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Copyright by Yu Jiang 2016 Copyright by Yu Jiang 2016 The Dissertation Committee for Yu Jiang Certifies that this is the approved version of the following dissertation: Nucleic acid circuit and its application in genetic diagnostic Committee: Andrew D. Ellington, Supervisor Jennifer S. Brodbelt Richard M. Crooks Eric V. Anslyn Ilya J. Finkelstein Nucleic acid circuit and its application in genetic diagnostic by Yu Jiang, B.S.; M.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin May 2016 Dedication I dedicate this work to my parents who have always been caring and supportive throughout my life and during my graduate studies. Acknowledgements I would like to thank my advisor Dr. Andrew D. Ellington who has always been supportive, encouraging, enlightening, and optimistic during my graduate study. I thank my committee members Dr. Jennifer S. Brodbelt, Dr. Richard M. Crooks, Dr. Eric V. Anslyn, and Dr. Ilya J. Finkelstein for their helpful comments and suggestions. I also thank past and current lab members for help in various ways. Special thanks to Dr. Xi Chen and Dr. Bingling Li for their guidance to me to this field. Many thanks to Dr. Sanchita Bhadra and Dr. Yan Du for their help on various lab techniques. v Nucleic acid circuit and its application in genetic diagnostic Yu Jiang, Ph.D. The University of Texas at Austin, 2016 Supervisor: Andrew D. Ellington DNA can execute programmed strand exchange reactions that process signals and information. In particular, toehold-mediated strand exchange, a process in which one strand of a hemi-duplex is replaced by another, single strand to create a more stable complex, is the basis for many DNA circuits. By engineering several strand exchange reactions in a systematic way, complex DNA circuits can be created that accomplish sophisticated control tasks, similar to an electronic circuit. In this dissertation, we will demonstrate how we engineered and improved a toehold-mediated strand exchanged-based reaction, catalytic hairpin assembly (CHA), to make it into a real-world nucleic acid diagnostic. While CHA has previously been shown to act as an excellent amplifier of nucleic acid signals, it can sometimes execute non-specifically even in the absence of catalyst, limiting signal-to-noise and limits of detection. By introducing two mismatched bases into a specific domain on the circuit, the background leakage can be greatly decreased and the signal-to-noise ratio can be improved from less than 10 to over 100. However, the improvement of the signal:background ratio still cannot increase sensitivity compatible to the enzyme-based nucleic acid amplification. Still, DNA circuits can improve upon background issues inherent in isothermal amplification reactions, which often produce spurious side products. We engineered DNA circuits that were vi thermostable from 37 °C to 60 °C and used these for the real-time detection of isothermal amplification reactions. These circuits in essence acted like an additional probe to measure the accumulation of correct, rather than spurious, amplicons. One isothermal amplification reaction, loop-mediated isothermal amplification (LAMP) combined with our DNA could detect particular alleles of M. tuberculosis RNA polymerase (rpoB) in sputum and of the melanoma-related biomarker BRAF. As one more step towards generating a true point-of-care (POC) test, we engineered DNA circuits to transduce amplicons into an off-the-shelf glucometer. Using these reactions and devices we could directly transduce Middle-East respiratory syndrome coronavirus (MERS) and Zaire Ebolavirus (Ebola) templates into glucose signals, with a sensitivity as low as 20-100 copies/µL. Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. vii Table of Contents List of Tables ........................................................................................................ xii List of Figures ...................................................................................................... xiii Chapter 1: Introduction and background .................................................................1 1.1 Introduction to strand exchange reactions and DNA circuits ...................1 1.2 Types of nucleic acid circuits ...................................................................4 1.2.1 Enzymatic-free DNA logic gates ..................................................4 1.2.2 Enzyme-free catalytic circuits .......................................................6 1.2.3 Strand-exchange based DNA nanowalkers ...................................8 1.3 Current analytical application of the non-enzymatic catalytic circuits ...11 1.3.1 Signal amplification ....................................................................12 1.3.2 Signal transduction......................................................................19 1.3.3 Circuit used for logic gates in multiplex-analysis .......................25 1.4 Conclusion ..............................................................................................30 Chapter 2: Mismatches improve the performance of strand-displacement nucleic acid circuits ...........................................................................................................31 2.1 Introduction .............................................................................................31 2.2 Materials and methods ............................................................................33 2.3 Result and Discussion .............................................................................34 2.3.1 The possible breathing sites ........................................................34 2.3.2 The signal-to-noise performance of different mismatched hairpin35 2.3.3 Different mismatches on the H2 .................................................41 2.3.4 Native PAGE characterization of the CircA-H2D2M2 reaction 45 2.3.5 MismatCHA performance with another set of circuit sequences46 2.4 Conclusion ..............................................................................................50 Chapter3: Real-time detection of isothermal amplification reactions with thermostable catalytic hairpin assembly .............................................................................52 3.1 Introduction .............................................................................................52 3.2 Material and methods ..............................................................................54 viii 3.2.1 Chemicals, oligonucleotides and oligonucleotide complexes. ...54 3.2.2 Real-time CHA fluorescence kinetic reading .............................54 3.2.3 Ligation of circular DNA ............................................................55 3.2.4 Real-time reading of RCA-CHA system ....................................55 3.2.5 Electrophoresis analysis of RCA reaction by agarose gel. .........56 3.3 Design principle of the CHA circuit and HT-CHA circuit. ....................59 3.3.1 CHA circuit workflow and design rule .......................................59 3.3.2 Design of HT-CHA .....................................................................62 3.4 Results and Discussion ...........................................................................62 3.4.1 Real-time CHA fluorescence kinetic reading .............................62 3.4.2 HT-CHA as a real-time detector of linear RCA .........................68 3.4.3 HT-CHA as a detector for SDA ..................................................70 3.4.4 LT-CHA as a detector for RCA and SDA ..................................72 3.5 Conclusion ..............................................................................................73 Chapter 4: Robust strand exchange reactions for the sequence-specific, real-time detection of LAMP amplicons ......................................................................75 4.1 Introduction .............................................................................................75 4.2 Material and methods ..............................................................................75 4.2.1 Chemicals and oligonucleotides. ................................................76 4.2.2 Preparation of the plasmids .........................................................78 4.2.3 OSD reporter design ...................................................................80 4.2.4 Initial testing of toehold-mediated strand displacement using synthetic target oligonucleotides .................................................81 4.2.5 Standard LAMP reactions and electrophoretic characterizations81 4.2.6 Real-time LAMP detection with either OSD or Evagreen .........82 4.2.7 Real-time multiplex LAMP detection with OSD ........................82 4.2.8 The pretreatment on the synthetic sputum. .................................83 4.3 Results and disscussion ...........................................................................84 4.3.1 OSD design .................................................................................84 4.3.2 Real-time gene detection with LAMP and OSDs .......................85 4.3.3 Real-time SNP discrimination with LAMP and OSDs ...............90 ix 4.3.4 Multiplex, real-time detection with LAMP and OSDs ...............95 4.3.5 LAMP-OSD-mediated detection of Mycobacterium
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