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Functionalized Aptamers for Detection of Small-Molecule Targets

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Functionalized Aptamers for Detection of Small-Molecule Targets Florida International University FIU Digital Commons FIU Electronic Theses and Dissertations University Graduate School 6-25-2019 Functionalized Aptamers for Detection of Small-Molecule Targets Haixiang Yu Florida International University, [email protected] Follow this and additional works at: https://digitalcommons.fiu.edu/etd Part of the Analytical Chemistry Commons, and the Biochemistry Commons Recommended Citation Yu, Haixiang, "Functionalized Aptamers for Detection of Small-Molecule Targets" (2019). FIU Electronic Theses and Dissertations. 4249. https://digitalcommons.fiu.edu/etd/4249 This work is brought to you for free and open access by the University Graduate School at FIU Digital Commons. It has been accepted for inclusion in FIU Electronic Theses and Dissertations by an authorized administrator of FIU Digital Commons. For more information, please contact [email protected]. FLORIDA INTERNATIONAL UNIVERSITY Miami, Florida FUNCTIONALIZED APTAMERS FOR DETECTION OF SMALL-MOLECULE TARGETS A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in BIOCHEMISTRY by Haixiang Yu 2019 To: Dean Michael R. Heithaus College of Arts, Sciences and Education This dissertation, written by Haixiang Yu, and entitled Functionalized Aptamers for Detection of Small-Molecule Targets, having been approved in respect to style and intellectual content, is referred to you for judgment. We have read this dissertation and recommend that it be approved. _______________________________________ Prem Chapagain _______________________________________ Yuan Liu _______________________________________ Alexander Mebel _______________________________________ Yi Xiao, Major Professor Date of Defense: June 25, 2019 The dissertation of Haixiang Yu is approved. _______________________________________ Dean Michael R. Heithaus College of Arts, Sciences and Education ______________________________________ Andrés G. Gil Vice President for Research and Economic Development and Dean of the University Graduate School Florida International University, 2019 ii © Copyright 2019 by Haixiang Yu All rights reserved. iii DEDICATION I dedicate this work to my parents Boyang Yu and Xuming Liu, and my grandmother Lijuan Xia with my deepest love. iv ACKNOWLEDGMENTS No accomplishment is achieved by a single pair of hands. My dissertation could have not been possible without the cooperation and support of numerous people. I want to express my most sincere appreciation to those who have helped me during my six years of study at FIU in my humble acknowledgment below. First and foremost, I would like to express my utmost gratitude to my major professor Dr. Yi Xiao, for her continuous effort and care throughout my Ph.D. years. Dr. Xiao has armed me with knowledge and skills, nurtured me with every opportunity and resource, ignited my dreams, and enlightened my way to future success. She has been a role model that encouraged me to conquer any obstacle, and is a beacon that gives me strength and persistence to overcome the darkest times. This artist carved and filed me into the current state I am now. I owe all my achievements to Dr. Xiao. I am most grateful to my dissertation committee, Dr. Prem Chapagain, Dr. Yuan Liu, and Dr. Alexander Mebel for their constant guidance and support during my graduate research and career development. They have made time for me whenever I needed help and have provided numerous valuable advices during the past six years. I would like to give special thanks to Dr. Liu, who is also the biochemistry graduate program director, for keep tracking my graduation progress during the years. I would also like to thank Dr. Xiaotang Wang, the former graduate program director, who admitted me into this great program and provided considerable help and support on my research and life in the past six years. I would like to acknowledge the Graduate Research Fellowship (2015-R2-CX-0034) from the National Institute of Justice, Office of Justice Programs, U.S. Department of v Justice, which not only funded my stipend and research between 2015 and 2018, but also supported me for training and presenting at several national research conferences throughout the years. I would like to acknowledge the Department of Chemistry and Biochemistry and Dissertation Year Fellowship from University Graduate School at FIU for the financial support during my Ph.D. research. I would also like to thank the Graduate and Professional Student Committee and the dean’s office of the College of Arts, Sciences and Education at Florida International University for travel funding. These supports were essential for me to acquire this Ph.D. I would like to thank all current and formal lab mates for their support. Particularly, I would like to thank Obtin Alkhamis, Bhargav Guntupalli, Juan Canoura, Pingping Liang, Yingzhu Liu, Yingping Luo, Zongwen Wang, Weijuan Yang for their direct contribution to my dissertation research. I could not accomplish my research without your help. I would also like to spread my gratitude to our collaborators for their generous help with my research. I would like to Dr. Milan Stojanovic and Dr. Kyung-Ae Yang at Columbia University for their training and guidance on aptamer isolation; Dr. Xinhui Lou and Dr. Shi Xu at Capital Normal University for their guidance on isothermal titration calorimetry; Dr. Yi Lu and Kang Yong Loh at University of Illinois Urbana-Champaign for their help for nanomaterial synthesis and characterization; and Dr. Paul Sharp at the FIU Department of Biological Sciences for assistance with the DNA sequencing process. I would also like to thank the faculty and staff members of the Department of Chemistry and Biochemistry for their help during my Ph.D. study. Special thanks to Magali Autie, Jackelyn Marcos, and Lilia San Miguel, who help me the most over the years. vi Last but not least, I would like to express my very profound gratitude to my parents Boyang Yu and Xuming Liu and my grandmother Lijuan Xia for their endless support, care, and encouragement throughout my years of study. They have been giving me strength for me to overcome even the hardest of times. I would also like to thank my other family members and friends for their support and encouragement; I must apologize as I could not mention each personally one by one. vii ABSTRACT OF THE DISSERTATION FUNCTIONALIZED APTAMERS FOR DETECTION OF SMALL-MOLECULE TARGETS by Haixiang Yu Florida International University, 2019 Miami, Florida Professor Yi Xiao, Major Professor Aptamers have recently gained considerable attention for small-molecule detection in diverse applications such as drug identification, medical diagnostics, and environmental monitoring. However, the performance of aptamer-based sensors has been greatly limited by the low target affinity and responsiveness of small-molecule binding aptamers. This dissertation describes several novel aptamer engineering and isolation strategies to remedy this problem. Specifically, we first develop a generally applicable strategy to engineer split aptamers containing two binding domains termed cooperative-binding split aptamers (CBSAs). CBSAs exhibit higher target binding affinity and are far more responsive in terms of target-induced split aptamer assembly compared to single-domain parent split aptamers from which they are derived. Using a cocaine-binding CBSA, we achieve specific fluorescence detection of as low as 50 nM cocaine in 10% saliva within 15 minutes. We then develop a general approach for creating rapid and sensitive CBSA- based enzyme-assisted target recycling (EATR)-amplified small-molecule sensors for sensitive target detection. Using this strategy, we develop a fluorescence assay for dehydroisoandrosterone-3-sulfate which achieves 100-fold enhanced target sensitivity viii relative to a non-EATR-based assay, and a colorimetric assay for visual detection of low- micromolar concentrations of cocaine. To simplify the sensor development process, we establish a novel and simple SELEX strategy for directly isolating aptamers with intrinsic dye-displacement functionality which transduce target-binding events into a change of dye absorbance. As a demonstration, we isolate an aptamer against the synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV) that can detect MDPV at concentrations as low as 300 nM in a label-free, rapid, and simple dye-displacement assay. To further control the target-binding spectra of aptamers, we employ a novel parallel-and-serial SELEX strategy to isolate an aptamer binding 12 synthetic cathinones with nanomolar affinity but not 11 non-target compounds that are closely related in structure. Using this aptamer, instantaneous visual detection of synthetic cathinones at nanomolar concentrations in biological samples is achieved. In summary, this work demonstrates the great potential of novel aptamer engineering and isolation strategies in generating functional signal-reporting aptamers for sensitive small molecule detection. Importantly, the strategies described here are generalizable and can be used to develop aptamer-based assays for virtually any small-molecule targets. ix TABLE OF CONTENTS CHAPTER PAGE 1: Introduction ......................................................................................................................1 1.1. Overview ................................................................................................................. 1 1.2. Motivation and goals ..............................................................................................
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