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Stranded Dna Aptamers That Bind to and Detect Bacteroides Fragilis Toxin Subtypes Bft-1 and Bft-2

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Stranded Dna Aptamers That Bind to and Detect Bacteroides Fragilis Toxin Subtypes Bft-1 and Bft-2 DESIGN AND CHARACTERIZATION OF CANDIDATE SINGLE- STRANDED DNA APTAMERS THAT BIND TO AND DETECT BACTEROIDES FRAGILIS TOXIN SUBTYPES BFT-1 AND BFT-2 By Payam Fathi A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science Baltimore, Maryland April 2017 ABSTRACT Enterotoxigenic Bacteroides fragilis (ETBF) is a diarrheal pathogen that secretes a small family of 20 kDa metalloproteases that have been designated B. fragilis toxins (BFT). BFTs bind to a currently uncharacterized receptor on the surface of epithelial cells resulting in the cleavage of host molecules that mediate cell-cell adhesion leading to loss of barrier integrity and chronic colitis. ETBF has also been associated with colorectal cancer and inflammatory bowel disease. Currently there are no well characterized and widely available molecular tools that are able to specifically label or inhibit BFTs. DNA aptamers are a promising class of versatile oligomers that can be designed to specifically bind to a vast array of target molecules. Herein, we identified and characterized ssDNA aptamers that show enhanced binding to the BFT subtypes BFT-1 and BFT-2. Ultimately, these aptamers may provide a relatively inexpensive way to label and possibly inhibit BFTs. A systematic evolution of ligands by the exponential enrichment (SELEX) approach employing magnetic beads was used to generate aptamer pools with enriched binding specificity to BFTs. A SYBR Green-based Real-Time PCR assay was developed to monitor the enrichment of aptamer pools against BFT. Next generation sequencing of aptamer pools using Illumina MiSeq-generated sequence data that were analyzed using publicly available graphical user interface (GUI)-based webservers. Sequence alignment and structure predictions were employed to identify and characterize eleven unique aptamers with promise for further work up. These experiments outline a lower cost and technically simple approach for the generation of ssDNA aptamers against bacterial protein toxins. Thesis readers: Cynthia L. Sears, MD and Alan L. Scott, PhD ii PREFACE I would like to acknowledge and thank my thesis advisor Dr. Cynthia L. Sears for all of her effort and support during my time at Hopkins. Dr. Sears hired me as a technician in her lab almost four years ago and has been a great mentor to me during my time in the lab. She was instrumental in fostering my interest in the human gut microbiome; an area of study which I hope to continue during my PhD years. I would like to also acknowledge Dr. Shaoguang Wu for her help, guidance, and tutoring while I have been at the Sears Lab. I am very appreciatative of Cindy and Shaoguang entertaining my ideas and enthusiasm for conducting pilot studies in the lab. I am sure there are few, if any, places where my opinions and thoughts would have held the same weight as they have in the Sears Lab. The degree of independence that I have been allowed while working with Cindy has taught me invaluable skills with respect to designing and conducting sound science, and for that I am forever grateful. Additionally, I would like to thank Dr. Christine Craig for her mentoring while she was at the Sears Lab. Thank you to Dr. Franck Housseau for writing me an outstanding recommendation letter that helped me gain acceptance to a PhD program. I would like to thank Dr. Alan L. Scott for serving as my MMI advisor and for always providing helpful advice. I’d like to also thank my colleagues in the Sears Lab for their help troubleshooting my experiments, commiseration, and for dealing with my general pessimistic grumpiness. iii TABLE OF CONTENTS ABSTRACT .................................................................................................................................................. ii PREFACE .................................................................................................................................................... iii LIST OF TABLES ..................................................................................................................................... vii LIST OF FIGURES ................................................................................................................................... vii INTRODUCTION ......................................................................................................................................... 8 A structurally analogous framework for the Gut Microbiome .................................................................... 8 Bacteroides fragilis and the Human Gut Microbiome .............................................................................. 11 Medical and Public Health significance of ETBF ..................................................................................... 14 Aptamers: A new-old method for the generation of molecules that bind to targets with high specificity 15 Aptamers as antibody-like molecules “Aptabodies” ................................................................................. 18 Aptamers as toxin neutralizing compounds .............................................................................................. 19 Purpose of Study ....................................................................................................................................... 20 Specific Aims ............................................................................................................................................ 21 CHAPTER 2: Biotinylation of BFT ........................................................................................................... 22 Purpose ..................................................................................................................................................... 22 Materials and Methods .............................................................................................................................. 22 B. fragilis Toxin BFT-1 and BFT-2 Purification .................................................................................. 22 NHS-Sulfo Biotinylation Reaction ....................................................................................................... 23 Dot Blot Analysis of Biotinylated BFT-1 and BFT-2........................................................................... 24 HT29/C1 Cell Rounding Assay ............................................................................................................ 24 Results ....................................................................................................................................................... 25 Discussion ................................................................................................................................................. 26 Chapter 2 Figures ...................................................................................................................................... 27 Streptavidin HRP Dot Blot visualizes the Biotinylation of BFT .......................................................... 27 Biotinylation of BFT-2 does not disrupt toxin activity ......................................................................... 28 CHAPTER 3: Optimization of SELEX PCR Conditions ........................................................................ 29 Purpose ..................................................................................................................................................... 29 Materials and Methods .............................................................................................................................. 30 Random Library and Primers ................................................................................................................ 30 PCR reactions and optimization conditions .......................................................................................... 31 Results ....................................................................................................................................................... 32 Discussion ................................................................................................................................................. 33 Chapter 3 Figures ...................................................................................................................................... 34 PCR optimization results in robust amplification of the Aptamer Library ........................................... 34 CHAPTER 4: Magnetic Bead SELEX for the Isolation of Aptamers Binding to BFT-1 and BFT-2 .. 35 iv Purpose ..................................................................................................................................................... 35 Materials and Methods .............................................................................................................................. 36 Buffer Preparation ................................................................................................................................ 36 Preparation of Pre-selection and Selection M280 Streptavidin Beads .................................................. 36 First SELEX Cycle ............................................................................................................................... 37 SELEX Cycles Two Through Ten ........................................................................................................ 38 FAM-labeled Aptamer Binding and FACS CALIBUR .......................................................................
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