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Design and Characterization of Heterochiral Strand Displacement Reactions
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The nature of Watson – Crick base pairing has enabled the rational design of complex and dynamic DNA/RNA-based molecular circuits capable of detecting nucleic acids in a sequence dependent fashion in vitro. Given the ease by which DNA can be programmed to interact with living systems, DNA-based molecular circuits provide an attractive avenue for the sequence-specific detection of RNA biomarkers in live cells. However, the stability of exogenous nucleic acids in biological environments remains a major concern. In order to overcome this limitation, modifications to the ribose backbone or within the phosphodiester bond have been employed to increase the resistance of DNA probes to nucleolytic degradation. Most DNA modifications in routine use alter the thermodynamic and kinetic properties of DNA/RNA hybridization, making it difficult to design complex reaction networks that function in the cellular environment. vL-DNA, the mirror image (i.e. enantiomer) of natural vD-DNA, represents a critically underexplored modification for this application. vL-DNAs have the same physical and chemical properties as their natural counterparts, but they are essentially ‘invisible’ to the stereospecific environment of biology. However, vL-DNA cannot form contiguous WC base pairs with vD-DNA/RNA, severely limiting its use in the development of sequence-specific probes for cellular nucleic acids (summarized in Chapter 1). Chapter 2 focuses on two potential solutions to this problem, both built on the well understood rules of DNA strand displacement reactions. We report a novel toehold-mediated strand displacement reaction utilizing achiral peptide nucleic acid (PNA)/vL- DNA duplexes and demonstrate the sequence-specific recognition of vD-DNA and vD-RNA inputs. An alternative strand displacement design is also reported whereby chimeric DDNA and vL-DNA duplexes are designed such that recognition of a vD-DNA or vD-RNA input causes the concomitant melting and release of an vL-DNA output. The work presented in this section represents first of their kind heterochiral strand displacements. Following these developments, we demonstrate the stability of these heterochiral strand displacements in living cells. Direct comparisons are made to vD-DNA components, as well as components containing the common 2′-O-methyl modification. This section underscores the potential stability of vL-DNA circuits, as well as the ‘plug-and-play’ utility of adapting vD-DNA circuit designs to vL-DNA. Finally, we design a model system in chapter 4 to thoroughly characterize heterochiral strand displacement. We show that strand displacements using PNA are generally slower than their all-DNA counterparts, and that in every case strand displacement occurs slower when the input and output chirality are not matched. Interestingly, this heterochiral barrier to strand displacement enhances the mismatch discrimination of heterochiral strand displacement systems. Overall, this work identifies dynamic molecular systems capable of sequence-specifically recognizing a vD-DNA input and generating an vL-DNA output. These heterochiral strand displacements are fast, biostable, and lay the foundation for the design of computational DNA systems capable of identifying endogenous nucleic acids inside live cells.
Young, Brian E. (2019). Design and Characterization of Heterochiral Strand Displacement Reactions. Doctoral dissertation, Texas A&M University. Available electronically from