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Design and Optimization of Nucleic Acid Probes for Intracellular Imaging and Biosensor Development

Permanent Link: http://ufdc.ufl.edu/UFE0022875/00001

Material Information

Title: Design and Optimization of Nucleic Acid Probes for Intracellular Imaging and Biosensor Development
Physical Description: 1 online resource (99 p.)
Language: english
Creator: Martinez Diaz, Karen
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: biosensors, dna, fluorescence, fret, molecular
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The detection of DNA and mRNA plays an important role in disease diagnostics, biomedical research and gene expression studies. Currently, there are many techniques that can help decipher some of the mechanisms occurring inside the cell including, in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and Northern or Southern blotting. These techniques are reliable and sensitive but are time consuming and often fail to give real time data. The key for rapid, selective and sensitive analysis is the use of molecular probes based on nucleic acids. However, currently there are limitations that hinder the use of molecular probes for bioanalysis, including low sensitivity, reduced selectivity, and poor stability. The focus of this work has been the development and optimization of molecular probes for intracellular imaging and biosensor applications. Recently, we introduced the hybrid molecular probe (HMP) as a novel probe for mRNA and DNA monitoring in solution and in living cells. A series of spectroscopic experiments was conducted to fully characterize and optimize HMP for both in vitro and in vivo analysis and as a tool for hybridization studies. The results proved that HMP enables very sensitive analysis at low concentrations and sample volumes. In addition, HMP was able to overcome some of the problems associated with traditional methods of gene expression analysis. Specifically HMP achieved a more than 20 fold signal enhancement compared to ~6 of linear probes, was very stable in a cell-like environment, and was less prone to generate false positive signal when compared with molecular beacons (MBs). MBs are well-known molecular probes that are currently used in bioanalysis. Although, MBs have previously been used for surface hybridization studies, their potential has not fully exploited, primarily because of the poor stability of the beacon after immobilization onto a surface, resulting in a low signal enhancement. By incorporating locked nucleic acid (LNA) bases into the MB sequence, a considerable improvement in the stability and therefore in the overall efficiency in the signal can be achieved for surface hybridization. As part of this research, locked molecular beacons (LMB) have been evaluated and compared to regular molecular beacons (RMB) in terms of selectivity, sensitivity, thermal stability, hybridization kinetics and robustness for the detection of target sequences. The experiments were performed using biotinylated beacons immobilized onto avidin-coated microscope slide surface. After incubation with the target DNA sequence, a 25-fold enhancement has been achieved for LMB, with detection limits extending down to the low nanomolar range. In addition, the LMB-based biosensor possesses better stability, reproducibility, selectivity and robustness when compared with the RMB. These results demonstrate the potential of the newly designed HMP and LMB probes as prospective tools in the development of DNA microarrays and for bioanalysis, disease diagnosis and biotechnology applications.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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 Karen Martinez Diaz.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Tan, Weihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022875:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022875/00001

Material Information

Title: Design and Optimization of Nucleic Acid Probes for Intracellular Imaging and Biosensor Development
Physical Description: 1 online resource (99 p.)
Language: english
Creator: Martinez Diaz, Karen
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: biosensors, dna, fluorescence, fret, molecular
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The detection of DNA and mRNA plays an important role in disease diagnostics, biomedical research and gene expression studies. Currently, there are many techniques that can help decipher some of the mechanisms occurring inside the cell including, in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and Northern or Southern blotting. These techniques are reliable and sensitive but are time consuming and often fail to give real time data. The key for rapid, selective and sensitive analysis is the use of molecular probes based on nucleic acids. However, currently there are limitations that hinder the use of molecular probes for bioanalysis, including low sensitivity, reduced selectivity, and poor stability. The focus of this work has been the development and optimization of molecular probes for intracellular imaging and biosensor applications. Recently, we introduced the hybrid molecular probe (HMP) as a novel probe for mRNA and DNA monitoring in solution and in living cells. A series of spectroscopic experiments was conducted to fully characterize and optimize HMP for both in vitro and in vivo analysis and as a tool for hybridization studies. The results proved that HMP enables very sensitive analysis at low concentrations and sample volumes. In addition, HMP was able to overcome some of the problems associated with traditional methods of gene expression analysis. Specifically HMP achieved a more than 20 fold signal enhancement compared to ~6 of linear probes, was very stable in a cell-like environment, and was less prone to generate false positive signal when compared with molecular beacons (MBs). MBs are well-known molecular probes that are currently used in bioanalysis. Although, MBs have previously been used for surface hybridization studies, their potential has not fully exploited, primarily because of the poor stability of the beacon after immobilization onto a surface, resulting in a low signal enhancement. By incorporating locked nucleic acid (LNA) bases into the MB sequence, a considerable improvement in the stability and therefore in the overall efficiency in the signal can be achieved for surface hybridization. As part of this research, locked molecular beacons (LMB) have been evaluated and compared to regular molecular beacons (RMB) in terms of selectivity, sensitivity, thermal stability, hybridization kinetics and robustness for the detection of target sequences. The experiments were performed using biotinylated beacons immobilized onto avidin-coated microscope slide surface. After incubation with the target DNA sequence, a 25-fold enhancement has been achieved for LMB, with detection limits extending down to the low nanomolar range. In addition, the LMB-based biosensor possesses better stability, reproducibility, selectivity and robustness when compared with the RMB. These results demonstrate the potential of the newly designed HMP and LMB probes as prospective tools in the development of DNA microarrays and for bioanalysis, disease diagnosis and biotechnology applications.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: 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 Karen Martinez Diaz.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Tan, Weihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022875:00001


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1 DESIGN AND OPTIMIZATION OF NUCLEIC ACID PROBES FOR INTRACELLULAR IMAGING AND BIOSENSOR DEVELOPMENT By KAREN MARTINEZ DIAZ A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Karen Martinez Diaz

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3 To my parents

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4 ACKNOWLEDGMENTS Many people supported, guided, helped and inspir ed me during my years at the University of Florida. I especially thank my advisor Dr Weihong Tan for his guidance, encouragement, ideas, and support. In addition, I would like to thank the many past and current members of the Tan research group for their support and encouragement. I thank my committee members; Dr. Benjamin Smith, Dr. Charles Cao, Dr. Thomas Lyons and Dr. Leonid L. Moroz, who in one way or anot her have made possible the completion of this dissertation. Also, I would like to thank Dr. Craig Aspinwall for all the advice in the early stages of my graduate studies. I am forever thankful to my family member s because they inspired me to continue. I especially thank my parents, Francisco Mart inez and Carmen Diaz, for their endless love, encouragement, unconditional support and guidance. I dedicate this disser tation to them because I know how much this work means to them, esp ecially my mom, who always has me in her prayers. I thank my older brother and sister, Rubier and Melissa Martinez and my little brothers Luis F. Cintron and Armando L. Cintron for thei r support and love every step of my way. They have significantly helped me b ecome the person that I am today, and for that I will be eternally grateful. I would like to take this opport unity to also thank the many friends and colleagues I have gained in and outside the group because each of them has made my experience in graduate school enjoyable and pleasant: Dr. Marie C. Vicens, Dr. Timothy J. Drake, Dr. Colin D. Medley, Dr. Joshua Smith, Dr. Chaoyong J. Yang, Dr. Alin a Munteanu, Dr. Jose Valle (aka bamby), Dalia Lopez, Dr. Prabodhika Mallikarachy (a ka PB), Meghan ODonahue, Dimitri Van Simaeys, Elizabeth Jimenez, Youngmi Kim, Jenni fer Martin, Dr. M-Carmen Estevez (aka la tia) and Roberto Reyes.

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5 My special thanks go to Dr. Colin D. Me dley, Dr. Chaoyong J. Yang and Yanrong Wu for helping me in my projects. Also, I would like to express my gratitude to Dr. M-Carmen Estevez for helping me in the completion of the biosensor project and teaching me so much about the true meaning of research. Life as a graduate student can be tough, but fortunately, I found great friends in the lab that kept things fun and enjoyable. I would like to thank Dalia Lopez and M-Carmen Estevez for being by my side in the toughest moments in the group. You are two of the most beautiful persons that I have met and I could not have asked for better friends. In addition, I would like to thank Mariela Rodriguez for her friendship, advice and for making me feel that I was not alone. Also, I especially thank Ramon Martinez for be ing such a good friend a nd for not allowing the distance affect our friendship. Last but not least, I am extremely grateful to my fiance Orlando Milians, for being my best friend and partner in crim e; and for his love, support, encouragement, motivation, patient and not letting me give up in the worst moment s. His suggestions and advice enabled me to continue and succeed. Without him I could not have come this far.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 ABSTRACT ...................................................................................................................... .............11 CHAPTER 1 INTRODUCTION .................................................................................................................. 13 Nucleic Acid Probes in Bioanalysis ....................................................................................... 13 Synthesis of Nucleic Acid Probes ...........................................................................................13 Fluorescence Spectrometry .....................................................................................................17 Quenching Mechanisms .................................................................................................. 19 Fluorescence Resonance Energy Transfer .......................................................................20 Organic Dyes ...................................................................................................................21 Principles of Selected Molecular Probes ................................................................................ 22 Nucleic Acids Monomers ................................................................................................ 23 Molecular Beacons .......................................................................................................... 26 Linear Fluorescent Probes ...............................................................................................31 Hybrid Molecular Probe ..................................................................................................31 DNA Based Sensors ............................................................................................................. ..32 Gene Expression Studies ........................................................................................................34 Intracellular Imaging Using MBs Probes ...............................................................................35 Challenges of Molecular Probes for Intr acellular Measurements and Bios ensor Development ................................................................................................................... ....37 Research Objective .................................................................................................................38 2 HYBRID MOLECULAR PROBE: DESI GN, SYNTHESIS AND IN VITRO CHARACTERIZATION ........................................................................................................ 39 Introduction .................................................................................................................. ...........39 Probe and Target Synthesis ....................................................................................................39 Fluorophores Selection ...........................................................................................................41 Proof of Principle for the novel HMP ..................................................................................... 42 Linker Effect in HMP .............................................................................................................44 Fluorophores Distance ............................................................................................................45 Selectivity of the HMP ...........................................................................................................47

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7 3 COMPARISON OF THE HMP WITH MB AND OTHER MOLECULAR PROBES FOR INTRACELLULAR MEASUREMENTS ..................................................................... 48 Introduction .................................................................................................................. ...........48 Probes and Target Synthesis ................................................................................................... 49 Fluorescence Measurements ............................................................................................51 Thermal Stability ............................................................................................................. 51 Cell Preparation ...............................................................................................................51 Fluorescence Imaging ...................................................................................................... 52 Comparison of HMP with and without PEG as a linker .........................................................53 Comparison Experiments between the HMP and MBs .......................................................... 56 Kinetics ...................................................................................................................... ......56 Selectivity of the HMP and MB ......................................................................................57 HMP and MBs reaction with Deoxyribonuclease Reaction ............................................ 58 Biological Stability .......................................................................................................... 60 Intracellular Measurements ....................................................................................................62 HMP for Surface DNA Hybr idization Studies .......................................................................65 Conclusion .................................................................................................................... ..........67 4 SURFACE HYBRIDIZATION STUDIES OF THE MO LECULAR BEACONS ................ 68 Introduction .................................................................................................................. ...........68 Materials and Methods ...........................................................................................................71 RMB and LMB for Surface Hybridization Studies ................................................................ 71 Immobilization of the Probe onto the Glass Surface .......................................................72 Stability of the MBs at Different Temperatures ..............................................................73 Stability in Complex Matrices ......................................................................................... 73 Fluorescence Imaging ...................................................................................................... 74 Results and Discussion ........................................................................................................ ...75 Molecular Beacon Designs and Surface Immobilization ................................................ 75 Sensitivity and Selectivity of the Immobilized MBs ....................................................... 78 Thermal Stability of the Beacons .................................................................................... 80 Hybridization Kinetics of the MBs ..................................................................................81 MB Sensitivity in the Presence of Complex Matrices ..................................................... 82 Conclusion .................................................................................................................... ..........84 5 SUMMARY AND FUTURE DIRECTIONS ......................................................................... 85 Designing Nucleic Acids Probes for Biosensor Applications ................................................ 85 Future Directions ....................................................................................................................87 LIST OF REFERENCES ...............................................................................................................92 BIOGRAPHICAL SKETCH .........................................................................................................99

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8 LIST OF TABLES Table page 1-1 Fluorophores used in this investig ation and their spectral properties ................................ 22 2-1 Sequence of HMP probe and their targets .........................................................................40 3-1 Sequences synthesized for molecular probes and targets .................................................. 50 4-1 Molecular beacons and target sequences ........................................................................... 72 4-2 Effect of different pol yethylene glycol units (PEG s) on the background intensity and the overall fluorescence change after target addition ........................................................77 5-1 Probe and Target Sequences for Multiplexing Analysis ....................................................88

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9 LIST OF FIGURES Figure page 1-1 Automated oligonucleotide synthesis achieved through phosphoramidite chem istry.. ..... 15 1-2 Absorption spectra of a DNA sequence.. ........................................................................... 17 1-3 Jablonski diagram that presents the absorption, fluorescence, phosphorescence, internal conversion and intersystem crossing pr o cesses form a singlet and triple state. ... 18 1-4 Energy transfer between donor (D) and acceptor (A).. ...................................................... 20 1-5 Comparison of the repetitive units structures of DNA, PNA, Morpholino, TNA and GNA. .......................................................................................................................... ........24 1-6 LNA and DNA structure. Left, LNA contai ns a me thylene bridge (red) that allows the ribose ring to be locked. Right, DNA structure. .......................................................25 1-7 Hybridization of MBs upon target addition.. ..................................................................... 27 1-8 Flow chart illustrating the MB design and synthesis process ............................................ 29 1-9 Working principle of the hybrid molecular probe (HMP). ..............................................32 2-1 Absorbance and emission spectra for FAM and Cy5.. ...................................................... 42 2-2 Hybridization of HMP-16 with the target cDNA and with a random DNA sequence ......43 2-3 Emission spectra of the HMP-16 before cDNA (tg) addition and after the equilibrium was reached .................................................................................................................. .....44 2-4 Effect of the PEG linker on the hybr idization of HMP with the target ............................. 45 2-5 Effect of distance of the Accep tor-Donor dye pair on I 665/I 520 ratio ............................ 46 2-6 Hybridization of 300nM HMP-16 to same concentration of their targets. ........................ 47 3-1 Concerns when using MBs for intracellular measurements .............................................. 49 3-2 Influence of the concentr ation of target in the hybridization with single strand probe (no PEG linker) and HM P (with PEG linker).. .................................................................. 54 3-3 Melting temperatures for HMP (with PEG) and single stranded probes (no PEG).. ......... 55 3-4 Hybridization rates of MBs and HMP upon target addition.. ............................................57 3-5 Comparison of selectivity of MB (blue) and HMP (red). ..................................................58

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10 3-6 Comparison of the intensity of MB and HMP in terms of DNase reaction.. ..................... 59 3-7 Response of MB and HMP to non-specific interactions. ...................................................60 3-8 Stability of HMP and MB in the pr esence of cell lysate, LDH and DNase I.. .................. 61 3-9 Real-time monitoring of the MBs and HMP to test the stability in side single cells.. ........ 63 3-10 HMP hybridized and unhybridi zed states inside the cell.. ................................................. 64 3-11 Immobilization of HMP on the gl ass surface for hybrid ization studies ............................ 65 3-12 Images show the hybridization of HMP w ith the target after immobilization onto the glass surface (left). Fluo rescence intensity change of the FAM and Cy5 after target addition (top right). Cy5/FAM fluorescence ra tio after hybridization with the target (bottom right) .....................................................................................................................66 4-1 Typical DNA biosensor components. Our work has focused on the use of channels, molecular probes and optical detection. ............................................................................. 68 4-4 Schematic representation of the mol ecular beacon biosensor immobilized on a glass surface. ...................................................................................................................... .........76 4-5 Comparison of the influence of target concentra tion in the hybridization of the RMBs and LMBs (gray and black respectively). ............................................................... 78 4-6 Normalized fluorescence intensity of LMB and RMB upon hybridization with the single base mi smatch (mismatch) and perfect complementary target (cDNA) ................. 79 4-7 Temperature effect on the stability of the MBs immobilized onto the surface. ................ 81 4-8 Hybridization kinetics of RMB (gra y) and LMB (black) immo bilized onto the surface after target addition (left). Initial hybridization rate of the immobilized beacons is shown on the right ............................................................................................ 82 4-9 Normalized fluorescence intensity of the imm obilized RMB and LMB after treatment with the target in fetal bovine serum (FBS), Cell lysate and PBS buffer solution ...................................................................................................................... .........83 5-1 Surface immobilization of MBs targeti ng mu ltiple genes and the addition of the target molecules .................................................................................................................89 5-2 Aptamer selection process: Systemat ic evolution of ligands by exponential enrichme nt (SELEX) .........................................................................................................91

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DESIGN AND OPTIMIZATION OF NUCLEIC ACID PROBES FOR INTRACELLULAR IMAGING AND BIOSENSOR DEVELOPMENT By Karen Martinez Diaz December 2008 Chair: Weihong Tan Major: Chemistry The detection of DNA and mRNA plays an important role in disease diagnostics, biomedical research and gene e xpression studies. Currently, ther e are many techniques that can help decipher some of the mechanisms occurring inside the cell including, in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and Northern or Southern blotting. These techniques are reliable and sensitive but are time consuming and often fail to give real time data. The key for rapid, selective and sensitiv e analysis is the use of molecular probes based on nucleic acids. However, currently there are li mitations that hinder the use of molecular probes for bioanalysis, including low sensitivity, reduc ed selectivity, and poor stability. The focus of this work has been the development and optim ization of molecular probes for intracellular imaging and biosensor applications. Recently, we introduced the hybrid molecula r probe (HMP) as a novel probe for mRNA and DNA monitoring in solution and in living cells. A series of spectroscopic experiments was conducted to fully characterize and optimize HMP for both in vitro and in vivo analysis and as a tool for hybridization studies. The results proved that HMP enables very sensitive analysis at low concentrations and sample volumes. In addi tion, HMP was able to overcome some of the problems associated with traditional methods of gene expression analysis. Specifically HMP

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12 achieved a more than 20 fold signal enhancemen t compared to ~6 of linear probes, was very stable in a cell-like environment, and was less prone to generate false positive signal when compared with molecular beacons (MBs). MBs are well-known molecular probes that ar e currently used in bioanalysis. Although, MBs have previously been used for surface hybrid ization studies, their potential has not fully exploited, primarily because of the poor stabil ity of the beacon after immobilization onto a surface, resulting in a low signal enhancement. By incorporating locked nucleic acid (LNA) bases into the MB sequence, a c onsiderable improvement in the stability and therefore in the overall efficiency in the signal can be achieved for surface hybridization. As part of this research, locked molecular beacons (LMB) have been ev aluated and compared to regular molecular beacons (RMB) in terms of sel ectivity, sensitivity, thermal stab ility, hybridization kinetics and robustness for the detection of target seque nces. The experiments were performed using biotinylated beacons immobilized onto avidin-coa ted microscope slide surface. After incubation with the target DNA sequence, a 25-fold enha ncement has been achieved for LMB, with detection limits extending down to the low na nomolar range. In addition, the LMB-based biosensor possesses better stabil ity, reproducibility, se lectivity and robustn ess when compared with the RMB. These results demonstrate the potential of the newly designed HMP and LMB probes as prospective tools in the development of DNA micr oarrays and for bioanalysis, disease diagnosis and biotechnology applications.

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13 CHAPTER 1 INTRODUCTION Nucleic Acid Probes in Bioanalysis Intracellular imaging and biosensor developm ent have becom e increasingly important in gaining knowledge processes inside the cell. These processes play a key role for cell function and keep the body working properly. Many tech niques have been developed to decipher intracellular mechanisms including Northern Hybridization, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Southern blotting. Th ese techniques are reliable and sensitive, but they are time consuming and fail to give a real -time data. Because these techniques involve many steps, their results may not give a true re flection of the targeted process. Therefore, methods capable of providing high sensitivity, selec tivity, speed and high signal to noise ratio are still needed. One of the most widely used methods for the design of molecular probes is based on nucleic acids (NAs) synthesis. NAs can be used as building blocks for the design and introduction of novel probes, mainly due to the st rong and specific base pairing interaction. In addition, the use of NAs has many attractive properties such as high solubility, nonimmunogenic, and convenient synthesis from the monomer nucleotides. Furthermore, chemically modified nucleotides can be included to produc e a wide variety of labeled molecular probes. These factors have led to wide popularity of molecular probes ba sed on nucleic acid bases in fields such as molecular biology, chem istry and biomedical sciences. Synthesis of Nucleic Acid Probes Nucleic acid probes usually containing10-50 nucleotides can be prepared by several me thods. However, the most common mechanism is based on the solid-phase synthesis. Because of the high efficiency of the automated o ligonucleotide synthesis, the use of DNA/RNA

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14 polymers have expanded to diverse areas of fundamental and applied biological research.1 Moreover, the different labels available allow the synthesis of the DNA sequences for a broad range of applications. Figure 1 depicts an automated DNA synthesis using phosphoramidite chemistry, which is one of the most widely used methods for oligonucleotides synthesis.2 The procedure involves four steps: detritylation, activ ation, coupling/capping and oxidati on. Mononucleotides are added, one at a time, to a starting m ononucleotide, conventionally the 3 end nucleotide, which is bound to a solid support or core pore glass (CPG). The advantage of using the solid support unlike liquid synthesis is that permits the reagents to be removed by filtration and eliminates the need of purification between base additions. The first step is detritylation, in which th e dimethoxytrityl (DMT) group is removed with trichloroacetic acid (TCA) or dichoroacetic acid (DCA) to free the 5'-hydroxyl for the coupling reaction. This ensures that the addition of the ne xt base will bind only to that site. Excess acid and by products are removed by washing the reaction column. The next monomer cannot be added until it has been activated; the activation is achieved by adding tetrazole to the phosphoramidite derivativ e of the nucleotide. Te trazole protonates the nitrogen of the phosphoramidite, which beco mes vulnerable to nucleophilic attack. The intermediate formed is reactive and the subsequent coupling step is complete in ~30 seconds. The active 5'-hydroxyl group of the preceding nucl eotide and the newly activated phosphorus bind to loosely join the two monomers forming an unstable phosphite linkage. The reaction column is then washed to remove any extr a tetrazole, unbound nucle otide and by-products. Ideally, the coupling reaction s hould have 100% yield, however, this is rarely the case. Therefore, a capping step is necessary to term inate any chains that did not undergo coupling.

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15 O B N-1 O~CPG DMTrO O B N-1 O~CPG HO O B N O DMTrO P N NCH2CH2CO + tetrazoleN N N H N 3. COUPLING or CAPPING 2. ACTIVATION 1. DETRITYLATIONO B N-1 O~CPG H3CCOO O B N O DMTrO P O OCH2CH2CN O B N-1 O~CPG O B N O DMTrO O NCH2CH2CO P O 4. OXIDATIONO B N-1 O~CPG Figure 1-1 Automated oligonucleotide synthesis achieved through phosphoramidite chemistry. There are four major steps involved in th e synthesis of DNA: (1) Detritylation, (2) Activation, (3) Capping or Coup ling, and (4) Oxidation. This step prevents any reaction of the failure product to react in latter additions of nucleotides which can be difficult to isol ate. Consequently, the unbound active 5-hydroxyl groups are capped by acetylation w ith a protective group that prohi bits the base from growing again. The reagents used for the capping step are acetic anhydride and N-methylimidazole,

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16 which react only with the residual 5'-hydroxyl groups. The column is then washed to remove any remaining capping reagents. The last step is the oxidati on, which is performed using iodi ne in water as the oxidizing agent in the presence of pyridine and tetrahydr ofuran (THF). The inte rnucleotide linkage is converted from a phosphite (less stable compound) to a highly stable pentavalent phosphate triester. The entire process is repeated in orde r to add more nucleotides and/or additional modifications. After all the nucleo tides have been added, the produc t is deprotected to separate the sequence from the CPG, precipitated with ethanol and purified either by solid phase extraction or by reverse phase high performance liquid chromatography (RP-HPLC). One of the biggest advantages of using RP-HPLC is that it separates the failed sequences from those that were successfully synthesized. This is a key step in obtaining good product because sequences that failed to complete the whole synthesis can significantly interfere with the analytical procedure. Furthermore, molecular probes with multiple labels are usually separated twice in order to make sure that a high quality and pure product is collected with minimum interferences. Nucleic acids sequences absorb in the 250nm to 300nm range, whereas other molecules used for labeling absorb in their unique ranges. Figure 12 shows a typical absorp tion spectra of targets DNA with no label. There are two major products that can be seen in the spectra; the truncated DNA or the failed sequences and the final produc t, which contains a higher retention time. Phosphoramidite chemistry synthesis offers fl exibility to introduce many functional groups at the 3' or 5' ends or in the middle of an oligonucleotide sequence. F unctional groups such as sulfuhydryl, carboxyl, amino, linkers, thiols, biotin groups, and a wide va riety of fluorophores can all be incorporated into oligonucleotides.

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17 Figure 1-2 Absorption spectra of a DNA sequence. The blue arrows show the failed sequences whereas as the red arrow, shows th e DNA sequence and/or final product. Fluorescence Spectrometry Designing nucleic acid probes for biosensor appl ications requ ires se veral considerations, such as the analytical method, choice of the labe ls, modifiers and the seco ndary structure of the sequence. There are many different techniques that can be selected for the analysis of nucleic acids, including electrochemistr y, mass spectrometry and fluorescence spectrometry. The latter is one of the most useful techniques, because it o ffers versatility via a wi de variety of fluorophores, sensitivity and it is non-destruc tive. Fluorescence spectroscopy is technique that is based on the analysis of the emission of photons from the samp le. The principles of fluorescence spectroscopy can be understood using Jablonski diagram (Figure 1-3). At room temperature most of the molecules occupy the lowest vibrational levels of the singlet ground state (S0, no unpaired electrons). When elect romagnetic radiation (usually UV light source) strikes the sample, the molecules are excited to a higher energy singlet state (S1). This absorption process is fast and usually take 10 -15 seconds to complete.

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18 S0S1T1 A F PIC ISCA-photon absorption F-fluorescence P-phosphorescence S-singlet state T-triplet state IC-Internal conversion ISC-intersystem crossing Figure 1-3 Jablonski diagram that presents the absorption, fluorescence, phosphorescence, internal conversion and intersystem crossing pr ocesses form a singlet and triple state. Once the molecules are in the excited electroni c state, they rapidly relax to the lowest vibrational level of S1 (10-12 seconds). This process is call ed vibrational relaxation and is generally due to the loss of energy of the molecule by intera ction with the environment. After vibrational relaxation has taken plac e fluorescence emission back to the S0 electronic state can occur with a lifetime of 10-10 to 10-8 second.3 As Figure 1-3 clearly shows, the emitted photons possess lower energy (longer emission wavelengt hs) than the absorbed excitation photons. Sometimes, molecules in the S1 state can undergo a change in a sp in to a triple state (2 unpaired electrons) via the process called intersystem crossing. This electron may return to the S0 without emission (internal conversion) or may undergo phosphorescence. The la tter process takes 10-3 to 1 second to occur and the proper ties of the molecules are quite different from that of the fluorescence. There are many processes and molecular in formation that can be obtained from fluorescence spectroscopy. The most common mol ecular parameters are fluorescence lifetime, anisotropy, quantum yield, fluores cence quenching, and resonance energy transfer (RET). In this work, the focus will be on fluorescence resonance energy transfer (FRET).

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19 Quenching Mechanisms Different processes, generally referred to as quenching, can decrease the fluorescence intensity. Th ere are several quenching mechanis ms: collisional quenching, static quenching, selfquenching as well as others non-radiative pathways. Collisional quenching involves the collision with other molecules in solution, resulting in loss of excitation energy as heat instead of light is emitted. This process deactivates the excited state of the fluorophore and is us ually present in the solution to some extent. For collisional quenching, the decrease in inte nsity is described by the well -known Stern-Volmer equation: F0/F = 1 + K[Q] = 1 + kq0 [Q] where K is the Stern-Volmer quenching constant, kq is the bimolecular quenching constant, 0 is the unquenched lifetime and [Q] is the quencher concentration.3 Molecules such as oxygen, halogens, amines, and electron deficient molecule s such as acrylamide can act as collisional quencher.3 The term static quenching refers to the interaction of the fluorophor e with a quencher to form a stable non-fluorescent complex. It occurs in the ground state and does not depend on diffusion or molecular collisions. There are many different molecular probes that are based on static quenchers. For example, the molecular beacon (MB) probes, discussed in this later chapter. Many different pairs of fluorophores and quenchers make this mechanism ideal for its use in molecular probes. Self-quenching is special type of the static quenching however in this case the fluorophore and quencher are the same species.

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20 Fluorescence Resonance Energy Transfer As described above, the emitted photon to longer wavelength at which abs orption occurs is the most important phenomenon of fluorescen ce. This phenomenon is called Stokes shift. Consequently, fluorescence can be extremely se nsitive due to the detection of emission photons that are spectrally separated from the excitation photons. Detection to low nanometers resolution and sensitivity down to single-molecule levels can be achieved. Fluorescence resonance energy transfer (FRET) is a distance-dependent physical process by which energy is transferred from an excited molecular fluorophore (the donor, D) to another fluorophore (the acceptor, A) by means of intermolecular long-range dipoledipole coupling.4 Even though, the energy transfer between th e donor and acceptor fluorophores occurs nonradiativelly a spectral overlap is required (see Figure 1-4). Acceptor absorption Donor emissionWavelength (nm)F l uor es c e nc e Intens i ty Figure 1-4 Energy transfer between donor (D) and acceptor (A). Diagonal lines represent the spectral overlap of the fluorophores.

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21 The extent of the spectral overlap and the distance between the fluorophores (usually between 15-100 ) determine the magnitude of th e energy transfer. The net result of FRET is that the donor fluorophore emits less energy than it nor mally would due to some of the energy being transferred to the acceptor, while the acc eptor emits more light energy at its emission wavelength, because it is obtaining extr a energy from the donor fluorophore. These aforementioned properties of fluorescence spectroscopy make this technique ideal to perform gene expression studies and biosensor developments. Organic Dyes Organic dyes are very important in application of optical spectroscopy, because of their wide range of spectral propertie s. The detection or im aging me thod for constructing high quality biosensors is in part determined by the physic ochemical properties of the chromophore used.5,6 Fluorophore properties affect the detection limit and the dynamic range of the method, the reliability of the readout for a particular target or event, and the suitability for multiplexing targets.7 There is wide variety of dyes from which to choose, but a suitable label should have the following characteristics: Excitation wavelength that does not interfere with the matrix Highly fluorescent Soluble Stability in buffer conditions Low cost High quantum yield Low toxicity In the last decade, there have been many in dyes for labeling compound labeling, nucleic acids, proteins, antibodies and nanomaterials Although, each dye has the specific labeling protocol, the procedure can be time consuming, and they are not amenable to bioanalytical

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22 applications. Synthesis of the molecular probes in this work, was restricted to fluorescein (FAM or FITC), Oregon green, Cyanine 3 (Cy3), and Cyanine 5 (Cy5) (see Table 1-1). These choices were based on several factors. First, these dyes are phosphoramidite chemistry compatible and therefore, are easy to couple w ith the desired nuclei c acid sequence. Secondly, the analysis is limited by the exitation sources and emission channels of the instruments in our laboratory. Third, the relative quantum yields of these fluorophores are acceptable for bioanalysis. Table 1-1 Fluorophores used in this inve stigation and their spectral properties Fluorophore Excitation (nm) Emission (nm) Quencher FAM 488 520 Dabcyl/BHQ1-543 Oregon Green 488 520 Dabcyl/BHQ1-543 Cy3 543 565 BHQ2-543 Cy5 645 665 BHQ3-579 Principles of Selected Molecular Probes This work focuses in the development of fl uorescence m olecular probes for the detection of mRNA and DNA in living cells and DNA microa rrays. These probes can potentially have a tremendous impact in drug discovery, disease dia gnosis, gene expression studies and biomedical fields.8-10 Molecular probes offer rapid analyses under homogenous conditions,11-15, as well as simultaneous detection of target molecule s allowing real-time analysis. Because DNA hybridization is very specific; these molecular probes permit th e detection of a single base mismatch.16,17 The following sections summarize the different nucleic acid analogs used for the synthesis of molecular probes.

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23 Nucleic Acids Monomers Nucleic acids are very large m olecules that consist of monomeric nucleotides sometimes referred to as bases. The most common nucle ic acids are deoxyribonucleic (DNA), which contains the genetic information, and ribonucleic acids (RNA), which can assume several roles in our body. There are 5 different nucleotides for DNA and RNA: adenine (A), thymine (T), cytosine (C) and guanine (G) and uracil (U is us ed instead of T for RNA). Complementary bases form hydrogen bonds called base pairs; A pairs with T (2 hydrogen bonds) whereas C with G (3 hydrogen bonds). These are also many artificial or man-made nuc leic acid analogs, called non-standard DNA bases that are use instead of DNA and RNA. Among them are: peptide nucleic acids (PNA), morpholino nucleic acids, glycol nucleic acids, (GNA), locked nuc leic acids (LNAs) and threose nucleic acids, all of which are distinguished fr om DNA or RNA by changes to the backbone of the molecule as shown in Figure 1-5. The rationale of using DNA analogs instead of regular DNA in biosensor design is the possibility of co mbining the inherent Watson and Crick base pair recognition with a more robust and accessible polymer synthesis. The backbone of a peptide nucleic avcis (PNA) is composed of repeating N-(2aminoethyl)-glycine units linked by peptides bones.18 The bases (A, C, G, and T) are linked to the backbone by methylene carbonyl bonds. All PNAs contain an N-terminus at the first position and the C-terminus at the right. These nucleic acids contain many attractive properties, such as lack of charged phosphate groups, which makes PNA/DNA binding is stronger than that of the DNA/DNA because electrosta tic repulsion is reduced. In additi on they are resist ant to enzymatic or nuclease degradation and are stable over a wi de pH range. However, because the uncharged backbone can induce self-aggregatio n which changes the secondary structure and interferes with

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24 the base-pairing process,19 PNAs used for biosensor developm ent and intracellular measurements is relatively limited. O HO O B P O OO O B P O OOR O DNAH2N N O B NH N O B O HNR O PNAO B H O P O -O O B H O P O -O OR ( S)-GNA O BH O P O -O O BH O P O -O OR ( R )-GNAO B O O P O -O O O B O O P O -O OR TNAN O B O PN O ON O B Morpholino Figure 1-5 Comparison of the repetitive units structures of DNA, PNA, Morpholino, TNA and GNA.

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25 Glycol nucleic acids (GNAs) are a nucleic acid analogs that follow canonical WatsonCrick base pairing schemes combined with an acyclic three-carbon propylene glycol phosphodiester backbone.20 Morpholinos, however, are synthetic oligonucleotides that contain a morpholino ring instead of a ribose ring.21 Morpholinos are resistant to nucleases, and are therefore very stable. They do not carry a negatively charged backbone, which minimizes the non-specifically interactions with othe r components if used inside the cell.22 Finally, one of the most promising non-standard nucleic acids is the locked nucleic acid (LNA), which are ribonucleotide analogues cont aining a methylene linkage between the 2 oxygen and the 4 -carbon of the ribose ring.23 Figure 1-6 shows the LNA and DNA structures. The bond in red is the methylene bridge, which a llows the ribose ring to be constrained in a locked 3 -endo conformation that permits high affin ity hybridization probably due to its close structure resemblance to RNA.24-31 Figure 1-6 LNA and DNA structure. Left, LNA contains a methylene bridge (red) that allows the ribose ring to be locke d. Right, DNA structure. In addition to the high binding affinity, LNAs offe r lack of toxicity, hi gh solubility and the possibility of synthesis using conventional phos phoramidite chemistry, and are therefore make ideal tools in therapeutics and diagnosis diseases.32

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26 Locked nucleic acid bases exhibit single-bas e mismatch discrimination equal to greater than that of the regular DNA. The properties of the LNA oligonucleotides has been evaluated in oligomers that range from 6 to 20 nucleot ides, with LNA/LNA, LNA/DNA and LNA/RNA mixtures.25,30,33,34 The stability that LNA bases bring to the oligonucleotides is reflected by an increase of the melting temperature (Tm) values up to +1 to +8 degrees against DNA and an increase of +2 to +10 against RNA.26,30,35,36 This increase, however, depends on the oligomer length and composition. It is impor tant to note that LNALNA base pairing is very strong and that the likelihood of self-aggrega tion is possible. Therefore, when using fully modified LNA or LNA DNA mixtures this problem mu st be taken into account. In this work, LNA bases were use to improve the detection capabilities of our probes, as discussed in Chapter 4. Molecular Beacons Molecular beacons (MBs), which were first introduced in 1996,37 they are single strand DNA probe molecules that have of a stem and loop structure.38-40 Figure 1-7 shows the molecular beacon structure and hybridization upon target ad dition. The loop sequence is complementary to the single strand target DNA.41 The stem portion consists of 5 to 7 base pairs complementary to each other, so that prior to binding the targ et DNA the structure is in the closed state.42 A fluorophore is covalently linked to the end of one arm (orange colo r), and a quencher is attached to the end of the other arm (blue color). Molecu lar beacons do not fluoresce when they are free in solution, because in the closed state form the fluorescence is quenched by the nearby quencher. However, when hybridizes to a nucleic acid strand containing th e target sequence, the stem break apart, distancing the quencher from the fluorophore, and therefore fluorescence is restored. Initially, MBs were used for DNA det ection but they can also be used for mRNA and molecules with higher complexity such as single DNA binding prot eins. The main advantages of these probes are: 1) the signal transduction mech anism, which provides high sensitivity, 2) target

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27 detection without further separation, 39 and 3) the ability to distinguish single base mismatches.38,39,42,43 Figure 1-7 Hybridization of MBs upon target addition. The MB consists of a stem and a loop structure that maintains the close proxim ity of the fluorophore (orange) and quencher (blue) moieties. Consequently, when the MB is free in solution, the fluorescence of the fluorophore is quenched by the quencher. However, upon addition of the target the loop sequence hybridizes with the complementary DNA and the beacon opens. The fluorophore and quencher moieties are no longer close, and fluorescence is restored. However, when designing MBs for biologi cal applications many factors must be considered. For example, the fluorophore-quencher pa ir selection is very important and is usually chosen according to the application and instrument s available (including thei r capabilities in term of excitation and detection wavelengths). Table 1-1 shows some of the available fluorophores/quencher pair in cluding the ones used in this wor k. One of the major considerations is the background fluorescence intensity of the MB in the close state, which can significantly affect the detection limits of the analysis. When considering the synthesis of MBs for bi ological applications, the selection of the nucleotide base adjacent to the fluorophore is im portant, due to the quenching properties of the nucleotides. The greatest quenching efficiency is given by guanosine (G), followed by adenosine (A), cytidine (C), and thymidine (T). This property is due to the electron-donating ability of G,

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28 that permits charge transfer from the nucleobase to the vicinity uorophore.44-46Since this additional quenching decreases the signal in the open form, guanosine is not usually bonded directly to the fluorophore. One of the most useful parameters to determin e the stability of the synthesized MBs is the melting temperature, which is the temperature of which half of the complementary DNA stands are hybridized and half are free in solution. Previ ous results have demons trated that GC rich sequences have more stability (therefore higher melting temperature) compared to non-GC rich sequences,47 because of the greater degree of H bonding between the G-C pairs compared to A-T pairs. MBs in solution with their target can exist in 3 states: 1) bound to target (opened state), 2) in the form of hairpin (closed state) and 3) random coil. The following formula explains these phenomenons:48 BT BOpen + T BClosed + T where BT is the probe-target duplex, BClosed is the molecular beacon in the form of a hairpin; BOpen is in the form of the random coil and T is the complementary target sequence. In addition, the sensitivit y and selectivity of the MB for its target can be optimized for the desire applicati on by adjusting the sequence to be more GC rich or deficient.49 Therefore, if a MB for single-base mismatch is needed for the analysis then a short loop sequence will provide best results. The same way, incr easing the length or strength of the stem of molecular beacons, increases the difference between melting temperat ure of the perfectly complementary sequence and the mismatched. Thus, changes in the stem allow further improvement in the molecular beacon selectivity, without altering experimental conditions. However, wh en the stem is too long, the hybridization kinetics is slow because the molecular beacons tend to remain closed. If

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29 this degree of selectivity is not required for an alysis longer loop sequence can be used. However, the longer the loop sequence, the more difficult it is for the stem to maintain the hairpin structure. Therefore, it is important to c onsider all the aforementioned properties when synthesizing a probe in order to make sure that it will hold some potential for bioanalysis. Molecular beacons have been used for intrace llular measurement with the ultimate goal of monitoring mRNA inside a single cell. This is possible because of the inherent signal transduction mechanism of the MB, which allows the detection of target molecules without the need of separation. However, the design of the MBs for this purpose requires a series of important steps as shown in Figure 1-8. First, an d perhaps the most important is to choose the appropriate target gene, which can be found in NBCI GenBank. The selected gene must be highly expressed inside the cell to increase th e sensitivity of the assay. Secondly, the possible mRNA secondary structure must be predicted, so that the most stable and accessible mRNA regions for probe binding can be chosen (can be done by using m-fold software package). Gene Selection mRNA structure Identify accessible target regions Design the loop and stem of the MBs Calculate the Tm for each loop and stem combination Predict secondary structures of the MB Select dye and quencher Test in vitro Test in vivo Figure 1-8 Flow chart illustrating th e MB design and synthesis process

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30 The complex secondary structures exhibited by the RNA sequences make this step critical. Usually, the best target site wi ll be a region that has the most single stranded RNA. These regions are used to create complementary loop sequences of the MBs with various lengths. Third, the number of base pairs in the stem and loop must be optimized. The stem is created with a high GC content (75 to100), which provides be tter stability and que nching efficiency. There are numerous Web-based tools to help designing optimal molecular beacons. For, example integrated DNA technology (www.idt.com) presents an excellent web-tool by providing secondary structure determinations, as well as the Tm for both the loop-target hybrids and stemloop conformation. The next and final steps are: dye and quencher selection, synthesis, and in vitro testing. In vitro testing is mainly performed because the R NA folding programs do not provide a reliable secondary structure therefore; di fferent probes are usually designe d and tested in buffer solution before further analysis. If sensitivity has been proved in vitro and in solution then experiments in vivo can be carry on. Intracellular delivery of the MBs for mRNA detection can be performed by different methods, which include, electroporation,50 cell-penetrating peptides, 51 Streptolysin O,52 and microinjection.53 The latter is the one effectively used in our lab and is, ther efore, the focus of this work. Microinjection of the probe achieve s high transduction efficiency in transductionchallenged cells, and it also controls deliv ery dosage and timing of delivery precisely.54 In addition, microinjection has lower cytotoxicity compared with chemical transfection or viral infection, especially in sensitive ce lls, such as human primary neurons.55,56 Microinjection has been widely used to deliver RNAs/interfering RNAs,57,58neutralizing antibodies,59,60 and

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31 nanoparticles.61 Although, there are only a few cells injected in a cell culture, the number of cell injected is sufficient to assure reasonable statistical analysis.54 In the last decade, MBs has been used to monitor and target mRNA inside cells.39,40,62-65 Moreover, multiple gene detection has been pos sible using MBs with different labels for a variety of mRNA sequences.53 Linear Fluorescent Probes Linear probes are labeled nuc leic-acid fragments that are co mplementary to the target sequence. Single-stranded DNA unlike MBs, do not fo rm a hairpin structure but they can have a quencher and a fluorophore attached at opposite ends They behave like random coils, creating a measurable signal change upon binding to the target These linear probes have been successfully tested to monitor RNA in tomato mosaic tobamovirus by coupling double donor fluorophores and one acceptor dye.66 Hybrid Molecular Probe In considering the requiremen ts of molecular probes for intracellular m RNA monitoring, we developed a hybrid molecular probe (HMP) for single living cell studies. This probe consists of two single strands of DNA with a polyethylene glycol (PEG) li nker used to tether these two sequences together. A fluorescen ce donor is attached to the 3 end of one DNA strand, and another fluorophore, acting as an acceptor, is atta ched to the 5 end of the other strand. In the random unhybridized conformation of the probe, the two fluorophores ar e spatially separated from each other. However, when the target sequence containing the complementary sequences to both probes at adjacent positions is added, each st rand binds to its corresponding target sequence. Thus, bringing the two fluorophores into close proximity, which allows fluorescence resonance energy transfer (FRET) to occur. It is this transfer of energy that result s in the quenching of the

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32 donor fluorescence and corresponding fluorescence enhancement of the acceptor fluorophore.67,68 The working principle of the probe is shown in Figure 1-9. Figure 1-9 Working principle of the hybrid molecu lar probe (HMP). HMPs consist of two single strands of DNA (green); a polyethylene glyc ol (PEG, in purple) linker is used to tether these two sequences together. A fluor escence donor is attached on the 3' end of one strand (green), and another fluorophore, acting as an accepto r, is attached to the 5' end of the other strand (orange). In th e random unhybridized conformation of the probe, the two fluorophores are apart from each other. However, when a target (orange strand) containing the complementary sequences to both probes at adjacent positions is added, each strand will bind to its corresponding target sequence bringing the two fluorophores into close proximity, which allows energy transfer to occur. DNA Based Sensors Nucleic acids hybridization can be combin ed with the sensitivity of optical, electrochemi cal or gravimetric transducer to form DNA sensors and DNA chips. DNA sensors are valuable tool in medical diagnostic, genetic screening, drug design, environmental and food analysis due to the rapid, simp le, low cost, sensitive and selective properties of these systems. DNA sensors are mostly based on the immobiliz ation of a DNA probe onto the transducer surface and the subsequent monitoring of the sign al generated due to hybridization with target molecules. There are many factors to consider when using DNA for hybridization studies onto a surface such as: type of surface, selection of the functional group, position of the functional group in the probe and label molecules. Glass, pl astic, and gold surfaces are the most well known surfaces for DNA based hybridization studies. There are different types of biosensors such as

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33 optical, electrochemical, DNA biochip, piezoelect ric, and colorimetric or strip type DNA sensors. In this report we will be focusing on the first three. Optical methods are the most frequently use d, however, requires a label for detection, which can be chosen based on the stability, and convenience. One example of an optical biosensor is that based on a fiber optic to tran sducer the emission signal of a fluorescence label.69 These biosensors can carry the light even at longer distances by using reflection. It requires the immobilization of a DNA probe at the end of the fiber and monitoring the changes in fluorescence upon hybridization with the target. Ev anescence wave have also been used as optical transduction because of the high sensitivity even at single molecule level. MBs have been used for this purpose and it will be further discussed in Chapter 4. Another example of optical biosensor is surface plasmon resonance (SPR), which measures the interaction of light with the metal surface (typically gold due to its stability). Under specific conditions the electrons at the surface ab sorb incident light photons and convert them into surface plasmon waves. Perturbations at th e surface, such as an interaction between probe molecules immobilized onto the surface and captured target molecules, induce a modification of resonance conditions, which are reported and measured as a change in reflectivity. The resonance conditions are influe nced by the material adsorbed onto the thin metal film.70 A linear relationship is found between re sonance energy and mass concentr ation of molecules such as proteins, sugars and DNA. Electrochemical biosensors are also used as DNA biosensors due to the fast and low cost analysis. They involve monitoring change s in conductance, electric potential and oxidation/reduction reactions. One of the biggest advantages of us ing electrochemical biosensors is that the detection can be performed on bot h label free and labeled probes. However, the

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34 immobilization step requires high probe orientation and accessibility for target hybridization. Methylene blue and ferrocenyl naph thalene diimide are some of the most used intercalator dyes in electrochemical biosensors. They contain high affinity fo r double stranded DNA whereas little or no signal is present for the single stranded probe. A large number of DNA probes immobilized in a known arrangement on a solid surface (e.g. glass, plastic or sili con) produce a DNA microarray.71 The probes can range from a few hundred to thousands of single-stranded oligonu cleotide sequences or other molecular probes (such as MBs) immobilized to discrete sensing regions on the solid substrate72-74 to allow the detection of different genes simultaneously. Fo r example, for two different samples the RNA must be isolated and labeled w ith two different fluorochromes (generally the cyanine 3 and 5, Cy3, Cy5) before being hybridized to with the immobilized probe ont o a glass microscope slide.70 A disadvantage of this method is that re quires prior knowledge of the genes to be assayed. However, this technology provides a fast, simple and sensitive method for the detection of genes that are highly expresse d. This is a novel technology promises to be at the vanguard of diseases diagnosis and therapy. Finally, forthcoming biosensors will require the development of novel devices or the improvement of the existing ones in order to allow superior tran sduction, amplification, processing, and conversion of the biological sign als as well as more compact and portable devices.70 Gene Expression Studies Genes that codes for protein are transcri bed to me ssenger RNA (mRNAs) in the cell nucleus. The mRNAs in turn are translated into proteins by ribosomes in the cytoplasm. The transcription level of a gene is taken to be the amount of its corresponding mRNA present in the cell. Traditional RNA techniques require mRNA pur ification from total cellular contents before

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35 analysis. However, mRNAs are difficult to work because are prone to degradation due to RNA digesting enzymes thus, to prevent the experime ntal samples from being lost, they are reversetranscribed back into more stable DNA form using reverse transcription polymerase chain reaction (RT-PCR). RT-PCR or qPCR is the gold standard methods for identifying and quantifying gene expression. The products of th is reaction are called cDNAs because their sequences are the complements of the original mRNA sequences. One of the disadvantages of using RT-PCR is that not all mRNAs are reverse transcribe with the same efficiency, which can change the relative amounts of cDNAs measured in each assay. On the other hand, the analysis saves time because there is no need to run a ge l, since the data is directly quantify by the instrument as the reaction proceed. In addition, can detect low abundance templates, which normally would not be visualized by conven tional-gel methods and reduces risk of contamination by using closed tubes. Another used approach for gene expression analys is is serial analysis of gene expression (SAGE). It is a powerful tool that allows the analysis of overall gene expression patterns with digital analysis.75,76 Because SAGE does not require a pr ior knowledge of the genes to be assayed (as with microarrays) it can be used to identify and quantitative new genes as well as known genes. SAGE requires a relatively high amount of input RNA required, consequently, several techniques have been developed to overcome this limita tion, such as microSAGE, which entails a new protocol for the analysis of mRNA. Intracellular Imaging Using MBs Probes Intracellular images using m olecular beacons have concentrated on the detection mRNA (qualitalive studies) rather than quantitative studies in a variety of cells systems. In 2001, MBs were used to detect and visualize -actin and -1 andrenergic mRNA in kangaroo rat kidney (PtK2) cells. In this study, a ne gative control MB (no compleme ntary sequence inside the cell)

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36 was used to prove with no doubts that the increa se in fluorescence was due to the hybridization with the target mRNA and not fr om non-specific interaction. This report was important because it demonstrated the real-time detection capabilities of MBs.39 In 2003, Tyagi et al used MBs to investigated mRNA in Drosophila melanogastar oocytes. This experimental design resulted in the visualiz ation of the distribution and trafficking of oskar mRNA. In this study, the use of binary MBs were used to hybridi ze adjacent regions of the same target resulting in FRET (the donor and acceptor fluorophore were brought within close proximity). This approach reduced the backgr ound of the MBs and gene rates a new signal to visualizing the mRNA distributi on. In addition, they were able to track the migration of the mRNA throughout the cell and even into adjacent cells in the oocyte. In this report, MBs demonstrated their used not only for the localizat ion of mRNA inside of single cells but it also that MBs could be used fo r tracking mRNA migration ev en into different cells.62 Recently, several studies have concentrated their efforts on decrease the false positive signal and the non-specific interactions of th e MBs. In our group, locked nucleic acids were used to engineer novel MBs for long term intracellular monitoring.77 These MBs showed high sensitivity, selectivity and biostability even after days of monitoring -actin and MnSOD mRNA. In a different study, MBs were linked to quantum dots (QDs) to preven t gaining entry into the nucleous and therefore, eliminating the non-sp ecific fluorescent signal. By using this method, Tsourkas et al was able to measure the expression of the endogenous proto-oncogene c-myc in MCF-7 breast cancer cells.78

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37 Challenges of Molecular Probes for In tracellular Measurements and Biosensor Development The aforem entioned molecular probes have been used for both intracellular measurements and biosensor development. However, one of th e biggest limitations of DNA probes is their low sensitivity. Thus, most applicatio ns have involved detection of highly expressed and stimulated genes.53,62 Moreover, the signal levels measured are not easily related to absolute quantities of target transcripts.79 The sensitivity of molecular probes is determined by many factors, including as mentioned above, dye selection. Every dye has different properties that may vary according to the application. For example, 6-carboxyfluores cein (FAM) is very sensitive, has a high fluorescence quantum yield, good water solubil ity, and excitation maximum (494 nm) that closely matches the 488 nm spectral line of the ar gon-ion laser. These properties make FAM an important fluorophore for confocal laser-s canning microscopy and flow cytometry, unfortunately, FAM has several limitations such as a high rate of photobleaching,80 pH sensitive fluorescence, a relative broad emission spectrum (limiting its utility in some multicolor applications), and a tendency toward que nching of its fluorescence on conjugation to biopolymers, particularly when each polymer molecule is substituted with many FAM.81 These characteristics can limit the sensitivity and application of this dye for intracellular measurement, where ultrasensitive de tection is required. In addition, molecular probes, es pecially MBs in their off or closed state form, can have high background intensity, which lowers the signa l/noise ratio and raises the detection limits. Moreover, DNA-based probes trend to suffer degradat ion inside the cell, wh ich can subsequently lead to false positive signal (especially for MB) and or a decrease in the overall sensitivity or selectivity of the assay.77,82

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38 Therefore, it is imperative to be able to construct reliable and stable nu cleic acid probes for both intracellular and bios ensor applications. Research Objective The overall goal of this research was the de velop ment of fluorescent molecular probes for both intracellular analysis and biosensor developmen t. As described earlier in this chapter, the main requirements for these molecular probes are: high stability, reproducibility, selectivity and signal-to-background ratio. Consequently, a hybri d molecular probe (HMP) was developed and optimized to overcome the major stability pr oblems of previous molecular probes for intracellular measurements. In addition, MBs were optimized with the ultimate goal of impr oving the selectivity, sensitivity and signal-to-background ratio for surf ace hybridization studies. In order to improve the existing MBs, locked nucleic acids (LNA) were used in the beacon design instead of regular DNA bases. Locked nucleic acids offer greater st ability, affinity and lower background intensity compared to MBs with regular DNA bases and are ideal for hybridization studies.

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39 CHAPTER 2 HYBRID MOLECULAR PROBE: DESI GN, SYNTHESIS AND IN VITRO CHARACT ERIZATION Introduction Gene expression studies in livi ng cells present a s ignificant challenge. As discussed in the previous chapter, one of the biggest difficultie s is the specificity, sensitivity and signal-tobackground ratio of the probes. These problems require not only careful design of the molecular probes, but also a better unders tanding of target accessibility and probetarget interactions.83 The central dogma of molecular biology reveal s important processes that maintain our bodies working properly. It states that the DNA contains all the genetic information, which is transcribed to mRNA, and fina lly translated to proteins.84,85 This overall process, called gene expression is down regulated according to the sp ecific needs of the individual. Therefore, measurements of changes in concentrations wi ll provide important information for molecular biology and medicine. In this chapter, a complete description of the design, synthe sis and characterization of the hybrid molecular probe (HMP) is reported (see Figure 1-5). The experiments will show that HMP has the potential not only for mRNA/DNA detection capabilities but also for future applications in biomedical fields becaus e of its specificity and sensitivity. Probe and Target Synthesis The -tubulin sequence fr om Aplysia genes, whic h are highly expressed within Aplysia cells, was used. All the DNA reagents for the sy nthesis of HMP-tubulin and targets shown in Table 2-1 were purchased from Glen Researc h. These probes and DNA targets were synthesized with an ABI3400 DNA/RNA synthesizer. FAM core pore glass (CPG) was used for all FAMlabeled probe synthesis, whereas Cy5 was labeled using Cy5 phosphoramidite. Specifically, spacer phosphoramidite 18 was used to incorporate varying lengths of PEG as a linker in the

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40 HMP probe, in order to investigate the role of the effect of th e number of monomer units: n= 8, 12, 16 and 20. Table 2-1 Sequence of HMP probe and thei r targets Name Sequence HMP Probe 5 -Cy5-CTC ATT TTG CTG ATG ACG-(PEG)n-CTG TCT GGG TAC TCC TCC-FAM-3 where n=8, 12, 16 and 20 units Target AT 5 -GCT CAT CAG CAA AAT GA G GGA GGA GTA CCC AGA CAG-3 Target AA 5 -GCT CAT CAG CAA AA A GAG GGA GGA GTA CCC AGA CAG-3 Target AG 5 -GCT CAT CAG CAA AA G GAG GGA GGA GTA CCC AGA CAG-3 Target AC 5 -GCT CAT CAG CAA AA C GAG GGA GGA GTA CCC AGA CAG-3 Random 5 -TCT GTG TAA TCA ACT GGG AGA ATG TAA CTG ACT AGC3 Target 1T 5 -GCT CAT CAG CAA AAT GAG T GG AGG AGT ACC CAG ACA G -3 Target 3T 5 -GCT CAT CAG CAA AAT GAG TTT GGA GGA GTA CCC AGA CAG -3 Target 5T 5 -GCT CAT CAG CAA AAT GAG TTT TT GGA GGA GTA CCC AGA CAG -3 Target 7T 5 -GCT CAT CAG CAA AAT GAG TTT TTT T GGA GGA GTA CCC AGA CAG -3 Letters in red represents a single base mismatch According to the manufacturer s recommendations sensitively labeled r eagents, such as Cy5, require the use of bases with special protecting groups. Therefor e, for all Cy5-labeled probes, monomers with phenoxyacetyl-protected (deoxyadenosine, dA), 4-isopropyl-phenoxyacetylprotected (deoxyguanidine, dG), and acetyl-protect ed (deoxycytidine, dC) were used for the synthesis. Subsequently, deprotection of these monomers, along with the Cy5-labeled phosphoramidite, was performed via overnig ht incubation with a mixture of 0.05 M K2CO3/methanol. The solutions that resulted from deprotection were then pr ecipitated in ethanol, and the precipitates were disso lved in 0.5 mL 0.1 M triethylammonium acetate (TEAA, pH 7.0) prior to purification with reve rsed-phase high-pressure liqui d chromatography (RP-HPLC) on a

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41 ProStar HPLC Station (Varian, CA) equipped with a fluorescent and phot odiode array detector. A C18 reversed-phase column (Alltech, C18, 5 M, 250 4.6 mm) was used for separation purposes. The product collected from the HPLC was vacuum dried and then mixed with TEAA for a second round of HPLC. The final product wa s collected and incubate d with acetic acid (80%) for 20 min, followed by 200 L of ethanol and subsequently vacuum dried. Quantification of the probes and targets were performe d using UV-Vis spectrometry. Fluorescence measurements were performed using a SPEX Fluo rolog spectrofluorometer from Horiba Jobin Yvon (Fisher Scientific Co., Pittsburgh, PA). The buffer used consisted of 20 mM Tris, 50mM of NaCl and 5 mM of MgCl2 with a pH of 7.5. Fluorophores Selection As described in Chapter 1, sele ction fluorophores plays an importa nt role in the design of any fluorescence probe. First, the efficiency of the energy transfer depends greatly on the selected dye, which is prim arily because each dye covers a specific spectral region. Second, the signal-to-background ratio can be si gnificantly improved by the proper selection of the dyes. In addition, sometimes it is necessary to evaluate th e experimental conditions, because factors such as pH and temperature can also affect the fluorescence characteristics of the fluorophores. Fluorescein (FAM, donor) and Cy5 (acceptor) we re selected for the HMP design. FAM will be the donor molecule and C y5 will be the acceptor dye. This dye pair was selected for the following reasons: the fluorescence emission of the two dyes is completely separated; the excitation of Cy5 caused by absorption of the FAM excitation light is negligible; and both dyes have a spectral overlap which allow the FRET. Figure 2-2 shows the absorbance and emission spectra for FAM and Cy5. The absorption and excitation wavelength of the dyes are: 488 nm and 520nm for FAM and 643 nm and 665 nm for Cy5.

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42 Figure 2-1 Absorbance and emission spectra for FAM and Cy5. Excitation of FAM occurs at 488 nm with an emission at 520 nm, a nd for Cy5 the excitation and emission wavelengths are 643 nm and 665 nm, respectively. Proof of Principle for the novel HMP The effect of PEG length in the new HMP wa s investigated in or der to understand the function of the polym er and therefore, to id entify if it interferes with the probe-target hybridization. As a proof of concept, an expe riment was conducted, using HMP-16 with both random DNA and a perfectly complementary seque nce as target (cDNA) in a Fluorolog-Tau 3 spectrometer. The buffer used for all the in vitro experiments consists of 20 mM Tris, 50mM of NaCl and 5 mM of MgCl2 with a pH of 7.5. Each solution contained 200 L buffer and adequate probe and target to make a final concentrati on of 300 nM. After mixing, the emission intensities at 520 nm (donor) and 665 nm (acceptor) were monitored as a function of time, and the results are shown in Figure 2-2. This experiment confirme d that the probe response was due to specific DNA hybridization. As expected, there was little or no change from the HMP when the random sequence was added, confirming that the hybridization was specifically due to the HMP-target hybridization.

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43 The graph shows the hybridization in terms of ratio versus the time taken for the probe to reach the equilibrium. The ratio repres ents the intensity of Cy5 and FAM (I665nm/I520nm) after target addition. Ratiometric measurements represent an enormous advantage for this probe because allow to compensate for light source fl uctuation. In addition, HMP was able to hybridize with the target without any separation step. This minimizes the assay time, and allows a real time detection of target molecules without the need of separating unbound probe Finally, this result not only showed that HMP hybridizes with the co mplementary target sequence but also is an indication of its selectivity since no si gnal was observed from the random DNA. 050100150200250300350400 0 2 4 6 8 10 12 14 16 18 20 22 24 Intensity Ratio Time (s) cDNA Random DNA665nm/520nmAddition of Target Figure 2-2 Hybridization of HM P-16 with the target cDNA and with a random DNA sequence Figure 2-3 shows the emission spectra before an d target addition and after equilibrium was reached (at 488 nm excitation and temperature of 25 C). The intensity changes show that FRET occurred only when the perfect complementary DNA target was added to the buffer solution. This phenomenon is mainly due to the close proximity of the dyes upon target hybridization. Therefore, the fluorescence signal of FAM at 520 nm decreased (donor molecule), while the Cy5

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44 fluorescence intensity increased at 665nm (acceptor molecule). This data provided evidence with no doubt that the system has undergoes FRET. Figure 2-3 Emission spectra of the HMP-16 before cDNA (tg) addition and after the equilibrium was reached. Linker Effect in HMP Design of HMP incorporates PEG for several reas ons: 1) it is compatible with the phosphoramidite chemistry (which al lows the integration of as many units as desired), 2) it is soluble, 3) it is non-toxic, 4) it provides mobility to the attached DNA sequences and 5) it lacks of interaction with the nucleotides bases.86 The main purpose of the linker is to tether the two DNA sequences together without in terfering with the hybridization with the target. It provides easy binding with the target, because only one whole molecule can be bound, instead of two separate DNA strands. Therefore, the polymer lengt h must be carefully optimized to keep the two sequences free to bind to their target, wh ile still allowing the two sequences to have relatively high local concentration to each other. The effect of li nker length was determined via the same experiment design described above (3 00 nM probe concentration) using HMP-8, 12, 16

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45 and 20 with cDNA as the target. As shown in Figur e 2-4, HMP-8 resulted in an intensity ratio of 7 compared to 12.5, 22 and 13 for HMP-12, 16 and 20 respectively. HMP8HMP 12 HMP16HMP20 0 5 10 15 20 25 Ratio(665nm/515nm) Figure 2-4 Effect of the PEG linker on th e hybridization of HMP with the target The low intensity ratio for HMP-8 can be attribut ed to the poor hybridization efficiency of the probe and target. Destabilization can also occur when the linker is too long. The low signal ratio of HMP-20 could be the result of multiple target binding. When the linker is too long it allows the two DNA strands to hybridize multiple targets and energy transfer is no t likely to occur from the donor to acceptor. In addition, a very large linker length decrea se the signal to the point that the advantage of using linki ng DNA fragments disappeared.86 Because the highest signal increase was found with HMP-16 this probe was used for the subsequent experiments. Fluorophores Distance Theoretically, FRET is a phenomenon that shou ld be involved in HMP; however, static quenching can play a significant role in the target hybridization and can decrease the signal

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46 enhancement. When HMP binds with the target it brings the two fluorophor es together, and this proximity allows FRET to happen. However, if the two fluorophores are too close to each other, static quenching can quench two fluorophores and decrease the overall signal enhancement. Therefore, the following experiments were pe rformed to optimize th e acceptor-donor distance and maximize FRET efficiency. The probe used was HMP-16 and the targets were synthesized by incorporating extra nucleoti des (thymidines) at the half way point between the perfect complementary sequences (targets 1T, 3T, 5T and 7T in Table 2-1). Figure 2-5 shows the results for this experiment where I 665/ I 520 nm ratio is plotted versus the number of bases added in the target. The graph shows that the energy tran sfer of the HMP is directly related to the fluorophores distance; incr easing the distance between the dyes, lowers the intensity ratio. As a result, energy transfer is lower when the distance between the dyes is increased. Thus, the highest intensity ratio resulted from the target with the perfect complementary sequence (0T bases). This experiment proved th at the fluorophores have to be in direct contact to obtain the highest FRET efficiency. Although, the scope of this experiment was not to determine the Frster distance, it was clear that the two fluorophores must be close in proximity. Figure 2-5 Effect of distance of the Accep tor-Donor dye pair on I 665/I 520 ratio

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47 Selectivity of the HMP For biological applica tions, it is very important for the HMP to be able to distinguish between the target m olecules and other molecules. In an attempt to observe the specificity of the HMP, target sequences were prepared with a si ngle base mismatch, as shown in Table 2-1. The degree of discrimination (selectivity) of the HM P is shown in Figure 2-6. As expected, the highest intensity ratio was produced by the target with the perfect complementary sequence, with the mismatched sequences showing varying results. Although, the signal di d not show significant differences, HMP has potential for monitoring a single-base mismatch in the target. This capability can be important for single polymorphism detection. The differences in signal could be due to the different affinities that each base has with a non-complementary base. AA AC AG AT 0 5 10 15 20 Intensity Ratio 665nm/520nm Figure 2-6 Hybridization of 300nM HMP-16 to same concentration of their targets. AT stands for the perfect matched target. Buffer used: 16mM Tris-HCl (pH7.5), 40mM NaCl, 4mM MgCl2 and 20% DMF

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48 CHAPTER 3 COMPARISON OF THE HMP WITH MB AND OTHER MOLECULAR PROBES FOR INTRACELLULAR ME ASUREME NTS Introduction Monitoring gene expression in li ving cells has long been of gr eat interest and a challenge for ma ny scientists in the field. As describe d in Chapter 1, well-known techniques such as Northern and Southern blot, in situ hybridization and RT-PCR, are time consuming and, in most cases, can yield only an average of millions of cells. To improve on methodologies, sensitive and stable nucleic acid probe s must be developed. Molecular beacons (MBs) and hybrid molecular probes (HMP) have been developed and promise to have a significant imp act in bioanalysis and biotechnology.8-10,68 The principles of MB design and use were described in Chapter 1. These new molecular probes offer several advantages over traditional DNA/mR NA techniques: 1) they have the ability to perform rapid and fully homogeneous analysis,13 2) recognition of the targ et and optical reporting occur simultaneously, which is an advantage for ho mogeneous high-throughput assays and real-time analysis; 3) as a result of DNA hybridization to complementary sequences, they are highly selective.17,87 On the other hand, the use of MBs for mRNA/DNA hybridization studies has several drawbacks. For example, it has been report ed that the response of MBs is not static and can fluctuate between different conformations.48 As shown in Figure 3-1, the effectiveness of MBs is also hindered by degradation from cyt oplasmic nucleases, protein interactions (with DNA binding proteins), destabiliza tion due to thermodynamics fluctuations, and sticky end pairing (SEP) all which can cause false positive signals.11,17 SEP occurs when 2 or more MBs are hybridized with the target and the stem of one MB hybridizes with other MB stem forming a double helix and quenching the fluorescence of th e MBs. This phenomenon can significantly decrease the overall fluorescence change in the assay.88 Finally, the closed state (off) form of the

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49 MBs is in dynamic equilibrium with the ope n (on) form. Depending on the favorability of spontaneous loop opening, the resulting backgrou nd signal can raise the detection limit. To address these limitations, an alternative probe for gene expression studies, the HMP, was developed and tested in vitro as described in Chapter 2. In this chapter, we report the investigation of the HMP for in vivo applications and compare it to other probes currently used for intracellular measurement. These experime nts demonstrate the advantages of HMPs over MBs and linear probes for use in either hybridiz ation studies or intrace llular analysis inside single living cells. Protein binding Sticky end pairing Enzymatic Cleavage Destabilization Figure 3-1 Concerns when using MBs for intracellular measurements Probes and Target Synthesis Molecular probes were designed based on the ma nganese superoxide dismutase (MnSOD) and -actin gene sequences. MnSOD is one of the cells primary defenses against free radical damage by regulating reactive oxygen species (ROS). Stimulated levels of MnSOD enzyme have developed to address increased free radical production during an inflammatory episode and as

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50 antiapoptotic agent.89,90 On the other hand, -actin is highly express within human cells, whereas -tubulin sequence from Aplysia gene is highly expressed within aplysia cells The synthesized sequences are shown in Table 3-1.The DNA reag ents for the synthesis of HMP-tubulin, HMP actin, HMP MnSOD, MB-tubulin, MB-MnSOD and targ ets were purchased from Glen Research, and an ABI3400 DNA/RNA synthesizer was used for the syntheses. Table 3-1 Sequences synthesized for molecular probes and targets Probe Name Sequence HMP Tub 5 -CY5-CTC ATT TTG CTG ATG ACG-(X)16-TGT CTG GGT ACT CCT CC-FAM-3 where X represent PEG HMP MnSOD 5 -Cy5-TCT TAC ATT GAC -(X)16-TTA GTT GAC CCC-FAM-3 HMP -actin 5 -Cy5-AGA GCG CCT CAG GGC-(X)16-GGA AGG AAG GCT GGA-Oregon green-3 MB Tub 5 -FAM-CGC ACC TCC TCC CTC ATT TTG CTG GGT GCG dabcyl-3 MB MnSOD 5 FAM-CCG AGC CAG TTA CAT TCT CC C AGT TGA TT G CTC GGdabcyl -3 MB control 5 -AF555-CCT AGC TCT AAA TCG CTA TGG TCG CGC TAG G BHQ2-3 Reference Probe 5 TCT AAA TCG CTA TGG TCG C-AF488-3 Target Name Sequence -tubulin 5 -GCT CAT CAG CAA AAT GA G GGA GGA GTA CCC AGA CAG-3 -actin 5 GCC CTG AGG CGC TCT TCC AGC CTT CCT TCC-3 MnSOD 5 -GTC AAT GTA AGA GGG TCA ACT AA3 Mismatch AA 5 GCT CAT CAG CAA AA A GAG GGA GGA GTA CCC AGA CAG-3 Mismatch AG 5 -GCT CAT CAG CAA AA G GAG GGA GGA GTA CCC AGA CAG-3 Mismatch AC 5 -GCT CAT CAG CAA AA C GAG GGA GGA GTA CCC AGA CAG-3 FAM CPG was used for all FAM-labeled probe synthesis, while Cy5 was labeled using Cy5 phosphoramidite. Spacer phosphoramidite 18 was used to incorporate PEG as the linker in HMP probes. The MBs were synthesized using dabcyl CPG and FAM phosphoramidite. Deprotection of the monomers and the Cy5 labeled phos phoramidite was performed using overnight incubation with 0.05M K2CO3/methanol. The solutions resul ting from deprotection were

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51 precipitated in ethanol. The precipitates were th en dissolved in 0.5mL 0.1 M TEAA (pH 7.0) prior to purification (twice) w ith RP-HPLC as described in Chapter 2. The reference probe, as well as the control MB, (based on publis hed mRNA sequences) were synthesized by Genomechanix (Gainesville, FL). Alexa Fluor 488 (AF488) and Alexa Fluor 555 (AF555) were purchased from Molecular Probes (Eugene, OR). The quencher used for the control MB was Blackhole Quencher 2 (BHQ2). Fluorescence Measurements The in vitro experime nts were performed using a SPEX Fluorolog spectrofluorometer from Horiba Jobin Yvon (Fisher Scientific Co., Pittsburgh, PA). The buffer for all in vitro hybridization experiments consisted of 20 mM Tris, 50mM of NaCl and 5 mM of MgCl2 with a pH of 7.5. The data was plotted and analyzed as the first derivative of the melting curve versus the temperature. The first derivati ve provides a mathematical formula tion of the rate of change in fluorescence over the range of temperatures. Thermal Stability The solutions were excited at 488nm and m onitored at 520nm and 665nm from 15 C to 95 C. Using a water bath (RTE-111 from Neslab), the melting temperatures were found for the respective hybridized probes. The instrument was settled in a way that increases one degree and holds for 3 mins, reading the fluorescence at 3 different channels afterwards. Cell Preparation MDA-MB-231 breast carcinoma cells (Am erican Type Culture Collection, Manassas, VA) were maintained in Dulbeccos Modification of Eagles Medium (DMEM, Fisher Scientific) with 10% fetal bovine serum (Invitrogen, Carlsb ad, CA) and 0.5 mg/ml Gentamycin (Sigma, St. Louis, MO) at 37oC in 5% CO2/air. Cells were plated on 35mm glass bottom culture dishes and grown to 80% confluency (MatTek Corp., Ashland, MA) for 48 hours prior to injection. To

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52 stimulate MnSOD mRNA expressio n, cells were incubated in 1 g/ml lipopolysaccharide (LPS) from E. coli serotype 055:B5 (Sigma, St. Louis, MO) for 4 hours prio r to injection. Fluorescence Imaging Fluorescence imaging was conducted with a conf ocal m icroscope setup consisting of an Olympus IX-81 inverted microscope with an Olympus Fluoview 500 confocal scanning system and three lasers, a tunable Argon Ion laser (458nm, 488nm, 514nm), a green HeNe laser (543nm), and a red HeNe laser (633nm) with three separate photomu ltiplier tubes (PMT) for detection. The cellular images were taken with a 40x 1.35 NA oil immersion objective. A Leiden microincubator with a TC-202A temperatur e controller (Harvard Apparatus, Holliston, MA) was used to keep the cells at 37oC during injection and mon itoring. An EXFO Burleigh PCS-6000-150 micromanipulator was used for positioning the injector tip. An Eppendorf Femtojet microinjector with 0.5 m Femtotips was used to inject the molecular probes and reference probe into the cells. The referen ce probe with AF488 was excited at 488nm and collected at 520nm. The control MB with AF 555 was excited at 543nm and collected at 570nm. The Oregon Green (donor)/Cy5 (acceptor) HMPs we re excited at 488nm and collected at 520nm and 665nm, respectively. Images were taken eith er every minute for 15 minutes, or every four minutes for 60 minutes, including an initial image after a brief focusing period to yield the highest intensity for the probe. The images were assigned color representations, which are not indicative of the actual emission wavelengths. Th e data collected from the confocal microscope were analyzed using the Fluoview analysis software. Ratiomet ric analysis of the HMP and single strands was performed by dividing the fluorescence emission of Cy5 at 665nm by the emission of Oregon green at 520nm: Ratio = I Cy5 665nm / I FAM 520nm

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53 where I Cy5 665nm and I FAM 520nm refer to the emission intensitie s of Cy5 and FAM dyes that were collected at 665 nm and 520 nm, respectively. The control MB data were analy zed using the following equation: Ratio = ( Sbeacon Bbeacon)/( Sreference Breference) where Sbeacon is the signal of the beacon in the open state form (570 nm), Sreference is the signal of the reference probe collected at 520 nm, and Bbeacon and Breference are the backgrounds of the beacon and reference probe, correspondingly. The Bbeacon and Breference signals were obtained from a region outside of the cell monitored in the 570 nm and 520 nm channels, respectively. Ratiometric analysis has the advantage of normalizing the fluorescent intensities by compensating for the instrumental and experimental variations inherent in intracellular analysis. Comparison of HMP with and wi thout PEG as a linker The main objective of this experiment was to investigate how the polymer linker affects HMP hybridization and equilibrium. To accomplish this, HMP -tubulin sequence from Aplysia genes was synthesized with a 16 units spacer (Table 3-1). Thes e genes are highly stable and highly expressed within Aplysia cells. Subsequently, the same sequence was used to synthesize the equivalent probes without PEG as a linker, i.e. two separate DNA probes. The I 664/I 520 nm ratio (Figure 3-2) was measured for both DNA probes (300 nM probe concentration) for different concentrations of targets. Figure 3-2 shows that HMP was able to de tect concentrations as low as 0.70 nM target solution, while the single stranded DNA probes without the PEG spacer required at least 7.2 nM target. Also, these probes we re found to differ in their respective dynamic ranges. Specifically, HMP (with the linker), has a larger dynamic range than the probe without the linker. This is explained in that the PEG linker keeps the two DNA strands in close proximity allowing the binding of the HMP to the same target even at high probe:target ratios. In contrast, the DNA strands without the PEG linker showed a d ecrease in fluorescence, even when the ratio

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54 of probe:target was one to one. This response oc curs because two single-strand probes tend to bind to different target molecules when the target is in excess. Th is decreases the energy transfer of the single strand probes. However, the response of HMP (with linker) shows that the tethering of the probes facilitates the hybrid ization with the target, because both ends of HMP bind to the molecule, and bring the energy tran sfer pair into close proximity. Figure 3-2 Influence of the concentration of targ et in the hybridization with single strand probe (no PEG linker) and HMP (with PEG linker). A) HMP shows better hybridization efficiency at higher concentrations of targ et. B) Hybridization of the probes at lower concentrations. Ratiometric analysis of the probes was performed by dividing the fluorescence signal of Cy5 at 665nm by that of FAM at 520nm. Three replicates were performed for each target concentration. An accurate and reliable way to predict the stability of DNA-based pr obes, targets and the resultant duplex is by evaluation of the dupl ex melting temperatures, Tm, which is the temperature at which one half of the duplexes have dissociated into the corresponds two single strands. The DNA probes were hybridized usi ng a one-to-one probe:t arget ratio at a concentration of 300 nM each. The probes were excited at 488 nm and monitored at 520 nm and 665 nm for both the HMP and the two single DNA strands. The fluorescence intensity for each

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55 probe was measured from 15 C to 95 C at interv als of 1C with holding times of 3 minutes. The melting temperature for both probe s is shown in Figure 3-3. 0 10 20 30 40 50 60 70 80 Single strands (no PEG)HMP (with PEG)Temperature (C) Figure 3-3 Melting temperatures for HMP (with PEG) and single stranded probes (no PEG). Higher melting temperature of the HMP indica tes higher stability of the duplex over the single strand probes without PEG. Calc ulation of the melting temperature values for each probe was determined as described in the materials and methods section. The melting temperatures of the HMP and th e probe with no linker were 67 and 30C, respectively. The higher melting temperature of the HMP indicates increased stability over the probe without the polymer linker, especially at physiologically relevant temperatures. Since PEG dramatically increases th e local concentration of one DNA stra nd to another, its importance as a linker is clearly demonstrated. Furthermore, th e local concentration e ffect facilitates probe hybridization and stabilizes the hybridized product. This is important in in tracellular applications taking place at 37C, because the duplex of the HMP and its target will be stable at that temperature while the duplex of the target a nd the probe with no linker will not be stable.

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56 Comparison Experiments between the HMP and MBs As me ntioned in the previous discussion MBs have already been used for hybridization studies with some degree of success. Therefore, a set of experiments was design to compare HMP to MBs on the basis of parameters such as kinetics and stability, both in vitro and in vivo In particular, and as a further test of their util ity, the performance of each type of probe inside single living cells will be explored. Kinetics Since the intensity ratio correlates to the overa ll mRNA expression, it is important that the signals reach equilibrium as quick ly as possible. If the probe requires too much tim e to reach equilibrium, the effectiveness of a probe for in tracellular measurements is compromised. Hence, for intracellular experiments, faster hybridizat ion kinetics is desirabl e. Experiments were conducted using 300 nM was used for the probes (H MP tubulin or MB tubulin) and the target in buffer solution and the intensity was meas ured as a function of time after mixing. As results in Figure 3-4 demonstrate, the HM P shows a faster hybridization rate compared to that of the MB. Hybridization of the HMP reached maximum intensit y (>90 %) in less than 400 seconds upon target addition. On the contrary, intensity of the MBs continued to increase, even at 600 seconds after target addition. Compared to MBs, the HMP exhibits a faster and more efficient hybridization mechanism, possibly because HMP, unlike MBs, ha s a flexible linker allowing its two strands to stay together in a single open state. In the MB, the stem portion of the pr obe must open prior the detection of the fluorescence si gnal. Consequently, MBs require additional time to transition from the closed state of the beacon to the open stat e in order to hybridize. This is not the case for the HMP, which is always in an open state, and thus requires less time to hybridize to its target and reach a state of equilibrium.

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57 02004006008001000 0 2 4 6 8 10 12 14 16 18 Signal EnhancementTime (s) MB HMP Figure 3-4 Hybridization rates of MBs and HMP upon target addition. A faster rate of the HMP was observed when compared to that of the MBs. Selectivity of the HMP and MB The selectivity of a probe is characterized by the ability to recognize mi nor differences in target sequences. To test sele ctivity, the equilibrium intensit y ratios were measured for hybridization to target sequence with the sing le nucleotide mismatch. HMP Tubulin and MB Tubulin, and the probe:target concentration were 300 nM. Figure 3-5 shows the response of MBs and HMP to targets with single base mismatch at position 15, AA, AC and AG (see Table 3-1). Under the experimental conditions, MBs showed about 8-fold signal enhancement with the perfect complementary sequences (AT). When T, was changed to A, G, and C, the signal enhancement were 5.5, 4.5, and 2.5 respectively. When the experiment was performed under identical conditions but with th e HMP instead of MB, the highes t signal enhancement was found with the perfect complementary sequence or AT, which resulted in a signal increase of 20. The single base mismatch targets: AA, AC, and AG targets showed a signal enhancement of about

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58 12.5, 11, and 14, respectively. Although the signal enhancement of HMP was higher than the MBs, the selectivity of the HMP was slightly lower than the MBs, especially when T was replaced by C or G. The advantage of the MB over the HMP leaves room for future probe optimization, and one possible change of the HMP can be the addition of a hairpin structure. AA AC AG AT 0 1 2 3 4 5 6 7 8 9 Signal Enhancement AA AC AG AT 0 5 10 15 20 Signal Enhancement Figure 3-5 Comparison of selectivity of MB (blue) and HMP (red). Both probes were hybridized under the same conditions. The selectivity of the probes to each target was normalized to the selectivity of perfect match target AT HMP and MBs reaction with Deoxyribonuclease Reaction There are different types of deoxyribonucleases (DNases) that cleave the backbone of the DNA. For example, so me DNases can cleave a si ngle stranded DNA, others just double-stranded DNA; some are able to cut anywhere along the chain, and others have very specific sequence requirements. Deoxyribonuclease I, which was used for this set of experiments, cleaves at the phosphodiester linkage adjacent to pyrimidine nuc leotides, yielding 5'-phosphate terminated polynucleotides with a free hydroxyl group on position 3'. The HMP Tub and MB Tub (300 nM) were mixed with either DNase or with the complementary -tubulin target (AT in Table 3-1) and the signal enhancement was monito red as a function of time. The results in Figure 3-6 show that MB hybridization with the complementary sequen ce resulted in a ~12 fold enhancement and the

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59 signal increased to ~16 as a resu lt of DNase cleavage. These results demonstrated that molecular beacons in the presence of DNase give false positive signals. This phenomenon can be explained because DNase cuts the beacon sequence and de stroys the integrity of the structure. Consequently, the fluorophore is no longer quenched by the quencher resulting in the fluorescence of the dye. The signal from MBs caused by DNase was actually higher than the signal with cDNA because the quencher and the fl uorophore separate comple tely in the case of DNase digestion, while the fluorophore and quenc her remain linked to each other by one fragment of the double stranded DNA in the cDNA case. 91 Conversely, HMP with the complementary sequence gave a signal enhancement of 16, and no false positive signal was observed for the DNase. However, a slight signal decrease was obser ved as a result of the linear probe degradation. The decrease can also be explained because the DNase cut the probe in pieces causing a degradation of the HMP and ther efore a decrease in the fluorescence intensity. 0100200300400500600700 0 2 4 6 8 10 12 14 16 18 Intensity (A.U.)Time (s) MB-cDNA MB-DNase HMP-DNase HMP-cDNA Figure 3-6 Comparison of the intensity of MB and HMP in terms of DNase reaction. Buffer: used: 20mM Tris, 50 mM NaCl, 5mM MgCl2 at a pH of 7.5. The performances of both HMP and MB were fu rther tested with cancer cell (CEM) lysate. However, MB Tub responded immediately after the addition of cell lysate. With no target

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60 sequence in the system, this false positive response could result only from the nuclease digestion or nonspecific protein binding. Conversely, the HMP Tub did not give any significant signal change when cell lysate was added. The false pos itive result from MB compromises its ability to detect intracellular nucleic acid targets, because it failed to di fferentiate cell lysate containing cDNA from cell lysate without c DNA (Figure 3-7). For the HMP, the cell lysate itself did not cause any significant signal, and the cell lysate with cDNA indu ced an intense and immediate response indicating that HMP can be used in intracellular studies. 0200400600800100012001400 0 25 50 75 100 125 150 175 200 225 250 (b)Fluorescence IntensityTime (Second) MBTub+Cell lysate MBTub+Cell lysate+cDNA050100150200250300350400450500 0.020 0.025 0.030 0.035 0.040 0.045 0.050 0.055 (c)Cy5/FAMTime (Second) HMPTub + Cell Lysate HMPTub+ Cell Lysate+cDNA Figure 3-7 Response of MB and HMP to non-spec ific interactions. Response of 300nM HMP MB Tub(Left) and Tub (right) to cell lysate with and without cDNA Biological Stability MBs and HMP were developed as tools for nucleic acid ta rget detection. These fluorescence probes have relatively good selectiv ity and are designed to recognized target molecules based on Watson-Crick base pairi ng. However, DNA-based probes can also be affected by a number of factors such as prot ein binding and nuclease de gradation in complex environments such as the inside of a cell. Conseq uently, we investigated the biological stability of the probes in the presence of deoxyribonucleas e (DNase), effects of protein binding, and interactions with cell lysate. Two different seque nces were synthesized and tested for the MBs and HMP to make sure the observed behavior is not sequence specific; tubulin and MnSOD (see

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61 Table 3-1). Experiments similar to those reporte d in Figures 3-6 and 3-7 were performed using probe and reactant concentrations of 300 nM. Figure 3-8 shows a comparison of the responses of MBs and HMPs to the target and a several proteins and compounds in buffer solution. The probes were evaluated with two proteins known to interact with DNA, DNase I, which was described above, lactate dehydrogenase (LDH), which catalyzes the reversible interconvers ion of lactate and pyruvate. The probes were also tested in cell lysate to ev aluate their overall st ability in a complex environment. In the case of HMP probes, there was very little response in the presence of cell lysate, LDH or DNase interaction. cDNACell LysateLDHDNase 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Normalized Intensity HMP Tub HMP MnSOD MB Tub MB MnSOD Figure 3-8 Stability of HMP and MB in the presence of cell lysate, LDH and DNase I. The fluorescence intensity was normalized respect to the maximum signal obtained in the presence of target at a concentration of 300 nM. Two different DNA sequences were studied: tubulin and MnSOD. However, addition of LDH to the MB solutio ns induced an increase in the fluorescence intensity resulting from the separation of the fluorophore and quencher. As long as the degradation or undesired binding of the MB results in the separation of the fluorophore and quencher, a fluorescent signal is produced that is indistinguishable from target hybridization.

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62 Although HMP may also interact with the some of th e proteins tested, it doe s not generate a false positive signal, which is a significant advantage for hybridization studies and/or intracellular measurements. Intracellular Measurements The experime nts reported above have inve stigated the behavior of the HMP only in vitro but it is more important to determine the performance of these probes in a cellular environment. Therefore, the next goal was to monitor the stability of the probes inside a living cell. In order to accomplish this, both the HMP and MB were delivered into human breast carcinoma cells and monitored every 4 minutes for one hour as shown in Figure 3-9. Neither the HMP nor the MB is complementary to a target inside the cell that is expressed at a detectable level. Ratiometric measurements were performed for the probes, preventing variability of the fluorescence signal due to scattering inside the cell, excitation so urce fluctuations, variation of cell volume and variation in microinjection delivery. A reference probe was inject ed along with the control MBs. The reference probe emits a stable and constant signal that acts as an internal standard for analyzing the signal from the MBs. Using this technique; signals originating from different cells can be directly compared. The probes had a concentration of 1 M, and the signals from the cytoplasm were then measured along with those of the background. In each channel, the background signal was subtracted from both the MB signal and the reference signal, after which the ratio values were calculated. This experiment was repeated multiple times, and the results are shown in Figure 3-9. In all the cells, the cont rol MB exhibited a significant increase in the fluorescence signal with time. As no target exists inside the cell to hybridize with the control MB, the resulting fluorescence signal must have resu lted from the degradation of the probe or by interaction with cellular protei ns during monitoring. As noted earlier, such conditions can result in false positive signals, whic h is a concern when using MBs for intracellular measurements.

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63 Unlike the control MB, HMP remained dark inside the cells, as no appreciable signal enhancement was observed in the absence of target Therefore, the high st ability of HMP inside the cells is yet another of the attr active features that this probes offers for intracellular imaging. Figure 3-9 Real-time monitoring of the MBs and HM P to test the stability inside single cells. HMPs showed high stability through out the monitoring period whereas MBs exhibited an increase in the fluorescence si gnal (false positive) due to degradation of the beacon structure or intera ction with cell components. In a subsequent experiment, our main goal was to investigate the potential of the HMP for hybridization studies inside a cell was studying using human breas t cancer cells. Figure 3-10A shows cellular measurements for the HMP -actin for hybridized (top) and unhybridized (bottom) states inside a cell. -actin was used as a target, because it is highly expressed in breast cancer cells (see Table 3-1). The im ages on the left side in Figu re 3-10A represent the channel for the detection of Oregon green, while the images on the right side are fo r the detection of Cy5 at 520 nm and 665 nm, respectively. In this expe riment, Oregon green was used instead of FAM because Oregon green is more stable in the cellular environment; however, both dyes have the same spectral properties. The control HMP was monitored to detect any non-specific binding; however, the signal remained stab le at very low levels throug hout the monitoring period in the absence of its complement. On the contrary, the injected HMP -actin showed an increase in the

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64 fluorescence signal in the presence of the highly expressed mRNA sequence. The negative response of the control HMP is significant, because it indicates that the signal produced from the -actin HMP inside the cell is a result of the hyb ridization with its mRNA complement and not a result of probe degradat ion inside the cell. 520 nm channel 665 nm channel 520 nm channel 665nm channel Figure 3-10 HMP hybridized and unhybridized states inside the cell. A) TOP: Confocal images of the HMPs before and after hybridizati on with target; Bottom: Control HMP with no target inside the cell; B) Bar graph representation of -actin HMP in the presence of the target (red), inducti on of HMP MnSOD (green) and control HMP (blue) where no induction was performed. If degradation had been taking place during the m onitoring period, then the control HMP, as well as the -actin HMP, would have been affected by equally producing the FRET signal. Figure 310B shows a bar graph for HMP hybridization using the average equilibrium values of five cells from the images. The enhancement of HMP with th e target resulted in more than 9-fold signal enhancement compared with the control HM P without the target. This result clearly demonstrates the effectiveness of HMP for use in hybridization studies in vivo To verify that the control HMP was functioning properly, its comple ment (MnSOD) was stimulated. The control

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65 HMP is complementary for Manganese Superoxide Dismutase (MnSOD). In cancer cells, the expression of MnSOD is significantly down-regulated and below the limit of detection for MBs and the HMP, but gene expressi on of MnSOD can be stimulated by incubating the cells with lipopolysaccharide (LPS). In the LPS-induced cells, the expression of MnSOD was readily detectable and showed a signal at least six times higher than that of the cells at basal expression levels. These experiments demonstrate that th e HMP can detect gene expression at the singlecell level, and that the HMP is a viable molecular probe for intracellular mRNA expression. HMP for Surface DNA Hybridization Studies One of the advantages of the hybrid molecu lar probe (HMP) over c onventional two-probe FRET system s is its larger dynamic range. Another advantage of using the HMP is that it can be used for surface hybridization ap plications, such as fiber op tic DNA sensors, DNA arrays, as well as microchannels for nucleic acid detection (Figure 3-11). glass surface Complementary Target glass surface Figure 3-11 Immobilization of HMP on the glass surface for hybr idization studies HMP probe was prepared with the same sequence as HMP tub except that there were two biotins inserted in the middle of the PEG li nker to improve the binding efficiency. The HMP tubulin sequence was 5 -Cy5-CTC ATT TTG CTG ATG ACG-(PEG)8Biotin2(PEG)8-CTG TCT GGG TAC TCC TCC-FAM-3 whereas the target corresponds to 5 -GCT CAT CAG CAA AAT

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66 GAG GGA GGA GTA CCC AGA CAG-3 Before immobilization onto a streptavidin-coated surface, a solution test was performed, and it showed similar signal responses of the biotinylated probe compared to the probe without biotin. This indicated that biotin in between the linker did not interfere the binding of probe to its ta rget. Figure 3-12 shows the response of the immobilized probe upon the addition of target DNA, including images of HMP before and after target addition. 0100020003000400050006000 100 200 300 400 500 600 700 800 Fluorescence IntensityTim e (Second) AM y5-50005001000150020002500300035004000 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Cy5/FAMTim e(Second) Cy5 C FAM F FAM CY5 FAM CY5 Before Hybridization After Hybridization Figure 3-12 Images show the hybridization of HMP with the target after immobilization onto the glass surface (left). Fluorescence intensity change of the FAM and Cy5 after target addition (top right). Cy5/FAM fluorescence ra tio after hybridization with the target (bottom right) The surface was excited at 488nm, and the images were monitored at the two emission channels specific for FAM and Cy5, respectively. Before the hybridization, the fl uorescence signal from FAM was strong and weak emission from Cy5 wa s observed. Immediatel y after addition of cDNA, the intensity of FAM diminished and the intensity of Cy5 increas ed as a result of

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67 hybridization. Overall, the fluorescent intensity ratio of Cy5/FAM increased dramatically. The intensity values themselves showed large fluctua tions due to disturbance of the detection system (Figure 3-12). By using the Cy5/FAM inte nsity ratio, the noise cancelled land a smooth hybridization result was observed. With ratiometric measurement capability, the new DNA probe design removes the internal fluctuations of the detection system and provides a more precise detection. Conclusion In this report, we have demonstrated the viability of using the HMP for hybridization studies both in solution and inside the cell. The probe was designed to take advantage of a fluorescen t signal transduction mechanism that enables a very sensitive analysis at low concentrations and sample volumes. This enables the HMP to overcome major problems associated with traditional methods of gene expression analysis. Specifically, the HMP signal enhancement can exceed baseline levels more than 20-fold. The HMP is very stable in a cell-like environment and has the further advantage of detection without separation. Although MBs have been used with relatively acceptable performance, their tendency to give false positive signals is a significant problem when used for intrace llular measurements, as it is impossible to differentiate between target hybridization and false positive si gnals. In contrast, we have established in this report that the HMP has far less propensity for false positive signals and thus performs better than MBs inside si ngle living cells. Overall, in th is work, we have characterized HMP and demonstrated its potential for both in vitro and in vivo analysis. Among its many advantages, the HMP has also exhibited the abilit y to detect mRNA expre ssion of different genes inside single cells from both basal and stimulated genes.

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68 CHAPTER 4 SURFACE HYBRIDIZATION STUDIES OF THE MOLECULAR BEACONS Introduction One of the most important aspects in the m ana gement of a disease is the detection of the disorder: the earlier the diagnos is, the better the chances for tr eatment and survival of the individual. Biosensors can be uti lized for this purpose. A biosenso r is an analytical device that incorporates a biological compone nt, a transducer element and a signal or electronic processor. Figure 4-1 represents a typical DNA biosensor, where the different components can be observed such as sample, surface, transducer element, a nd the signal processor. Biosensors, such as DNA microarrays and/or DNA chips, have gained popula rity because they allow the measurement and detection of a high number of sample s in a fast and simple set-up. Figure 4-1 Typical DNA biosensor components. Our work has focused on the use of channels, molecular probes and optical detection. Moreover, biosensors allow for real time de tection of DNA molecule s and gene expression changes.69,92-97 However, these results can only be achieved by efficient, reproducible, and stable immobilization onto specific surfaces.69,92,95-97 Most DNA biosensors are based on the detection of the DNA-DNA and DNA-RNA h ybrids with the DNA probe immobilized on a transducer surface. Of the many varieties of DNA sensors th e most common are: i) electrochemical-based sensor detection, which involves immobilization of the probe ont o an electrode surface, and

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69 target molecules are detected by changes in electron transfer98,99 and ii) fluorescence-based sensor detection, where immobilization is usua lly performed on glass surface, and target molecules are detected by changes in the fluorescence intensity.69,100,101Molecular Beacons is an example of a fluorescence-based probe (MB).37,100,102 These probes have been widely studied as DNA-based probe in DNA sensors with the aim of overcoming some drawbacks inherent in single-stranded DNA probes69,103-105 and DNA array applications.17,69,94,95,106,107 Specifically, Due to their inherent signal transduction mechanism, MBs provide superior sensitivity and selectivity over single-stranded DNA probes. In essence, this capability is a function of MB structure, as previously described in Chapter 1. In solution, MBs possess many attractive propertie s such as high sensitivity, selectivity and DNA detection that can be performed in real -time as the unbound probes do not need to be separated from the bound probes. However, the us e of MBs for surface hybr idization has been limited, mainly because enhancement of the i mmobilized MB decreases significantly when compared to that of the same MB in solu tion. Thus, whereas MBs can typically achieve fluorescence enhancements above 25-fold in solution,37,108 once immobilized these values drop to 2 to 5-fold.17,69,104,105,107 This behavior is mainly due to in teractions of the MB with the surface which partially disrupt the loop and stem st ructure and can cause inefficient quenching,107 which is reflected in high fl uorescence background signal.104,105 Some published studies have reported ways of improving MB performance on surfaces. For example, in order to have a more liquidlike environment instead of liquid-solid interf ace, polyacrylamide and agarose gel have been used for MB immobilization.17,93 In other reports, investigators have achieved limited improvement by adjusting physico-chemical parameters such as pH and ionic strength, as well as the distance of the MBs to the surface. Despite these advances, further development of new DNA

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70 probes having simple designs, low background signa ls, and high hybridizati on efficiencies, are still needed to match the performance and sensi tivity of MBs in solution. In addition, improving MB performance on surfaces will be easily transferable to biotechnologies based on immobilization of nucleic acid probes on surfaces, such as DNA array and protein arrays-based on aptamers. In this study, we have modified and optimi zed the MB design by incorporating locked nucleic acids (LNA) in order to improve the pe rformance of these probe s on a solid surface for hybridization studies. As described in Chapter 1, LNA is a nucleic acid analogue containing a bicyclic furanose unit locked in an RNA mimicking sugar conformation.24 The methylene bridge that connects the 2'-oxygen and the 4'-carbon of th e ribose ring confers higher structure rigidity to the LNA base pair, therefore preventing po tential interaction with the surface and other molecules that might be present in solution. Furthermore, LNAs ha ve a structure resemblance to RNA, which allows superior affi nity and specificity towards the target strand when compared to that of DNA. 23 Other attractive properties of the LNA bases are high resistance against degradation and thermostability. All of these performance charact eristics are reflected in low background signals, and effici ent target hybridization.109,110 Despite these advantages the hybridization kinetics of the fu lly LNA modified MB are slow compared to that of DNA MBs, due to the slow stem dehybridization rate.110 Therefore, in this work, we have synthesized a MB with both regular DNA bases and with a combin ation of DNA and LNA bases. The ultimate goal was to compare their performances after immobili zation onto a glass surface in terms of stability, background signal, thermodynamics, kinetics, select ivity, and sensitivity. This newly design MB can potentially be used for a more sensitive DNA array detection and other biotechnological applications.

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71 Materials and Methods RMB and LMB for Surface Hybridization Studies The MBs sequences, compleme ntary DNA and mism atch targets are and listed in Table 41. All the DNA and LNA reagents for the synthesi s of the beacons and targets were purchased from Glen Research. The probes and DNA complementary targets were synthesized with an ABI3400 DNA/RNA synthesizer (A pplied Biosystems, CA). Blackhole quencher 2 (BHQ2) core pore glass (CPG) was used for all MB synthese s; Cy3 phosphoramidite was used to label the probes with Cy3, and Spacer phosphoramidite 18, was used to incorporate PEG as linker in the beacons. Biotin phosphoramidite was introduced for labeling at the 5 terminus of the MBs. MBs containing LNA bases were synthesized using LNA phosphoramidites. LMBs were prepared by alternating LNA and DNA bases in both the stem and the loop (every other base). This design was chosen to take advantage of LNA bases without compromising the hybridization rate. In addition, the targets were prepared in such a way that the complementary sequence was not only complementary to the target but to the stem as well to make sure the beacons open in the presence of the target (shared stem target). Deprotection of the pr obes was performed using overnight incubation with ammonium hydroxide at room temperature. The solution resulting from deprotection was precipitated in cold ethanol. Subsequently, the precipitates were then dissolved in 0.5 mL 0.1 M TEAA fo r further purification with RP -HPLC. The product collected from the HPLC was vacuum dried and then mi xed with TEAA for a second round of HPLC. The final product was collected and incubated with acetic acid (80%) for 20 min, followed by 200 L of ethanol, and was then, vacuum dried. Quantifi cation of the probes and targets were performed using UV-Vis Spectrometer as previous ly described in earlier chapters.

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72 Table 4-1 Molecular beacons and target sequences Regular Molecular Beacon (RMB) No PEG 5-Biotin Cy3 CCT AGC TCT AAA TCA CTA TGG TCG CGC TAG G -BHQ2-3 6 PEG units 5-Biotin PEG (X)6 Cy3 CCT AGC TCT AAA TCA CTA TGG TCG CGC TAG G -BHQ2-3 12 PEG units 5-Biotin PEG (X)12 Cy3 CCT AGC TCT AAA TCA CTA TGG TCG CGC TAG G -BHQ2-3 Locked Molecular Beacon (LMB) No PEGa 5-Biotin Cy3 C C T A G C T C T A A A T C A C T A T G G T C G C G C T A G G -BHQ2-3 6 PEG units 5-Biotin PEG (X)6 Cy3 C C T A G C T C T A A A T C A C T A T G G T C G C G C T A G G -BHQ2-3 12 PEG units 5-Biotin PEG (X)12 Cy3 C C T A G C T C T A A A T C A C T A T G G T C G C G C T A G G -BHQ2-3 Target 5-GCG ACC ATA GT G ATT TAG AGC TAG G-3 Mismatch 5-GCG ACC ATA GTG A A T TAG AGC TAG G-3 a Red letters represents LNA bases b Blue letter represents the mismatch base Immobilization of the Probe onto the Glass Surface The mi croscope slides and cover slips used in the experiments were obtained from Fisher (optical borosilicate glass w ith a size of 18 x 18mm and 0.13 to 0.17mm thick). The surfaces were cleaned with a 3:1 ratio of conc. H2SO4 to 30% H2O2 (30%) to remove organic impurities. Subsequently, they were washed thoroughly with deionized water and dried with compressed nitrogen. Strips of double-sided ta pe (3M) were placed 3 mm apart on a microscope slide, and a cover glass was placed on top. Channels were f illed by capillary action. Solution exchange was performed by simultaneously pipe tting solution at one end and w ithdrawing fluid from the other end with P8 filter paper (Fisher).111 The channels were washed twice with 10 mM phosphate buffer (PBS) at pH 7.4 before use. Next, 5 L of 1 mgmL-1 avidin was incubated in the channels for 5 min. The excess avidin was removed w ith PBS washes (20 L, three times). The biotinylated MB (2.5 M in PBS buffer) was incu bated with the avidin treated channels for 10

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73 min, and the excess of MB was removed with wash ing steps of PBS buffer. A solution of the complementary DNA (cDNA, in 20 mM Tris, 50mM NaCl and 5 mM MgCl2, pH 7.5) was incubated with the immobilized MB for 10 min, and finally, the surface was washed with PBS (20 L, 3 times) to remove unbound reagents. Severa l images were collected at every step to obtain an average fluorescence, and the procedur e was repeated and measured several times. Stability of the MBs at Differe nt Temperatures The stability of the MBs at different temp eratures (from 20 to 50 C) was studied by incubating the glass slide on a surface block dr y bath (Barnstead Thermolyne Type 17600 DriBath). The samples tested at 4C were kept in the refrigerator prior to use to achieve the desired temperature. The probes were covered with alum inum foil to prevent any light damage to the probes. All the steps were performed under the co nditions previously described and washes with PBS buffer (at the same temperature under study was used). Images of the glass slide were taken at every step as earlier detailed. Stability in Complex Matrices The stability of the system in complex matrices was studied by performing the experiments in cell lysate and serum media. Cell lysate solution was prepared according to commercial specifications from Cell Signaling Technology, Inc. (Danvers, MA) and was kept at -20 C until analysis. Fetal Bovine Serum (FBS, heat-ina ctivated, GIBCO) was obtained from Invitrogen (Carlsbad, CA). For this set of experiments, buf fer, cell lysate, and FBS were incubated for 10 min with the immobilized MBs. In addition, target solutions were prepared in buffer, cell lysate or FBS for a final concentration of 10 M and were subsequently incubated with the immobilized beacon. Then, fluorescence measur ements were performed following the same immobilization conditions previously described.

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74 Fluorescence Imaging Fluorescence imaging was perform ed with a co nfocal microscope setup consisting of an Olympus IX-81 inverted microscope with an Olympus Fluoview 500 confocal scanning system and a green HeNe laser (543 nm), with a photom ultiplier tube (PMT) for detection. The images were acquired with a 10x/0.30 NA objective. Cy3 labeled MBs were excited at 543 nm and the emitted light was collected at 560 nm with a lo ng pass filter. The data collected from the confocal microscope were analyzed us ing the Fluoview analysis software. To make sure the images were obtained from the brightest point of the channel, the microscope was focused onto the surface of the microslide, followed by a zeta section scan to capture the brightest image at a specific zeta position. Subsequently, all the images from avidin, immobilized MB and target addition were obtai ned at the same position for that particular channel. This position may vary for each channe l; therefore, the zeta position was calculated for each immobilization. The data collected from the images were analyzed and the fluorescence enhancement was calculated using the following equation: Fluorescence Enhancement = ( SMB open Bavidin)/(SMB close Bavidin) where SMB open is the signal of the beacon in the open state form (hybridized with the target), SMB close is the signal of the MB in the close state form (unhybridized) and Bavidin is the background fluorescence intensity corresponding to the immobilized avidin in buffer solution. An average of the fluorescence was determined by taking severa l images from the same channel. Kinetics experiments were performed using confocal microscopy by taking images of the hybridization with the ta rget every 3 seconds for 10 min. Each image was analyzed by taking the average fluorescence intensity at the determined zeta position.

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75 Results and Discussion Molecular Beacon Designs and Surface Immobilization In previous reports, fully modified LNA beacons have shown higher sensitivity and relatively slower hybridization ra tes than the counterpart DNA MBs.109,110 By decreasing the percentage of LNA bases in the stem and the loop LMB can retain the affinity and the stability of the LNA bases without compromising the kinetic s and hybridization rate of the assay. Thus, LMBs with alternating LNA and DNA bases in both the stem and the loop (every other base) were utilized for this study. In addition, it is we ll known that LNA bases have a strong affinity for complementary LNA bases.23 Therefore, targets were prepar ed such that the cDNA sequence was complementary not only to the loop of the MB s, but to the stem as well (also known as a shared stem target). In this study, MBs were immobilized on a glass surface via an avidin-biotin interaction. Its strong affinity and stability, as well as the easy incorporation of the biotin group into oligonucleotide sequence make it ideal for surfa ce immobilization. As shown in Figure 4-4, the biotin molecule was attached at the 5' end of the MB followed by a PEG linker to increase the distance of the MBs to the glass surface. Advant ages of PEG include its great solubility and hydrophilicity and easy incorporation into DNA sequences using standard phosphoramidite chemistry. Furthermore, PEG does not possess any ch arge and therefore, has limited electrostatic interactions, and does not precipitat e or aggregate at the glass surface.112 Finally, since PEG adds flexibility to an otherwise rigid oligonucleotid e structure, the PEG linker facilitates efficient immobilization onto the solid surface and furt her hybridization with target molecules. The length of the PEG was a key factor in the MB design, and care was taken to ensure that the linker was neither too short nor too long, either of which could affect hybridization efficiency. Therefore, sets of LMBs and RMBs with different distances between the biotin group

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76 and the sequence were initially designed by incor porating 0, 6 and 12 units of PEG, as shown in Table 4-2. Figure 4-4 Schematic representation of the mo lecular beacon biosensor immobilized on a glass surface. The images on the right represent MB hybridization before and after addition of cDNA The initial background fluorescen ce of the beacons in the ab sence of complementary DNA, as well as the fluorescence change after target addition were evaluate d, and the results are summarized in Table 4-2. For the regular mo lecular beacon (RMB) th e background fluorescence slightly increased when 6 units of PEG were in corporated in the sequence. However, for the RMB with 12 PEG the resulting background was ~5 -fold higher than RMBs containing zero and 6 PEG linker units. On the other hand for the LMB the background intensity remained similar and stable regardless of the le ngth of the linker, and most impo rtantly, lower than that of RMB (around 2.5 times). Moreover, the signal was compar able to the one obtained from the system background (avidin-coated surface, ~160 10). These data support previous reports, which concluded that destabilization of regular DNA-based-MB occurs after immobilization, resulting in inefficient quenching, that is reflected in high background signal.69,107 This is one of the major limitations of using MBs for surface immobilization. However, the addition of LNA bases into the beacon for hybridization studie s brings stability and minimizes surface-glass interactions.

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77 Moreover, LNA bases in the stem keep the fl uorophore/quencher moieties in close contact, a phenomenon reflected in the low background signal. Table 4-2 Effect of different polyethylene glycol units (PEGs) on the background intensity and the overall fluorescence change after target addition Probes Background intensity (a .u.) Fluorescence change RMB 0 PEG 510 14 2.8 0.4 6 PEG 608 113 7.9 0.6 12 PEG 2540 220 1.2 0.1 LMB 0 PEG 200 14 2.8 0.6 6 PEG 258 6 25 5 12 PEG 239 28 6.1 2 On the other hand, a higher fluorescence cha nge of the RMB after target addition was obtained when RMB contained a PEG linker between the biotin and the sequence, with a maximum signal enhancement when 6 PEG units we re added. This result proves that the distance provided by PEG facilitates the accessibility of the molecular beacon for an efficient hybridization with the target. Similarly, LMB with 6 PEG units produced the highest fluorescence enhancement upon target addition, a f actor of 25 signal enhancement compared to 2.8 and 6.1 for 0 and 12 PEG units, respectively. Thus, considering the similar background intensities of the LMB with different PEG units, we can conclude that the enhancement of the signal comes in this case from a more efficient hybridization when a linke r with 6 PEG units is used, which may represent the optimum probe-t o-surface distance. The addition of more PEG units (12 units) results in a l onger and perhaps a more flexible linker, but the extended length and higher bulkiness of the linker can, at the same time, hinder hybridizat ion with the target, resulting in the overall lower si gnal enhancement. Considering th ese results, the probes with 6 PEG units were selected for further experiments.

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78 Sensitivity and Selectivity of the Immobiliz ed MBs In DNA arrays and biosensors, it is important to determine the mini mum concentration of target molecules that can provide a measurable signal. Thus, in the following experiment, we investigated the ability of the immobilized MBs to detect differe nt concentrations of target, ranging from 1 nM up to 100 M, with the same concentration of MB s immobilized (initial concentration of 2.5 M). However, it should be not ed that only the initia l concentration of MBs used for immobilization is known. As expected, the signal enhancements for both the LMB and RMB were observed to be proporti onal to the target concentra tion, and the saturation point for each beacon was 5 M, as shown in Figure 4-5, w ith detection limits (DL) for RMB and LMB of 25 nM and 7.5 nM, respectively. 0 5 10 15 20 25 0 20 40 60 80 100 Concentration of Target (M) RMB LMB DL=7.4 nM DL=25 nM0 2 4 6 8 10 12 0100200300400500 Concentration of Target (nM) Figure 4-5 Comparison of the influence of target concentration in the hybridization of the RMBs and LMBs (gray and black respectively). Le ft graph: calibration curve of the MBs upon hybridization with target from 0-100 uM Right graph: the detection limits for both MBs at lower concentrati ons of target (0-500 nM) The lower background intensity of the LMBs compared to that of the RMBs allows the detection of the target, even at low target concentrations. On the contrary, the major drawback of the RMBs is the high background intensity that increases the amount of target needed for detection. In addition, it has been document ed that LNA bases possess higher affinity23 for DNA bases than DNA itself, an important factor in the lower DL of the LMBs.

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79 Biosensor accuracy is critical particularly in DNA-based diagnostic devices. Therefore, the ability of molecular beacons to discriminate the perfect complementary target from other sequences is essential. For example, this discri mination ability is importa nt in the diagnosis of genetic diseases, where detec tion of a single base mismatch (SBM) is needed. Thus, the selectivity of MBs immobilized onto the glass surface was also inve stigated. For this purpose, a sequence with a single base mismatch was synt hesized and evaluated. It is well known that the location of the mismatch can affect the selectivit y of the probe. In this analysis, the mismatch was positioned in the loop at a nucleotide complementary to a DNA base and not a LNA base. The signal enhancement was obtained for the perfect match and mismatch targets to compare the detection capabilities of RMB and LMB. The re sults in Figure 4-6, only the LMB was capable of detecting the mismatch, whereas the RMB mism atch signal was still within one standard deviation of the perfect match signal. Similar be havior was observed in solution, where the LMB showed selectivity superior to that of the RMB.109 The excellent discriminatory capability of the LMB can be directly attributed to the high affinity of the LNA bases towards DNA target molecules.113 0.0 0.2 0.4 0.6 0.8 1.0 Mismatch cDNANormalized Fluorescence Intensity the RMB LMB Figure 4-6 Normalized fluorescence intensity of LMB and RMB upon hybridization with the single base mismatch (mismatch) and perfect complementary target (cDNA)

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80 Thermal Stability of the Beacons In order to demo strate the improved stability and robustness of the sensor using LNA bases, the influence of inc ubation temperature was inves tigated. Figure 4-7 shows the background intensities of the beacons before target addition and the signal enhancements of the MBs after addition of th e target sequence at 4, 20, 30, 40, and 50 C. As can be observed in the graph, the highest signal enhancement for RMBs was recorded at 20 C, with a 9.4-fold enhancement. Subsequent temperature increases caused a decrease in the signal enhancement of the RMBs to 3.4-fold, as a resu lt of a significant and gradual increase in the overall background signal at higher temperatures. These results suggest thermal destab ilization of the beacon structure, which compromises the stability of th e probe-target complex. In addition, this increase in temperature is likely to unzip the bases of th e stem and thereby decrease the rigidity of the RMB, forcing the beacon to melt and form fluorescence random coils.108 The random coil forces the fluorophore and quencher to separate, which causes an increase of b ackground intensity. In contrast, LMBs showed better stability, as can be observed from the steady background throughout the entire array of temp eratures studied. This resulted in a stable enhancement signal over the entire range of temper atures, which in all cases was ~22, and was also clearly higher than the results for RMB, as demonstrated in the previous experiments. This improved robustness is a result of the intrinsic stability of LNA bases, bringing rigidity to the probe, which is necessary to maintain the beacon in the closed state form, even at higher temperatures. By the addition of the LNA bases in the beacon design, we have demonstrated that MBs with higher stability and reproducibility can be obtained, thus increasing the versatility of the sensor for extended applications.

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81 Figure 4-7 Temperature effect on the stability of the MBs immobilized onto the surface. Black straight line: fluorescent enhancement in LMB; black dashed line: background intensity of LMB; grey stra ight line: fluorescent enhancem ent in RMB; grey dashed line: background intensity of RMB Hybridization Kinetics of the MBs The kinetic properties of the LMBs are relati vely different from those of the RMBs. The percent of LNA bases in the MBs can greatly aff ect the hybridization efficiency, usually slowing the hybridization rate with th e target DNA. In order to st udy and compare the hybridization kinetics of both immobilized molecular beacons the fluorescent enhancement was monitored using the confocal microscope every 3 seconds fo r 10 min after addition of the target. As shown in Figure 4-8, the fluorescence signal of RMB r eached hybridization equilibrium within 3 min, whereas the LMB signal slowly in creased over time. This behavior already observed in previous work, can result from the presence of a LNA-LNA base-pair in the stem of the LMB.110 The LMB has three LNA-LNA pairs in the stem (50%), which provide the beacon with great stability and affinity with itself, and make the st em dehybridization difficult and unfavorable. In order to know how the rates of LMB and RMB hybridization w ith target vary, we determined the initial rate for the hybridization of the beacons. The initial rate can be calculated from the slope of the

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82 linear parts of the relativ e enhancement versus time plots with in the initial 20 seconds (Figure 48 right). The slope of the RMB is ~7 times higher and, therefore, faster than that of the LMB. Interestingly, this hybridization rate can be dr astically accelerated by using more DNA bases in the stem of the LMBs or by decreasing the number of LNA bases in the stem. This can potentially reduce the energy barrier and speed up the opening of the beacon, but at the same time it decreases the stability of the LMB.110 Therefore, we believe th at the hybridization rate exhibited by the LMB is still very reasonable for nucleic acid-based biosensors, because of the outstanding stability and high signal enhancement that the LNAs bring to the beacon structure. 0 4 8 12 16 20 24 0.0 2.0 4.0 6.0 8.010.0Time (min) RMB LMB m = 61.1 m = 9.2 0 4 8 12 16 0 5 10 15 20 Time (s) LMB RMB Figure 4-8 Hybridization kinetics of RMB (gray) and LMB (black) immobilized onto the surface after target addition (left). Initial hybridization rate of the immobilized beacons is shown on the right. The date points corres pond to the average of three independent experiments MB Sensitivity in the Presence of Complex Matrices Another im portant aspect in the study of RMBs and LMBs for use in surface immobilized biosensors is the ability of probes to detect a target in complex matrices. This is especially important in the early detection of various genetic diseases where rapid and sensitive detection is needed. Ideally, the MB signal shou ld arise only by the detection of target molecules. However, this is not always the case, since degradati on of the MB can occur because of cellular DNA

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83 binding proteins and nucleases, resulting in fals e positive signals. Moreover, these proteins can hinder the ability of the MB to bind with the targets, thereby decreasing overall signal enhancement. Therefore, it is imperative to ev aluate the performance of the molecular beaconbased biosensor in complex matrices. Two diffe rent media, fetal bovine serum (FBS) and cell lysate, were selected, and the results are summari zed in Figure 4-9. These matrices were selected because they contain many proteins, amino-acids, sugars and lipids that can potentially interfere with the hybridization efficiency and/or degrad e the MB. As expected, the data revealed a decrease in the signal enhancement of the RMB in the presence of either FBS or cell lysate. 0.0 0.2 0.4 0.6 0.8 1.0 FBS Cell lysate BufferNormalized fluorescence intensity RMB LMB Figure 4-9 Normalized fluorescen ce intensity of the immobilized RMB and LMB after treatment with the target in fetal bovine serum (F BS), Cell lysate and PBS buffer solution Because the background intensity of the RMBs did not change significantly (data not shown), the decrease in signal enhancement must be due to matrix components, which impede RMB binding with the target. Also, it is possi ble that degradation of the loop sequence has taken place, since

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84 the DNA bases in the loop are more exposed and, th erefore, more prone to degradation than the DNA bases in the stem. This effect is not as si gnificant in LMBs, sin ce the signal enhancement appears to be unaffected after treatment with FB S and cell lysate. The degr adation of the loop is less likely because of the inherent resistance of the LNA bases to the degradation typically caused by nucleases. Nonetheless, endogenous materi als can still block hybridization with the target, which, to some degree, explains the redu ction of the signal enhancement observed for the LMB. Conclusion We have developed a locked m olecular beac on, which possess superior stability for surface hybridization studies. The incorp oration of LNA bases into MB design allows us to take advantage of both the sensitivity of the MBs and the high binding affinity and stability of the LNA bases. Moreover, the incorporation of a PEG linker with the optimum length has considerably improved the target hybridization. Overall, the introduction of these modifications in the molecular beacon sequence has made it po ssible to achieve a signal-to-background of 25fold, considerably better than the previously be st reported values of onl y 5.5-fold when regular MBs were used as immobilized probes. The bi osensor developed with the probes was highly stable and robust, selective and sensitive, resulting in a promising design for use in a wide variety of biological and bi otechnology applications. Future investigation of these probes will ad dress the immobilization of multiple probes targeting different gene sequences. This will minimize the cost of the analysis by allowing the detection of multiple genes simultaneously. Furt hermore, the development of DNA microarrays can permit clinical laboratories to utilize probe s such as LMBs for diagnostic purposes and sensitivity assays for drugs obtained from human samples.

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85 CHAPTER 5 SUMMARY AND FUTURE DIRECTIONS Designing Nucleic Acids Probes for Biosensor Applications The use of molecular probes to help decipher pr ocesses at sub -cellular levels has been an important task for researchers over the last decade. However, only a few years after the completion of the Human Genomic Project, ther e is still only limited knowledge of the specific interactions of DNA and RNA leading to protei n synthesis. Nevertheless, the knowledge gained so far has the impacted the biomedical field by shifting attention to the development of methods and techniques for the early detection and identi fication of disease biomarkers. The ultimate goal is to increase clinical success rates and reduce disease-related mortality in patients. However, in order to do so, we must have ultrasensitive methods and/or probes that are able to locate small amounts of DNA, RNA, proteins or other small molecules in body fl uids and nucleic acid probes are playing key roles in this endevor. Although mo lecular probes can suffer from low sensitivity, selectivity and poor stab ility in a cellular environment; nucle ic acid probes are excellent tools, which allow rapid, fast, simple and low cost analysis. Moreover, the development of novel fluorescence dyes and improvements in the instrume nt detection capabilities will continue to allow the construction of molecular probes w ith increased stability and sensitivity for bioanalytical applications. The work performed in this thesis focuses on the development and optimization of molecular probes for biosensor applications a nd intracellular measurements. The rationale behind that project was to crea te a nucleic acid probe capable of overcoming some of the limitations of molecular beacons and linear probes. A hybrid molecular probe (HMP) was designed with that purpose. The first expe riments performed using the HMP involved the optimization of the probe in vitro discussed in Chapter 2. After optimization of the HMP,

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86 experiments were designed to compare the stab ility, sensitivity and selectivity of HMP with those of MBs and linear probes. When HMP was compared with linear probes, the former resulted in a larger dynamic range and lower lim its of detection (0.7 nM for HMP and 7.2 nM for linear probes). In addition, HMP has greater thermal stability compared to linear probes as indicated by the higher melting temperature (67 C for HMP compared to 30 C for linear probes). Furthermore, in comparison with molecular beacons, HMP resulted in no false positive signals and performed better than MB inside single living cells. Th e intrinsic signal transduction mechanism of the HMP facilitates a very sensitiv e analysis at low samples volumes. This probe can reach signal-to-background ratios above 20-fold. In addition, this probe is very stable inside cells, which is a great importance when using nucleic acid probes fo r intracellular measurements. Overall, in Chapter 3 of this work, we have ch aracterized an HMP and demonstrated its potential for both in vitro and in vivo analysis and as a tool for hybridiz ation studies. In addition, the HMP has exhibited the ability to dete ct the mRNA expression of differe nt genes inside single cells from both basal and stimulated genes. Chapter 4, on the other hand, focused on the a pplication of molecular probes for surface hybridization studies. The main purpose was to impr ove the existing MB in terms of sensitivity and overall stability after immobilization onto a gl ass surface. Therefore, we redesigned the MB by incorporation of LNA bases in the beacon desi gn. This allowed us to take advantage of both the sensitivity of the MBs and the high binding affinity and stability of the LNAs. Whereas previous articles have reported a maximum si gnal-to-background ratio of only 5.5 when regular MBs (RMBs) where used as immobilized probes, here we report a locked MB that offers a signal-to-background ratio of 25. Furthermore, the immobilized using the locked nucleic acids MBs (LMB) is highly stable, select ive, and sensitive indicating in a promising design to be used

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87 in a wide variety of biological and biotechnology applications. In addition, the LMBs have lower background signals compared to RMBs, due to the rigid structure given by the LNA bases. The LMB was optimized in terms of the distance of the beacon to the surface by the addition of PEG linker units of varying lengths The RMB showed signal-to-background ratio of 7.9 compared to 25 for the LMB, because incorporation of LNAs minimizes the interaction with the surface and increases the stability of the beacon structure. Moreover, the immobilized LMBs have much greater thermal stability. The LM Bs showed no change in the b ackground signal upon heating to 50 C, unlike the background signal increases observed for RMBs. Additionally, the LMBs are sufficiently stable on the surface that they show mi nimal or no signal variation in the presence of FBS and cell lysate components. Although the ra te of hybridization with target can be compromised for the LMB, this factor can be ea sily manipulated by the removal of certain LNA bases in both the stem and the loop. On the ot her hand, replacement of LNAs with DNAs can influence the stability of the beacon on the glass surface. Overall, however the hybridization rate exhibited by the LNA-based MB is very reasonabl e for nucleic acid based biosensors, given that regular DNA bases tend to interact with the surface resulting in in efficient quenching. Future Directions The monitoring of m ultiple genes has been lim ited primarily to a few techniques such as reverse transcritase polymerase chain reac tion (RT-PCR), Western blot analysis, and in situ hybridization studies. However, Medley et al (2005) developed MBs for multiple genes in single cell in breast cancer cells.53 In spite of this accomplishment, Medley et al used microinjection for MB delivery, which limited the analysis to only few cells. The use of DNA microarrays alleviates this burden and promises to be an efficient solution fo r a high throughput, rapid, sensitive and low cost analysis as a diagnosis tool for diseases a nd biomedical research.

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88 Therefore, the aim of the presen t work is the application of the improved LMBs for hybridization studies in the detection of multiple genes in vivo. The synthesis of -actin MB, MnSOD and a control be acon will be performed according to the designs as shown in Table 51. As described in previous chapters, these gene sequences are ideal for this purpose because of the housekeeping gene -actin sequence is highly expressed within cancer cells and MnSOD can be down-regulated by the us e of LPS. Figure 5-1 shows a schematic diagram of the MBs (targeting multip le genes) immobilized onto a glass surface before and after the addition of the target sequ ence. Note that the beacons will be synthesized (with FAM, Cy3 and Cy5 as labels) and tested in vitro and then in vivo Monitoring will be performed using the protocol previous ly described earlier in Chapter 4. Table 5-1 Probe and Target Seque nces for Multiplexing Analysis Name Sequence -actin 5 -Biotin PEG6 Cy5 C T C A TC T T G T T T T C T G C G C C C C G A T G A G BHQ3-3 MnSOD 5 -Biotin PEG6 Cy3 T A T A C C A C T A C A A A A A C A G G C A C G G G T A T A BHQ2-3 Control or 226 MB 5 -Biotin PEG6 FAM C C T A G C T C T A A A T C A C T A T G G T C G C G C T A G G BHQ1-3 -actin Target 5 -GGG GCG CAG AAA ACA AGA TGA G-3 MnSOD Target 5 -CGT GCC TGT TTT TGT AGT GGT ATA-3 Control Target 5 -GCG ACC ATA GTG ATT TAG AGC TAG G-3 Red letters indicate ba ses containing LNAs The cell line that will be used for this analysis is MDAMB-231 breast carcinoma cells. To stimulate MnSOD mRNA expression, cells will be incubated in 1 g/ml lipopolysaccharide (LPS) from E. coli serotype for 4 hours prior to preparati on of the cell lysate. The cells will be treated with TRIzol Reagent in orde r to isolate the RNA of the cells. Another area for future investigation is the possibility of immobilization of multiple LMBs targeting different and more significant gene sequ ences. An example of this is the use of micro

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89 RNA (miRNA) sequences, which have caught the attention of resear chers in the last few years. Micro RNAs are single-stranded RNA molecules containing a bout 21-23 nucleotides that regulate gene expression.114 Figure 5-1 Surface immobilization of MBs targeting multiple genes and the addition of the target molecules A deep understanding of miRNA will elucidate information a bout the different pathways of gene expression, since it is believed that ex pression of 30% of human genes may be regulated by miRNA.114 In addition, the mechanisms responsible for the effects of miRNA in animals cells are important research topics.115 The design of LMBs targeting miRNA sequences will aid in investigating the role of miRNA in cancergenesis. In addition, future applications of these probes involve their use in the study of disease states to investigate gene expression in single ce lls, including medical re levant areas, such as investigating the effects on gene expression in human breast carcinoma cells after treatment with chemotherapeutic drugs. Other issues of biol ogical importance involve investigating mRNA expression patterns in single ne uron cells as a means of explor ing the processes involved in

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90 memory and learning. Furthermore, plans to incor porate nuclease-resistant bases, such as LNAs into HMPs will allow extended measurements inside living cells, on the order of hours or even days. Ultimately, utilizing HMPs linked to cell-penetrating peptides may allow the delivery of HMPs to thousands of cells simultaneously. Development of such techniques may eventually permit clinical laboratories to utilize HMPs in fluorescent plat e readers for diagnostic and drug sensitivity assays in cells obtai ned from human biopsy specimens. Another important development that has emerged in recent years is the use of aptamers as biomarkers for cancer cells. Briefly, aptamers are single stranded oligonucleotides having a definite 3D structures, selective to many target molecules (ions, proteins, organic molecules, cells) and are highly stable. Aptamers are select ed from oligonucleotide pools by a process called Systematic Evolution of Ligands by EXponential enrichment or SELEX (Figure 5-2).116 Aptamers can be slightly modified with LNAs bases for surface immobili zation onto the glass. The goal is to establish a protocol that will a llow the rapid, sensitive and reliable detection of cancer cells. In addition, protein detection can be performed, if the sensitive si gnal transduction mechanism of MBs is combined with the specif icity of aptamers to create a MB aptamer.117 Furthermore, modification of the MB aptamer can be performed by adding non-standard bases, such as LNAs to improve the performance of the probe in vivo Finally, the aforementioned systems can be used for investigating mRNA as means of exploring highly important biological processes. This will minimize the cost of the analysis by allowing the detection of multiple genes simulta neously. Furthermore, the development of DNA microarrays can permit clinical laboratories to utilize probes such as LMBs for diagnosis and drug sensitivity assays.

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91 Figure 5-2 Aptamer selection process: System atic evolution of ligands by exponential enrichment (SELEX)

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99 BIOGRAPHICAL SKETCH Karen Martinez was born in Humacao, Puerto Ric o, and was raised in Las Piedras, a small town in Puerto Rico, (Las Piedras) where sh e had a pleasant childhood. Karen was educated at the public prim ary, secondary and high schools of her hometown. In high school Karen was part of a group of talented students who received adva nced classes in mathematics, Spanish, English and the sciences. It was there th at she discovered great passion for science. She then, attended the University of Puerto Rico, Cayey campus, where she graduated magna cum laude with a B.S. in chemistry in 2001. After working at one of the largest biotechnology companies in Puerto Rico for a year, Karen realized that she could do more than a technician level jobs. In 2003, she joined the doctoral program in ch emistry at the University of Flor ida under the supervision of Dr. Weihong Tan. While working on her PhD, Karen pur sued a masters degr ee in pharmacy and pharmaceutical Sciences, which she completed in August 2008. She received her Doctor of Philosophy degree in analytical chemistry from the University of Florida in December 2008.