rep and cap and a p lasmid with the required helper functions encoded by the natural auxiliary virus. All 3 plasmids are co transfected into permissive cells, and then packaged into rAAV2 virions containing only the therapeutic vector genomes In the absence of helper virus, AAV infected cells cannot produce any progeny virus and enters a latent state. The AAV genome is integ rated into a specific site, referred t o as AAVS1, found on the chromosome 19. In rAAV viruses, the viral genes of rep are deleted, so the virus ca n n ot integrate in the AAVS1 locus, which ensures that the treatment is innocuous and there is no risk of insertional mutations. When a latently infe cted cell encounters superinfection by any of the helper viruses, the integrated AAV genome undergoes productive lytic cycle. The tropism of the vector can be altered by genetic modification of AAV capsid. It has been shown that several sites within the AA V2 capsid protein are amenable to the insertion of small peptides. But, the genetic insertion of foreign sequences reduces

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124 vector titer or DNA packing efficiency or has other effects on vector biology. Also, some peptide epitopes are inefficiently or inapp ropriately displayed when engineered into AAV capsid. labeled with biotin and further conjugated to biotin labeled aptamers via biotin streptavidin interaction to control the vira l tropism Materials and Methods Chemical Conjugation of the Aptamer C apsid For the g eneration of ch emically biotinylated AAV2 EGFP, 10 10 particles AAV EGFP w ere mock treated or incubated with 5 mg/mL sulfo NHS biotin in PBS buffer (pH7.4) for 1 hr at room temperature, purified by ultrafiltration and analyzed by Western Blot with AAV antibody B1 ( Figure B 1, A ) and streptavidin HRP ( Figure B 1, B ). After purification, the AAV was incubated with 1/10000 excess strepta vidin PBS at room temperature Excess streptavidin was removed by ultrafiltration using 150 KDa molecular weight cut off filters. About 10 12 streptavidin labeled AAV virions were further conjugated with 1/100 excess biotin aptamer (Sgc8) L PBS for 15 min at room temperature The final AAV aptamer complex was purified by ultrafiltration. The aptamer conjugated AAV was tested for transfection of non permissive cells as explained below. Construction of AAV BAP for biotinylation of AAV in cell s The AAV virus was genetically modified to insert a biotin accepto r peptide (BAP) sequence in the three capsid proteins. The BAP was biotinylated in cells expressing biotin ligase (encod ed by BirA gene from bacteria). The metabolically biotinylated virus was purified by ultrafiltration and analyzed by Western Blot. AAV was further modified with streptavidin and biotin labeled aptamer as explained above.

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125 Aptamer M ediated AAV I nfection of CEM C ells Aptamer AAV2 mediated expression of EGFP in CCRF CEM suspens ion cells. About 105 CCRF CEM cells were transduced with biotinylated AAV2 or aptamer biotin streptavidin biotin AAV complex at 104 particles/cell. The cells were analyzed for the detection of GFP using fluorescence microscopy. Cells were also labeled with DAPI. Results Chemical C onjugation of the A ptamer C apsid In order to control the viral tropism, the aptamer Sgc8 was conjugated to the AAV viral capsid. The AAV viral capsid proteins were chemically labeled with sulfo NHS biotin. The conjugation was conf irmed by Western Blot (Figure B 1). The presence of the three AAV capsid proteins was confirmed by Western Blot using the antibody B 1 (Figure B 1 A), while the presence of biotin on the viral capsid was confirmed using streptavidin HRP (Figure B 1 B). Aft er biotinylation, the AAV was further labeled with streptavidin and biotin labeled Sgc8. Construction of AAV BAP for B iotinylation of AAV in C ells In order to produce metabolically biotinylated AAV, a biotin acceptor peptide sequence (BAP) was introduced i nto the AAV plasmid inside the CAP gene (Figure B 2) and the virus was produced in cells expressing biotin ligase. The biotinylation of the virus was confirmed by Western Blot (Figure B 3). Aptamer M ediated AAV I nfection of CEM C ells In order to verify if the viral tropism can be controlled by the conjugation of an aptamer to the AAV capsid proteins, AAV2 was chemically conjugated to the aptamer Sgc8. The AAV contains a GFP coding gene. Therefore, AAV transfected cells will express GFP, while non transfect ed cells remain GFP negative. The conjugation of the

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126 Sgc8 to the AAV was achieved by chemically biotinylating the AAV capsid using Sulfo NHS biotin and further conjugating the AAV to biotin labeled Sgc8 via streptavidin biotin interaction. The aptamer modi fied aptamer was then tested for the transfection of CEM cells, which express the Sgc8 target, PT K7. Sgc8 modified AAV and unmodified virus were incubated with CEM cells. The unmodified AAV was unable to transfect CEM cells (Figure B 4, Top). On the other hand, aptamer modified AAV was able to transfect CEM cells (Figure B 4, bottom). Conjugation of the AAV virus via metabolically introduced biotin was also explored. Metabolically biotinylated AAV was conjugated to Sgc8 via streptavidin biotin interaction a s explained above. The aptamer modified aptamer was then tested for the transfection of CEM cells. Sgc8 modified AAV and unmodified virus were incubated with CEM cells. The unmodified AAV was unable to transfect CEM cells (Figure B 5, Top). On the other ha nd, aptamer modified AAV was able to transfect CEM cells (Figure B 5, bottom). Concluding Remarks AAV tropism could be achieved by the conjugation of the capsid proteins to an aptamer. AAV vectors can be conjugated with cell specific aptamers through bioti n strep t avidin interaction. Biotinylated AAV can be generated in a test t ube using chemical reagents, or in culture cells using genetically modified AAV vectors. Once conjugated, the cell specific aptamers can guide the AAV into target cells for expression of transgenes. This approach offers a proof of principle for development of novel cancer gene therapies The incorporation of a suicide gene, such as saporin, into the AAV plasmid can lead to the development of a targeted cancer gene therapy. Furthermore, as new aptamers are selected showing selective binding to other disease cells, this approach can be applied for the delivery of therapeutic genes specific for the disease.

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127 A B Figure B 1: Chemical biotinylation of the AAV capsid. The chemical reagent s ulfo N hydroxysuccinimide (NHS) B iotin (Thermo Fisher Scientific) was used to label AAV serotype 2 with biotin, and confirm biotinylation of AAV part icle by Western blot analysis. (A) B1 anti b ody recognizing all the three capsid proteins of AAV; Control: n o sulfo NHS Biotin in reaction. (B) Streptavidin HRP detects the biotinylated AAV capsid. Figure B 2: Construction of AAV BAP for biotinylation of AAV in cells

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128 Figure B 3 : Metabolic biotinylation of the AAV capsid. B iotinylation of AAV part icle was confirmed by Western blot analysis. (A) B1 anti b ody recognizing all the three capsid proteins of AAV; Control: wild type virus (B) Streptavidin HRP detects the biotinylated AAV capsid.

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129 Figure B 4: Transfection of CEM cells using aptamer labeled AAV. Ch emical biotinylation of the AAV capsid proteins.

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130 Figure B 5: Transfection of CEM cells using aptamer labeled AAV. Metabolic biotinylation of the AAV capsi d proteins.

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143 BIOGRAPHICAL SKETCH Dalia L pez Col n was born in San Juan Puerto Rico. She obtained her bachel or s degree in science (2005) in i ndustr ial c hemistry from the University of Puerto Rico, Humacao campus. During her undergraduate studies, Dalia performed research on the synergistic effect of ultrasound and antitumor quinones for four years under the supervision of Dr. Antonio E. Alegra Furthermore, she participated in the University of Research Experience for Undergraduates ( REU ) program in 2003, when she coated silica membranes as templates. Th e following year, she participated in the REU study abroad program at the Universit Louis Pasteur, where she worked with Dr. Laurence Sabatier to study of Drosophila mellanogaster the University of Florida in 2005 and receive d her Ph D in Biochemistry in 2011 under the tutorage of Dr Weihong Tan.