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Optimizing Acyclic Identification of Aptamers Syracuse University SURFACE Dissertations - ALL SURFACE December 2015 Optimizing Acyclic Identification of Aptamers Caitlin M. Miller Syracuse University Follow this and additional works at: https://surface.syr.edu/etd Part of the Physical Sciences and Mathematics Commons Recommended Citation Miller, Caitlin M., "Optimizing Acyclic Identification of Aptamers" (2015). Dissertations - ALL. 398. https://surface.syr.edu/etd/398 This Dissertation is brought to you for free and open access by the SURFACE at SURFACE. It has been accepted for inclusion in Dissertations - ALL by an authorized administrator of SURFACE. For more information, please contact [email protected]. Abstract The minimal human alpha-thrombin binding aptamer, d(GGTTGGTGTGGTTGG), has previously been successfully identified from a DNA library of randomized hairpin loop, m=15, with the High Throughput Screening of Aptamers (HTSA) technique, later termed Acyclic Identification of Aptamers (AIA). AIA eliminated the need for multiple cycles of in vitro evolution typically used for aptamer discovery by employing libraries with an over- representation of all possible sequences and high throughput sequencing. Although the method was successful at identifying the thrombin binding aptamer, improvements to partitioning and sample preparation inconsistencies encountered during replication attempts were necessary in order to maximize its value. Subsequent revisions and variations of the protocol improved and streamlined the work-flow such that the thrombin binding aptamer (TBA) and multiple variants were identified as high affinity sequences using the ligation based m=15 DNA hairpin loop library mentioned above. The revised AIA protocol was also successful for identifying TBA and multiple variants in a nuclease resistant, modified 2’-OMe RNA/DNA chimera library. Sample throughput was increased with the introduction of indexed adapters containing sequence “barcodes” that facilitate multiplexing during high throughput sequencing. To eliminate the constraints of the hairpin loop library structure, a library based on direct amplification, the “adapter” library was designed. The m=15 DNA and m=15 2’-OMe RNA/DNA chimera adapter libraries were unsuccessful at identifying TBA from the library pool, likely due to interference from the flanking adapter regions required for PCR amplification. A secondary PCR product identified during sample work-up was also characterized. This prompted a significant interest in creating the ability to accurately assess whether or not a sample should be sequenced prior to consuming valuable resources. To accomplish this, the capture of a minimally flanked library (pACAC-m15-CACA) with full length adapters and no requirement for amplification was optimized. The library provides greater flexibility in secondary structure formation and was shown to successfully identify TBA from an over-represented library pool. Amplification-free AIA introduced the unique ability to predict relative maximum sequence frequencies based on the quantity of recovered library, initial degree of over-representation, and anticipated data output. The ability to predict whether or not a sequence could be counted above background was used to assess whether that sample would be sequenced, ultimately saving time and money. To expand the applicability of the tailed libraries and amplification free protocol, a novel partitioning method that eliminated the requirement for protein immobilization was developed. Reversible formaldehyde cross-linking in conjunction with Electrophoretic Mobility Shift Assay was used to successfully identify TBA above background in proof of concept experiments using amplification free sample preparation. The ability to perform AIA partitioning in solution provides greater flexibility in target selection, including mixtures of proteins. The method also effectively reduces the aptamer off-rate to zero by covalently linking high and moderate affinity sequences to the protein target during selection, an advantage over protein immobilization for partitioning where loss of some DNA from a reversibly bound complex is inevitable. The sample preparation techniques that evolved over the course of this work offer superior control and predictability in the outcome of high throughput sequencing data. This was aided by absolute quantification with qPCR CopyCountTM software that effectively improved library quantification, distribution of indices, and cluster quality, which is crucial for maximization of data output on Illumina sequencing platforms. Consistent, high quality data eliminates the potential for costly resequencing. Future experiments will capitalize on the breadth of improvements to the AIA method described in this work. OPTIMIZING ACYCLIC IDENTIFICATION OF APTAMERS By Caitlin M. Miller B.S. Seton Hall University, 2009 Dissertation Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Syracuse University December 2015 Copyright © Caitlin M. Miller 2015 All Rights Reserved Acknowledgements I would like to thank my advisor, Professor Philip N. Borer, for his support, guidance and knowledge. Most importantly, I am thankful for the freedom and encouragement he provided, allowing me to become a more independent and thoughtful scientist. I would like to thank those whose efforts contributed to the success of this work, including Dr. Gillian Kupakuwana and Dr. Lei Chen, for their many years of research on the original high throughput screening projects. I would like to thank Dr. Mark McPike for his collaborative efforts and most helpful knowledge, and James Crill for his insightful advice and friendship. Profound thanks are given to Dr. Damian Allis for assistance in data analysis. His knowledge, effort and patience contributed immensely to the success of this project. Also, members of the Borer group, including Dr. Deborah Kerwood, Dr. Wei Ouyang, Dr. Collin Fischer, Raghuvaran Iyer and Nan Thuzar Myint. I am also grateful to Dr. Tom Duncan of SUNY Upstate for use of the ForteBio Octet RED96 system and Vicki Lyle of the SUNY Upstate DNA sequencing facility. Also, Dr. Frank Middleton of the SUNY Upstate Molecular Analysis Core Facility for use of the MiSeq and Karen Gentile for running the experiments. Thank you to Dr. Liviu Movileanu for serving as the chair of my dissertation committee and to Dr. Joseph Chaiken, Dr. Robert Doyle, Dr. Bruce Hudson and Dr. James Hougland for serving on my dissertation committee. v I am especially grateful to the Graduate School for the many opportunities afforded to me through the Future Professoriate Program, Women in Science and Engineering Future Professoriate Program, and Teaching Mentor program. I am most grateful for the opportunity to engage my passion for science education while working on this project. I would like to thank my family and friends for their love and confidence over the years, especially my husband, Sean, for his encouragement, humor, lighthearted sarcasm and patience while I continued my education. Finally, my daughter, Leah, who has inspired me in ways I could never have imagined. vi For my daughter, Leah. vii Table of Contents Abstract ... ....................................................................................................................................... i Acknowledgements ....................................................................................................................... v Table of Contents ....................................................................................................................... viii List of Tables and Figures ............................................................................................................ x Chapter 1: Introduction ............................................................................................................... 1 The Origin of Aptamers .............................................................................................................. 2 The SELEX Protocol ................................................................................................................... 3 Improving the SELEX Method ................................................................................................... 8 Aptamer Applications ............................................................................................................... 16 Chapter 2: Acyclic Identification of Aptamers ........................................................................ 31 Chapter Summary ...................................................................................................................... 31 Library Design and Target Selection ........................................................................................ 32 Replicating Aptamer Selection for Thrombin ........................................................................... 35 Partitioning at Other Protein: Library Ratios ............................................................................ 41 High Throughput Sequencing ................................................................................................... 42 Results and Discussion .............................................................................................................. 45 Chapter 3: AIA Improvements and Application of a 2’-OMe RNA/DNA Chimera Library ..................................................................................................................................... 64 Chapter
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