Cellular RNA labeling strategies based on bioorthogonal chemical reactions are much less developed in comparison to glycan, protein and DNA due to its inherent instability and lack of effective methods to introduce bioorthogonal reactive functionalities (e.g. azide) into RNA. Here we report the development of a simple and modular posttranscriptional chemical labeling and imaging technique for RNA by using a novel toolbox comprised of azide-modified UTP analogs. These analogs facilitate the enzymatic incorporation of azide groups into RNA, which can be posttranscriptionally labeled with a variety of probes by click and Staudinger reactions. Importantly, we show for the first time the specific incorporation of azide groups into cellular RNA by endogenous RNA polymerases, which enabled the imaging of newly transcribing RNA in fixed and in live cells by click reactions. This labeling method is practical and provides a new platform to study RNA in vitro and in cells.

Dynamic interplay between peptide synthesis and membrane assembly would have been crucial for the emergence of protocells on the prebiotic Earth. However, the effect of membrane-forming amphiphiles on peptide synthesis,...

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Direct incorporation of azide groups into RNA oligonucleotides by in vitro transcription reactions in the presence of a new azide-modified UTP analogue, and subsequent posttranscriptional chemical labeling of azide-modified oligoribonucleotide transcripts by click and Staudinger reactions are described. This postsynthetic labeling protocol is robust and modular, and offers an alternative access to RNA labeled with biophysical probes.

Numerous biophysical tools based on fluorescence have been developed to advance the understanding of RNA–nucleic acid, RNA–protein, and RNA–small molecule inter-actions. In this regard, fluorescent ribonucleoside analogues that are sensitive to their local environment provide sensitive probes for investigating RNA structure, dynamics, and recognition. Most of these analogues closely resemble the native ribonucleosides with respect to their overall dimension and have the ability to form canonical Watson–Crick (WC) base pairs. Therefore, it is possible to place these probes near the point of interaction in a target nucleic acid with minimum structural perturbations and gain insight into the intricacies of conformational changes taking place in and around the interaction site. Here, we provide a concise background on the development and recent advances in the applications of base-modified fluorescent ribonucleoside analogue probes. We first present various base-modified fluorescent ribonucleoside analogues, their photophysical properties, and methods to incorporate these analogues into oligoribonucleotides. We then discuss the established spectroscopic techniques, which make use of the fluorescence properties of these emissive ribonucleoside analogues. Finally, we present the applications of base-modified fluorescent ribonucleoside analogues used as probes incorporated into oligoribonucleotides in investigating RNA structures and functions.

This protocol describes the detailed experimental procedure for the synthesis of an azide-modified uridine triphosphate analog and its effective incorporation into an oligoribonucleotide by in vitro transcription reactions. Furthermore, procedures for labeling azide-modified oligoribonucleotides post-transcriptionally with biophysical probes by copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) and Staudinger reactions are also provided. This post-transcriptional chemical modification protocol is simple and modular, and it affords labeled oligonucleotides in reasonable amounts for biophysical assays. The procedure for enzymatic incorporation of the monophosphate of azide-modified UTP into an oligoribonucleotide transcript takes ∼2 d, and subsequent post-transcriptional chemical functionalization of the transcript takes about 2 d.

The development of robust tools and practical RNA labeling strategies that would facilitate the biophysical analysis of RNA in both cell-free and cellular systems will have profound implications in the discovery of new RNA diagnostic tools and therapeutic strategies. In this context, we describe the development of a new alkyne-modified UTP analog, 5-(1,7-octadinyl)uridine triphosphate (ODUTP), which serves as an efficient substrate for the introduction of a clickable alkyne label into RNA transcripts by bacteriophage T7 RNA polymerase and mammalian cellular RNA polymerases. The ODU-labeled RNA is effectively used by reverse transcriptase to produce cDNA, a property which could be utilized in expanding the chemical space of a RNA library in the aptamer selection scheme. Further, the alkyne label on RNA provides a convenient tool for the posttranscriptional chemical functionalization with a variety of biophysical tags (fluorescent, affinity, amino acid and sugar) by using alkyne-azide cycloaddition reaction. Importantly, the ability of endogenous RNA polymerases to specifically incorporate ODUTP into cellular RNA transcripts enabled the visualization of newly transcribing RNA in cells by microscopy using click reactions. In addition to a clickable alkyne group, ODU contains a Raman scattering label (internal disubstituted alkyne), which exhibits characteristic Raman shifts that fall in the Raman-silent region of cells. Our results indicate that an ODU label could potentially facilitate two-channel visualization of RNA in cells by using click chemistry and Raman spectroscopy. Taken together, ODU represents a multipurpose ribonucleoside tool, which is expected to provide new avenues to study RNA in cell-free and cellular systems.

<p>The prelude to the origin of cellular life on Earth would have involved a fundamental step, that of protocell formation. This involves the coming together of two crucial processes; abiotic synthesis of informational and catalytic polymers, and the assembly of membrane compartments. Mutual interactions between these processes would have likely affected the emergence and early stages of protocell evolution. Previous investigations have predominantly focused on cooperative interactions, often neglecting any competitive behavior that might ensue as ‘counterproductive cross-talk’. However, in a realistic scenario, both cooperative and competitive reactions would have occurred simultaneously in a complex prebiotic soup, generating a plethora of chemical species with their own prebiotic implications. In this study, we followed a systematic and unbiased approach to explore this interdependence. We used a lipid-amino acid system to demonstrate the above-mentioned phenomenon wherein we investigated the effect of a membrane-forming amphiphile on peptide synthesis, under prebiotically plausible conditions. </p><p><br></p> <p>Interestingly, our study shows the formation of a hitherto unobserved reaction product that could have played a significant role during the emergence of life on the early Earth. We do show that peptide synthesis occurs but with a decrease in the yield. This is due to another concurrent and competing reaction, wherein an amino acid covalently interacts with a phospholipid to generate new amphiphilic species called N-acyl amino acids (NAAs) via an ester-amide exchange process. These NAAs are thermostable and, hence, persistent even at high temperatures. Furthermore, this protoamphiphile is also able to self-assemble into vesicles at acidic pH. <i>Au contraire</i>, fatty acids, a widely accepted constituent of prebiotic compartments, have been shown to generate vesicles only at neutral to alkaline pH. Thus, NAAs could have had a selective advantage over fatty acids to form thermostable protocell compartments under acidic geothermal pool-like conditions, a niche that has gained prominence as one of the important geological settings where life could have originated. Our study underlines the importance of an unbiased exploration of the complex interactions between prebiotic processes, which could potentially open new avenues to solving the origin of life conundrum. </p> <p> </p>