Nucleic Acid Templated Reactions: Consequences of Probe Reactivity and Readout Strategy for Amplified Signaling and Sequence Selectivity

Abstract

DNA- and RNA-templated chemical reactions can serve as a diagnostic means for the detection of nucleic acids. Reaction schemes that allow amplified detection are of high interest for polymerase chain reaction (PCR)-free DNA and RNA diagnosis. These reactions typically draw upon the catalytic activity of the template, which is able to trigger the conversion of many signaling molecules per template molecule. However, the design of reactive probes that allow both sensitive and selective nucleic acid detection is a challenge and requires insight into three major parameters: a) reactivity of functional groups involved, b) affinity of probes for the template, and c) the readout system. In this study we used peptide nucleic acid (PNA)-based probes to investigate in detail the signaling power and the selectivity of a transfer reaction derived from a native chemical ligation. We show that subtle variations of the thioesters involved had a tremendous impact on the sensitivity and selectivity of the reaction system. The results suggest that reactions at turnover conditions require low rates of non-templated reaction pathways to provide high target selectivity and sensitivity. On the other hand, very high rates of templated reactions should be avoided to allow mismatched probe-template complexes to dissociate prior to bond formation. Furthermore, the temperature dependence of the DNA-catalyzed transfer reaction was studied and provided insight into crucial strand-exchange processes. Further improvements of selective signaling were achieved through a new readout based on pyrene-transfer reactions. This method reduces background signals and enables significant increases in the signaling rates compared with previous fluorescence-based methods.

Reducing Product Inhibition in Nucleic Acid‐Templated Ligation Reactions: DNA‐Templated Cycligation

“…4 h vs. 24 h) than the reaction under isothermal conditions. Though the data clearly indicate that product cyclization speeds up ligation reactions when substoichiometric template is used, the observed efficiency still is inferior to the efficiencies reported for templated reactions without ligation, such as acyl transfer, [25,26,[58][59][60]62] Wittig transfer, [61] transition metal catalysis, [64,65] or nitrogen release reactions. [24,44,45,47,49,50,52,56,57] We performed melting experiments to assess the template affinities of the molecules involved in the templated ligation-cyclization reaction (Figure 4).…”

Fluorescence coupled in-capillary DNA hybridization assay and its application to identification of DNA point mutation

“…Due to the shorter mixing and reaction time of the in-capillary assay, the detection limit was not satisfied at this time. We have realized that the detection limit may not be one of the advantages for this assay compared to other available approaches mediated by Förster resonance energy transfer (FRET) [38] or nanoparticles [37,39]. As a future direction to our studies, we will optimize the experimental conditions and explore the possibility to label two ssDNA with different dye simultaneously in generating a FRET signal, which will greatly increase the detection limit.…”

Dual Sensing of Hairpin and Quadruplex DNA Structures Using Multicolored Peptide Nucleic Acid Fluorescent Probes

“…(32)(33)(34)(35)(36) We recently reported the use of fluorogenic PNA probes for sensing Gquadruplex formation. Upon binding of both probes to the single-stranded flanking arms of a quadruplex, formation of a trimethine cyanine dye from two non-fluorescent precursors was templated.…”