Enzyme-Free, Isothermal Noncovalent DNA Catalytic Reactions
Creators: Xu, Jing Yang
Publisher: name: University of Alberta Library
Year: 2024

<p>MicroRNA is a class of endogenous, noncoding, short RNA molecules and the aberrant expression of microRNA is related to the development of a variety of human diseases. The detection of microRNA in living cells is vital to understanding its dynamic functions and profiles, diagnosing diseases, and developing microRNA-based therapy. My thesis research focuses on the development of enzyme-free, isothermal amplification techniques applicable to the detection of nucleic acids such as microRNA.<br>
Non-covalent DNA catalytic reactions represent a recent major advance of dynamic DNA nanotechnology. These reactions can produce multiple output DNA strands from a single nucleic acid input through cycling toehold-mediated strand displacement and/or toehold exchange reactions. The programmability and the signal amplification capability of these catalytic reactions have been utilized for building DNA nanomachines, realizing molecular computation, and detecting disease biomarkers. These DNA catalytic reactions can achieve amplified detection of nucleic acids without the need of any enzymes and washing steps at room temperature and 37 °C. <br>
Three main DNA catalytic reactions have been reported previously, including hybridization chain reaction (HCR), catalytic hairpin assembly (CHA), and entropy-driven DNA catalysis (EDC). However, the application of these reactions is limited by their complexity in design and low amplification efficiency. The primary objective of my thesis research was to develop improved DNA catalytic reactions by destabilizing the substrate of the toehold exchange catalytic reaction to make the forward reaction energetically favorable. The toehold exchange catalytic reaction, requiring only a dsDNA substrate with a toehold and a ssDNA fuel, is chosen for its simplicity. I hypothesize that the substrate can be destabilized by introducing DNA structures, mismatches or chemically modified nucleotides into the substrate. Modification of the substrate serves several purposes. First, it raises the free energy of hybridization of the substrate (〖ΔG〗<em>(f</em>Substrate)^o) making the overall reaction energetically favorable, 〖ΔG〗<em>OR^o&lt;0. Secondly, raising the 〖ΔG〗</em>(f<em>S)^o makes the formation of intermediates more energetically favorable. Finally, the modification of the substrate can affect the kinetics of the reverse reaction to favor the formation of products.<br>
 I developed a three-way-junction (TWJ) mediated DNA catalytic reaction. Without the TWJ, the products of the toehold exchange catalytic reaction is identical to the reactants, and the reaction free energy change, 〖ΔG〗</em>OR^o, is zero. The TWJ disrupts the base-stacking interaction and destabilizes the substrate, making the 〖ΔG〗<em>OR^o  negative. Compared to without TWJ, the introduction of TWJ improved amplification efficiency of the DNA catalytic reaction by approximately 13-fold within one hour. To further improve the amplification efficiency, we cascaded two TWJ-mediated DNA catalytic reactions, where the output DNA of the first reaction acted as the input DNA for the second reaction. This two-layer reaction technique afforded &gt;250-fold amplification in 1 hour and lowered the limit of detection (LOD) to 0.3 pM.<br>
Inspired by these results, a second technique was developed by introducing DNA mismatches into the DNA substrate, which offers two advantages compared to using TWJ: (1) the structure of substrate is simpler because mismatch-aided catalytic reaction uses a simple double-stranded duplex containing a mismatch as its substrate, whereas the substrate of TWJ-mediated catalytic reaction needs three strands, or the output needs to contain a stem-loop structure; (2) the use of mismatch allows for modulation of the stability of the substrate. I estimated 〖ΔG〗</em>(f<em>Substrate)^o , 〖ΔG〗</em>(f<em>Intermediate)^o , and 〖ΔG〗</em>(f<em>Product)^o values and designed substrates with different 〖ΔG〗</em>(f<em>Substrate)^o, and studied the relationship between 〖ΔG〗</em>(f<em>Substrate)^o and both the catalyzed reaction and uncatalyzed reaction, representative of amplification efficiency and background, respectively. The substrate had the largest catalyzed to uncatalyzed rate ratio when 〖ΔG〗</em>(f<em>Substrate)^o≈〖ΔG〗</em>(f<em>Intermediate)^o&gt;〖ΔG〗</em>(f_Product)^o. The mismatch-aided DNA catalytic reaction achieved a limit of detection of 2.3 pM and a linear range from 5 pM to 500 pM of a short nucleic acid strand. <br>
A third technique using modified nucleotides instead of DNA mismatches was then developed. Three types of nucleotide modifications, including abasic site, anuleotide site, and phosphorothioate linkages were compared. Replacing a nucleotide with an anucleotide site resulted in both high amplification efficiency and low background. This modified nucleotide-aided DNA catalytic reaction successfully demonstrated the detection of microRNA 10b both in buffer solutions and in MB-231 cell lysate samples. <br>
The DNA catalytic reactions reported here opens avenue for future developments, such as intracellular detection, point-of-care testing, DNA computation, and dynamic molecular circuits.</p>