A Kinetic and Fluorogenic Enhancement Strategy for Labeling of Nucleic Acids

Abstract

Chemical modification of nucleic acids in living cells can be sterically hindered by tight packing of bioorthogonal functional groups in chromatin. To address this limitation, we report here a dual enhancement strategy for nucleic acid‐templated reactions utilizing a fluorogenic intercalating agent capable of undergoing inverse electron‐demand Diels–Alder (IEDDA) reactions with DNA containing 5‐vinyl‐2′‐deoxyuridine (VdU) or RNA containing 5‐vinyl‐uridine (VU). Reversible high‐affinity intercalation of a novel acridine–tetrazine conjugate “PINK” (KD=5±1 μM) increases the reaction rate of tetrazine–alkene IEDDA on duplex DNA by 60 000‐fold (590 M−1 s−1) as compared to the non‐templated reaction. At the same time, loss of tetrazine–acridine fluorescence quenching renders the reaction highly fluorogenic and detectable under no‐wash conditions. This strategy enables live‐cell dynamic imaging of acridine‐modified nucleic acids in dividing cells.

Hybridization-Sensitive Fluorescent Probes for DNA and RNA by a Modular “Click” Approach

“…The DNA base pairs adjacent to the fluorescent probe typically influence the spectroscopic properties of the dye. ,, For this reason, the first experiments aimed to evaluate the sequence dependence of the fluorescent readout and identify the optimal sequence environment(s) representatively for the combination of the linker 1 with the ( S )-configuration and the dye from azide 10 in the center of short DNA pieces (abbreviated with “ X ” in Figure ). This dye is a blue-green-emitting analogue of our recently developed clickable dyes with very high photostability. , The other parts of the sequence of the oligonucleotide probes were chosen randomly.…”

“…In this set of DNA probes, only DNA-S-16 and DNA-R-16 showed a significant light-up effect of 6.7 and 4.7, respectively, if at all. Since thiazole orange was a very often used dye in hybridization-sensitive DNA probes, ,,,, we also tested three thiazole orange-derived dye azides, 17 – 19 , bearing spacers of two different lengths. Unexpectedly, those three dyes did not show any light-up effect when attached to DNA probes, which we cannot explain based on their structures.…”

“…In this set of DNA probes, only DNA-S-16 and DNA-R-16 showed a significant light-up effect of 6.7 and 4.7, respectively, if at all. Since thiazole orange was a very often used dye in hybridization-sensitive DNA probes, 24,47,48,55,56 we also tested three thiazole orange-derived dye azides, 17−19, bearing spacers of two different lengths.…”

“…However, those fluorescent probes were not hybridization sensitive and require intensive washing steps for mRNA detection which limits their sensitivity and application to fixed cells. There are several promising approaches for RNA labeling in vitro and in cells based on click reactions. , This includes the enzyme-directed incorporation of non-natural nucleotides, for instance, artificial nucleosides as “stealth” fluorescent labels, nucleosides with bio-orthogonally reacting groups for metabolic labeling, − and chemo-enzymatic approaches for labeling RNAs for post-synthetic modifications . Herein, we report new fluorescent oligonucleotide probes that are hybridization-sensitive and were prepared in a modular approach with a structurally simple building block using the click post-synthetic modification strategy.…”

Two‐Factor Fluorogenicity of Tetrazine‐Modified Cyanine‐Styryl Dyes for Bioorthogonal Labelling of DNA

“…used this concept for nucleic acid‐templated ligations and phospholipids [18] . The tetrazine quenching concept was applied just once for nucleic acid labelling in living cells (not fixed cells) by means of a different strategy [19] …”