Second-Generation Chiral Amino Acid Derivatives Afford High Stereoselectivity and Stability in Aqueous RNA Acylation.

Activated acyl species have proven versatile in the esterification of 2'-OH groups in RNA, enabling structure mapping, caging, profiling, and labeling of the biopolymer. Nearly all reagents developed for this reaction have been achiral; however, a recent study reported that simple chiral amino acid acylimidazole derivatives could yield diastereoselective reactions at RNA 2'-OH in water, enabling up to 4:1 selectivity in screening. Here, we investigated the effect of steric bulk on the stereoselectivity of RNA reaction and on the stability of adducts with a library of 36 chiral acylimidazole scaffolds with increasing steric demand. The results document the highest stereoselectivity yet achieved in RNA acylation reactions, with as high as >99:1 diastereoselectivity at >70% conversion. Also notably, the bulky adducts were found to have markedly improved stability on RNA.

Selective Arylation of RNA 2'-OH Groups via SNAr Reaction with Trialkylammonium Heterocycles.

Small-molecule reactions at the 2'-OH groups of RNA enable useful applications for transcriptome technology and biology. To date, all reactions have involved carbonyl acylation and mechanistically related sulfonylation, limiting the types of modifications and properties that can be achieved. Here we report that electron-deficient heteroaryl species selectively react with 2'-OH groups of RNA in water via SNAr chemistry. In particular, trialkyl-ammonium (TAA)-activated aromatic heterocycles, prepared in one step from aryl chloride precursors, give high conversions to aryl ether adducts with RNAs in aqueous buffer in ~2-3 h. Remarkably, a TAA triazine previously used only for reaction with carboxylic acids, shows unprecedented selectivity for RNA over water, reacting rapidly with 2'-OH groups while exhibiting a half-life in water of >10 days. We further show that a triazine aryl species can be used as a probe at trace-level yields to map RNA structure in vitro. Finally, we prepare a number of functionalized trialkylammonium triazine reagents and show that they can be used to covalently label RNA efficiently for use in vitro and in living cells. This direct arylation chemistry offers a simple and distinct structural scaffold for post-synthetic RNA modification, with potential utility in multiple applications in transcriptome research.

Chimeric d/l-DNA Probes of Base Excision Repair Enable Real-Time Monitoring of Thymine DNA Glycosylase Activity in Live Cells.

The base excision repair (BER) pathway is a frontline defender of genomic integrity and plays a central role in epigenetic regulation through its involvement in the erasure of 5-methylcytosine. This biological and clinical significance has led to a demand for analytical methods capable of monitoring BER activities, especially in living cells. Unfortunately, prevailing methods, which are primarily derived from nucleic acids, are mostly incompatible with intracellular use due to their susceptibility to nuclease degradation and other off-target interactions. These limitations preclude important biological studies of BER enzymes and many clinical applications. Herein, we report a straightforward approach for constructing biostable BER probes using a unique chimeric d/l-DNA architecture that exploits the bioorthogonal properties of mirror-image l-DNA. We show that chimeric BER probes have excellent stability within living cells, where they were successfully employed to monitor relative BER activity, evaluate the efficiency of small molecule BER inhibitors, and study enzyme mutants. Notably, we report the first example of a fluorescent probe for real-time monitoring of thymine DNA glycosylase (TDG)-mediated BER of 5-formylcytosine and 5-carboxylcytosine in living cells, providing a much-needed tool for studying DNA (de)methylation biology. Chimeric probes offer a robust and highly generalizable approach for real-time monitoring of BER activity in living cells, which should enable a broad spectrum of basic research and clinical applications.

Integration of chemically modified nucleotides with DNA strand displacement reactions for applications in living systems.

Watson-Crick base pairing rules provide a powerful approach for engineering DNA-based nanodevices with programmable and predictable behaviors. In particular, DNA strand displacement reactions have enabled the development of an impressive repertoire of molecular devices with complex functionalities. By relying on DNA to function, dynamic strand displacement devices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation in living systems has been a slow process due to several persistent challenges, including nuclease degradation. To circumvent these issues, researchers are increasingly turning to chemically modified nucleotides as a means to increase device performance and reliability within harsh biological environments. In this review, we summarize recent progress toward the integration of chemically modified nucleotides with DNA strand displacement reactions, highlighting key successes in the development of robust systems and devices that operate in living cells and in vivo. We discuss the advantages and disadvantages of commonly employed modifications as they pertain to DNA strand displacement, as well as considerations that must be taken into account when applying modified oligonucleotide to living cells. Finally, we explore how chemically modified nucleotides fit into the broader goal of bringing dynamic DNA nanotechnology into the cell, and the challenges that remain. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Diagnostic Tools > Biosensing.

Direct Comparison of d-DNA and l-DNA Strand-Displacement Reactions in Living Mammalian Cells.

To overcome technical challenges associated with the use of DNA strand-displacement circuits in vivo, including degradation by cellular nucleases, researchers are increasingly turning to bio-orthogonal l-DNA. Although enhanced stability and improved performance of l-DNA-based circuits within living cells are often implied, direct experimental evidence has not been provided. Herein, we directly compare the functional stability and kinetics of d-DNA and l-DNA strand-displacement in live cells for the first time. We show that l-DNA strand-displacement reaction systems have minimal "leak", fast reaction kinetics, and prolonged stability inside living cells as compared to conventional d-DNA. Furthermore, using "heterochiral" strand-displacement, we demonstrate that biostable l-DNA reaction components can be easily interfaced with native DNA inside cells. Overall, our results strongly support the broader adoption of l-DNA in the field of DNA molecular circuitry, especially for in vivo applications.

A Mirror Image Fluorogenic Aptamer Sensor for Live-Cell Imaging of MicroRNAs.

Development of biocompatible tools for intracellular imaging of RNA expression remains a central challenge. Herein, we report the use of heterochiral strand-displacement to sequence-specifically interface endogenous d-miRNAs with an l-RNA version of the fluorogenic aptamer Mango III, thereby generating a novel class of biocompatible miRNA sensors. Fluorescence activation of the sensor is achieved through the displacement of an achiral blocking strand from the l-Mango aptamer by the d-RNA target. In contrast to d-Mango, we show that the l-Mango sensor retains full functionality in serum, enabling a light-up fluorescence response to the target. Importantly, we employ a self-delivering version of the l-Mango sensor to image the expression of microRNA-155 in living cells, representing the first time l-oligonucleotides have been interfaced with a living system. Overall, this work provides a new paradigm for the development of biocompatible hybridization-based sensors for live-cell imaging of RNAs and greatly expands the utility of fluorogenic aptamers for cellular applications.