Temporal Labeling of Nascent RNA Using Photoclick Chemistry in Live Cells.

We report the first cellular application of a photoclick SPAAC reagent to label azide-functionalized RNA. 350 nm irradiation of a cyclopropenone caged oxo-dibenzocyclooctyne (photo-ODIBO) biotin yields formation of the SPAAC reactive species, which rapidly forms adducts with RNA containing 2'-azidoadenosine (2'N3-A). Photo-ODIBO was found to be highly stable in the presence of thiols, conferring greater stability relative to ODIBO. Light activated photo-ODIBO enabled tagging of cellular RNA, in addition to fluorescent imaging as well as enrichment of RNA in cell subpopulations via selective irradiation.

Cell-Selective Bioorthogonal Metabolic Labeling of RNA.

Stringent chemical methods to profile RNA expression within discrete cellular populations remains a key challenge in biology. To address this issue, we developed a chemical-genetic strategy for metabolic labeling of RNA. Cell-specific labeling of RNA can be profiled and imaged using bioorthogonal chemistry. We anticipate that this platform will provide the community with a much-needed chemical toolset for cell-type specific profiling of cell-specific transcriptomes derived from complex biological systems.

Progress and challenges for chemical probing of RNA structure inside living cells.

Proper gene expression is essential for the survival of every cell. Once thought to be a passive transporter of genetic information, RNA has recently emerged as a key player in nearly every pathway in the cell. A full description of its structure is critical to understanding RNA function. Decades of research have focused on utilizing chemical tools to interrogate the structures of RNAs, with recent focus shifting to performing experiments inside living cells. This Review will detail the design and utility of chemical reagents used in RNA structure probing. We also outline how these reagents have been used to gain a deeper understanding of RNA structure in vivo. We review the recent merger of chemical probing with deep sequencing. Finally, we outline some of the hurdles that remain in fully characterizing the structure of RNA inside living cells, and how chemical biology can uniquely tackle such challenges.

RNA structure: merging chemistry and genomics for a holistic perspective.

The advent of deep sequencing technology has unexpectedly advanced our structural understanding of molecules composed of nucleic acids. A significant amount of progress has been made recently extrapolating the chemical methods to probe RNA structure into sequencing methods. Herein we review some of the canonical methods to analyze RNA structure, and then we outline how these have been used to probe the structure of many RNAs in parallel. The key is the transformation of structural biology problems into sequencing problems, whereby sequencing power can be interpreted to understand nucleic acid proximity, nucleic acid conformation, or nucleic acid-protein interactions. Utilizing such technologies in this way has the promise to provide novel structural insights into the mechanisms that control normal cellular physiology and provide insight into how structure could be perturbed in disease.

Metabolic Incorporation of Azide Functionality into Cellular RNA.

Real-time tracking of RNA expression can provide insight into the mechanisms used to generate cellular diversity, as well as help determine the underlying causes of disease. Here we present the exploration of azide-modified nucleoside analogues and their ability to be metabolically incorporated into cellular RNA. We report robust incorporation of adenosine analogues bearing azide handles at both the 2'- and N6-positions; 5-methylazidouridine was not incorporated into cellular RNA. We further demonstrate selectivity of our adenosine analogues for transcription and polyadenylation. We predict that azidonucleosides will find widespread utility in examining RNA functions inside living cells, as well as in more complex systems such as tissues and living animals.

A Bump-Hole Strategy for Increased Stringency of Cell-Specific Metabolic Labeling of RNA.

Profiling RNA expression in a cell-specific manner continues to be a grand challenge in biochemical research. Bioorthogonal nucleosides can be utilized to track RNA expression; however, these methods currently have limitations due to background and incorporation of analogs into undesired cells. Herein, we design and demonstrate that uracil phosphoribosyltransferase can be engineered to match 5-vinyluracil for cell-specific metabolic labeling of RNA with exceptional specificity and stringency.

Expanding the Scope of RNA Metabolic Labeling with Vinyl Nucleosides and Inverse Electron-Demand Diels-Alder Chemistry.

Optimized and stringent chemical methods to profile nascent RNA expression are still in demand. Herein, we expand the toolkit for metabolic labeling of RNA through application of inverse electron demand Diels-Alder (IEDDA) chemistry. Structural examination of metabolic enzymes guided the design and synthesis of vinyl-modified nucleosides, which we systematically tested for their ability to be installed through cellular machinery. Further, we tested these nucleosides against a panel of tetrazines to identify those which are able to react with a terminal alkene, but are stable enough for selective conjugation. The selected pairings then facilitated RNA functionalization with biotin and fluorophores. We found that this chemistry not only is amenable to preserving RNA integrity but also endows the ability to both tag and image RNA in cells. These key findings represent a significant advancement in methods to profile the nascent transcriptome using chemical approaches.