<%BANNER%>

Selection, Target Identification and Application of Aptamers Selected for Poxvirus Infected Cells

Permanent Link: http://ufdc.ufl.edu/UFE0025003/00001

Material Information

Title: Selection, Target Identification and Application of Aptamers Selected for Poxvirus Infected Cells
Physical Description: 1 online resource (131 p.)
Language: english
Creator: Parekh, Parag
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: alphascreen, aptamers, cowpox, flow, glycosylation, hemagglutinin, imaging, infectious, poxviruses, selex, smallpox, vaccinia
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: Infectious diseases are a subject of public health safety. In case of events such as bioterrorism or food samples tainted with a disease causing bacteria or virus the standard traditional methods of detection of viral or bacterial detection are too slow. We have developed molecular probes known as ?aptamers? to detect infection with high specificity and sensitivity. Aptamer, a word derived from Latin ?aptus? meaning ?to fit?; are molecular probes which are generated using nucleic acids which recognize and bind their target with a very high affinity and specificity. Aptamers are evolved in vitro in a test tube for its target. Aptamers are generated using a screening process known an SELEX, which stands for Systematic Evolution of Ligands by Exponential Enrichment. A library of 10 14 to 10 16 unique sequences is synthesized. These sequences are fractionated based on interactions with the target for which the aptamer is generated. The weaker binding sequences are weeded out after each successive round of incubation leaving sequences with high affinity for the target. PCR is performed after each round of selection. This process leads to aptamers which bind the target. The three dimensional folding of the aptamer around the ligand involves interactions such as hydrogen bonding and Van der Waals forces to provide high affinity and specificity. A viral infection causes molecular change on the cell surface of the infected cell. We applied an infected cell-based SELEX strategy to utilize cell surface markers expressed differentially on vaccinia infected cells, a model for smallpox to generate a panel of aptamer probes. This method does not require previous knowledge of cell surface molecules. We successfully generated aptamers recognizing only the vaccinia infected cells. The target identification of the aptamer can be used as cell-surface biomarkers and be used as a diagnostic marker for infection. The virally encoded protein Hemagglutinin(HA) was identified as the target of aptamer. This aptamer was tested for various applications. The aptamer can be used to detect infection not only in laboratory buffers but also complex biological environment such as serum, plasma and even whole blood. The aptamers were tested for their antiviral properties using various assays but at the concentrations used in these assays we did not detect any significant antiviral activity. We have selected aptamers for vaccinia virus infected cells, identified the target of one aptamer and used that aptamer for different 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 Parag Parekh.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Tan, Weihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-10-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0025003:00001

Permanent Link: http://ufdc.ufl.edu/UFE0025003/00001

Material Information

Title: Selection, Target Identification and Application of Aptamers Selected for Poxvirus Infected Cells
Physical Description: 1 online resource (131 p.)
Language: english
Creator: Parekh, Parag
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: alphascreen, aptamers, cowpox, flow, glycosylation, hemagglutinin, imaging, infectious, poxviruses, selex, smallpox, vaccinia
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: Infectious diseases are a subject of public health safety. In case of events such as bioterrorism or food samples tainted with a disease causing bacteria or virus the standard traditional methods of detection of viral or bacterial detection are too slow. We have developed molecular probes known as ?aptamers? to detect infection with high specificity and sensitivity. Aptamer, a word derived from Latin ?aptus? meaning ?to fit?; are molecular probes which are generated using nucleic acids which recognize and bind their target with a very high affinity and specificity. Aptamers are evolved in vitro in a test tube for its target. Aptamers are generated using a screening process known an SELEX, which stands for Systematic Evolution of Ligands by Exponential Enrichment. A library of 10 14 to 10 16 unique sequences is synthesized. These sequences are fractionated based on interactions with the target for which the aptamer is generated. The weaker binding sequences are weeded out after each successive round of incubation leaving sequences with high affinity for the target. PCR is performed after each round of selection. This process leads to aptamers which bind the target. The three dimensional folding of the aptamer around the ligand involves interactions such as hydrogen bonding and Van der Waals forces to provide high affinity and specificity. A viral infection causes molecular change on the cell surface of the infected cell. We applied an infected cell-based SELEX strategy to utilize cell surface markers expressed differentially on vaccinia infected cells, a model for smallpox to generate a panel of aptamer probes. This method does not require previous knowledge of cell surface molecules. We successfully generated aptamers recognizing only the vaccinia infected cells. The target identification of the aptamer can be used as cell-surface biomarkers and be used as a diagnostic marker for infection. The virally encoded protein Hemagglutinin(HA) was identified as the target of aptamer. This aptamer was tested for various applications. The aptamer can be used to detect infection not only in laboratory buffers but also complex biological environment such as serum, plasma and even whole blood. The aptamers were tested for their antiviral properties using various assays but at the concentrations used in these assays we did not detect any significant antiviral activity. We have selected aptamers for vaccinia virus infected cells, identified the target of one aptamer and used that aptamer for different 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 Parag Parekh.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Tan, Weihong.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-10-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0025003:00001


This item has the following downloads:


Full Text

PAGE 1

10

PAGE 2

10

PAGE 9

Vaccinia virus

PAGE 12

May 10 aptus in vitro

PAGE 14

Infectious Diseases Smallpox History of Smallpox

PAGE 15

Cause and Clinical Course of Smallpox Variola major and Variola minor V major V minor ena n them a papules

PAGE 16

Diagnosis of Smallpox Poxviruses and Classification of P oxviruses

PAGE 17

Vaccinia Virus Vaccinia virus

PAGE 18

Vaccinia Virus Lifecycle and Mo rphogenesis

PAGE 19

Aptamers i n vitro in vitro

PAGE 20

Screening of Aptamers using SELEX Aptamer -Target Molecular Recognition and Interactions

PAGE 21

Advantages of Aptamers Compared to Antibodies in vitro

PAGE 22

Synthesis and Modification of DNA Library and Aptamers

PAGE 24

Biomarker Discovery with Aptamers Biomarkers in vivo

PAGE 25

Biomarker Discovery and Target Identification

PAGE 27

Applications of Aptamers

PAGE 28

Analytical Applications of Aptamers

PAGE 30

Aptamer B ased Bioassays Fransciella Tularensis

PAGE 31

Therapeutic Aptamers

PAGE 32

Targeted D elivery with Aptamers Aptamers in V irology

PAGE 33

Research Objective

PAGE 34

Vacc inia virus

PAGE 40

Introduction

PAGE 42

Principle of SELEX

PAGE 43

Different Methods of SELEX

PAGE 44

Materials and Methods Cell Culture

PAGE 45

Virus Growth v accinia virus

PAGE 46

Virus Titer Infection Protocol Adherent HeLa

PAGE 47

Suspension HeLa S3 DNA Library S ynthesis

PAGE 48

Chemicals an d Reagents Polymerase Chain Reaction

PAGE 49

Taq

PAGE 50

Gel Electrophoresis ssDNA Preparation

PAGE 51

Cell SELEX P rotocol

PAGE 52

Flow C ytometry

PAGE 53

Cloning and Sequencing Taq Escherichia coli

PAGE 54

E.coli E.coli Resu lts and Discussions Selection of Aptamers

PAGE 56

Alignment

PAGE 57

Dissociation Constants

PAGE 58

Competition Experiments

PAGE 59

Conclusion

PAGE 67

TTTTGCA TTTTGCA TTTTGCA

PAGE 69

2D Graph 2 f = Bmax*abs(x)/(Kd + abs(x))X Data 0 200 400 600 Y Data 0 2 4 6 8 10 12 14 16 18 x column vs y column Col 1 vs Col 2 2D Graph 3 f = Bmax*abs(x)/(Kd + abs(x))X Data 0 100 200 300 Y Data 0 5 10 15 20 25 30 x column vs y column Col 1 vs Col 2 2D Graph 6 f = Bmax*abs(x)/(Kd + abs(x))X Data 0 20406080100120 Y Data 6 8 10 12 14 16 18 20 x column vs y column Col 1 vs Col 2 2D Graph 2 f = Bmax*abs(x)/(Kd + abs(x))X Data 0 100200300400500 Y Data 0 2 4 6 8 10 12 x column vs y column Col 1 vs Col 2

PAGE 72

Introduction

PAGE 73

Proteinases and Glycosylation Inhibitors viz.

PAGE 74

Alp hascreen

PAGE 75

Materials and Methods Cell Culture Viruses

PAGE 76

Flow Cytometry Different cell lines Proteinase

PAGE 77

Different Viruses Glycosylation Inhibitors Alphascreen

PAGE 78

Protein Gel

PAGE 79

Western Blot Results and Discussions Flow Cytometry

PAGE 80

viz. cowpox vaccinia IHD -J, vaccinia IHD -W rabbitpox

PAGE 82

AlphaScreen

PAGE 83

Conclusions

PAGE 93

Introduction Detection of Infection

PAGE 94

A ntiviral Aptamers Analytical Applications of the Selected A ptamer

PAGE 95

in vitro in vivo Materials and Methods Imaging Infected C ells Imagi ng Infected T issues

PAGE 96

Flow Cytometry

PAGE 97

Bionanotechnology Assays to Determine the Antiviral Effect of Aptamer Luminescen ce assay

PAGE 98

Plaque inhibition assay Southwestern Blot with Aptamers

PAGE 99

Results and Discussions Imaging Infection Detection of Infection Using Flow Cytometry

PAGE 100

in vitro in vivo

PAGE 101

ex vivo Antiviral Effect of Aptamer

PAGE 102

Southwestern Blot with Aptamer Conclusions ex vivo

PAGE 103

in vivo

PAGE 116

Summary in vitro

PAGE 117

Future Directions Generation of Functional Antiviral Aptamers

PAGE 118

In -vivo Monitoring of the P rogr ess of Disease

PAGE 119

Primary Health Care now more than ever The Global Epideimology of Infectious Disease Bulletin of the History of Medicine 65 The Greatest Killer: Smallpox in History Smallpox:the fight to eradicate a global scourge Medical Aspects of Chemical and Biological Warfare BUMC Proceedings 17 Medical microbiology and immunology; 8 Edition Primary Care Update for OB/GYNS 9 Smallpox Journal of Laboratory and Clinical Medicine 142

PAGE 120

N Engl J Med 346 J. Clin. Microbiol. 42 J. Cl in. Microbiol. 33 Microscopy and Microanalysis 12 Orthopoxviruses Pathogenic for Humans Microbiological Reviews 55 Nature Reviews Microbiology 3 Poxviruses Virology 389 Journal of General Virology 86 Journal of Virology 80 Annual Review of Cell and Developmental Biology 14 Science 320 Trends in Microbiology 16 346

PAGE 121

Science 249 405 Biochemistry 44 Biochemical and Biophysical Research Communications 366 Biochemistry 33 Current Opinion in Structural Biology 9 Journal of Biological Chemistry 275 Rna Biology 6 Journal of the American Chemical Society 129 Mol. BioSyst. Advance Aricle J. Virol. 71 Nature 355 Journal of Biological Chemistry 273

PAGE 122

Analytical Chemistry 79 Analytical Chemistry 80 Nucl. Acids Res. 37 Biotechnology Letters 28 Science 287 Nature 382 Chemical Communications Electrophoresis 27 Clinical Chemistry 45 Current Opinion in Chemical Biology 10 J. Clin. Invest. 106 Clin Pharmacol Ther 69 Lancet 351 British Journal of Clinical Pharmacology 44 Canc er 57

PAGE 123

New England Journal of Medicine 350 Cancer Research 64 Molecular & Cellular Proteomics 3 Trends in Biotechnology 22 Nature Biotechnology 24 Molecular & Cellular Proteomics 5 Proteomics 6 Electrophoresis 27 Analytical Chemistry 76 Electrophoresis 27 Journal of Chromatography B -Analytical Technologies in the Biomedical and Life Sciences 845 J. Am. Chem. Soc. 125

PAGE 124

Nucl. Acids Res. 31 Anal. Chem. 73 Anal. Chem. 75 Chem. Comm 103 Bioorganic & Medicinal Chemistry 9 Anal. Chem. 74 Anal. Chem. 76 Analytical Chemistry 77 Angewandte Chemie -Internati onal Edition 39 Current Opinion in Chemical Biology 8 Biochemical and Biophysical Research Communications 292 Chembiochem 4 Journal of the American Chemical Society 125 Proceedings of the National Academy of Sciences of the United States of America 102

PAGE 125

Chembiochem 6 Journal of the American Chemical Society 131 Analytical Chemistry 81 Protein Protein Interaction 110 Laboratory Investigation 86 Nature Biotechnology 14 Proteomics 4 20 O phthalmology 112 Nature Reviews Drug Discovery 5 Pharmaceutical Research 17 Biochimie 87 Nature Biotechnology 22

PAGE 126

Journal of Molecular Biology 272 Nature 419 Proceedings of the National Academy of Sciences of the United States of America 100 Antisense & Nucleic Acid Drug Development 10 Journal of Clinical Investigation 106 Aaps Journal 7 J Nucl Med 47 Journal of Neuroscience 27 Proceedings of the National Academy of Sciences 105 Nature Biotechnology 24 Gene Therapy 14 Biosensors & Bioelectronics 15 Journal of General Virology 88 Proceedings of the National Academy of Sciences of the United States of America 89

PAGE 127

J. V irol. 76 Genes to Cells 5 J Gen Virol 87 Clinical Infectious Diseases 38 Biosensors and Bioelectronics 24 Essentials of diagnostic virology Poxviridae: the poxviruses Clinical Chemistry 53 Clinical Chemistry 50 Cold Spring Harbor Symposia on Quantitative Biology 51 Nucl. Acids Res. 34 Biotechniques 32 Nucleic Acids Research 33 Biochemical and Biophysical Research Communications 291

PAGE 128

Journal of Biotechnology 81 Bioorganic & Medicinal Chemis try 9 Accounts of Chemical Research 41 20 Current Opinion in Chemical Biology 6 Bioinformatics 23 BMC Bioinformatics 9 Infect Dis Clin North Am 20 Virology 11 Trends in Immunology 26 J. Virol. 66 Genome Research 11 Clin Chem 46 J Gen Virol 86 Virology 196

PAGE 129

J Gen Virol 45 Virus Genes 5 J Virol. 31 J. Exp. Med. 170 Journal of Virology 65 Virology 306 Cell 33 Molecular and Cellular Biology 6 J. Virol. 73 J. Virol. 81 J. Virol. 82 Biosensors and Bioelectronics 22 J. Virol. 66 J. Virol. 65

PAGE 130

Virology Virology 50th Anniversary Issue 344 Cancer Res 63 Proceedings of the National Academy of Sciences 105 Analytical Chemistry 80

PAGE 131

first class m 'o c bMay 2010