Critical Analysis of Rate Constants and Turnover Frequency in Nucleic Acid-Templated Reactions: Reaching Terminal Velocity

Abstract

Nucleic acid-templated reactions have attracted significant attention for nucleic acid sensing and imaging. The level of signal amplification obtained from templated reactions is a function of the template turnover, wherein the template acts as the catalyst. Herein, we report the application of a pyridinium linker that immolates upon photocatalytic reduction with a ruthenium complex to yield the fastest nucleic acid templated reaction reported to date. We show that the templated reaction turnover is limited by the duplex dissociation kinetics beyond probes longer than a 6-mer and proceeded fastest for a 5-mer PNA probe. Using a beacon architecture that masks the catalytic template, we show that this methodology can be used for nucleic acid sensing extending the analyte recognition beyond a 5-mer. The system proceeds with a catalytic efficiency of 10 M s and achieves turnover frequency of >100 h.

Turn On of a Ruthenium(II) Photocatalyst by DNA‐Templated Ligation

“…We reasoned that our system could be extended to proceed at low substrate concentration, if an overhang sequence was adjacent to the catalyst for substrate hybridization. We had previously shown that a pentamer PNA was sufficient for PNA–PNA templated reactions . This was achieved using a bifunctional bipyridine bearing a carboxylic acid group at the 5‐position and a Fmoc‐protected glycine conjugated to the 5′‐amino group (Figure ).…”

“…We have shown that the photocatalytic activity of [Ru(bpy) 3 ] 2+ type of complexes can be harnessed to reduce different immolative linkers, enabling substrate turnover with only one reagent exchanging on the template. However, these studies clearly point to the fact that substrate turnover is rate limiting . Alternatively, a templated reaction could be used to generate an active catalyst.…”

A Bifunctional Fluorogenic Rhodamine Probe for Proximity‐Induced Bioorthogonal Chemistry

“…Expectedly, k app significantly increased for norbornene 14mer‐ 15 , the most reactive dienophile used in this study, to the highest herein determined values ( k app [10 −4 s −1 ]=[22±1] to [46±3]). The determined k app values are in accordance with reported first order rate constants for DNA‐templated reactions . The half‐lives ranged from 46±6 min (14mer‐ 11 ) to 2.5±0.9 min (14mer‐ 15 ) in DA inv with 29mer‐ g1 as template (Table S4).…”

Nucleic Acid Templated Reactions for Chemical Biology

“…The 5‐mer PNA was inserted in a beacon architecture composed of an 8‐mer l ‐γ‐PNA for DNA recognition and a 5‐mer d ‐γ‐PNA to ensure sufficient specificity for nucleic acid sensing. The interaction with sub‐stoichiometric DNA template and light irradiation at 455 nm induced hairpin opening and triggered photocatalytic reduction of the pyridinium linker …”