Chemical methods for measuring RNA expression with metabolic labeling

Abstract

Tracking the expression of RNA in a cell-specific manner is a major challenge in basic and disease research. Herein we outline the current state of employing chemical approaches for cell-specific RNA expression studies. We define the utility of metabolic labels for tracking RNA synthesis, the approaches for characterizing metabolic incorporation and enrichment of labeled RNAs, and finally outline how these approaches have been used to study biological systems by providing mechanistic insights into transcriptional dynamics. Further efforts on this front will be the continued development of novel chemical handles for RNA enrichment and profiling as well as innovative approaches to control cell-specific incorporation of chemically modified metabolic probes. These advancements in RNA metabolic labeling techniques permit sensitive detection of RNA expression dynamics within relatively small subsets of cells in living tissues and organisms that are critical to performing complex developmental and pathological processes. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Evolution and Genomics > Ribonomics RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry

Labeling and sequencing nucleic acid modifications using bio-orthogonal tools

“…Detection RNA in cell is not only a fundamental study, but is also relevant to disease research. 88 Nucleoside analogs are wildly used for detecting nascent RNA and RNA-binding proteins, and bio-orthogonal methods combined with relative nucleoside analogs enrich target RNA from total RNA. [89][90][91][92][93] 4-thio-uridine (4SU) was used as nucleoside analog since 1978, 94 Mark Helm et al attached alkyne group in target RNA, based on the selective reaction between 4SU and 4bromomethyl-7-propargyloxycoumarin (PBC).…”

“…Detection of RNA in cells is not only a fundamental study, but is also relevant to disease research. 88 Nucleoside analogs are widely used for detecting nascent RNA and RNA-binding proteins, and bio-orthogonal methods combined with relative nucleoside analogs enrich target RNA from total RNA ( Fig. 6 ).…”

Synthesis of 4-thiouridines with prodrug functionalization for RNA metabolic labeling

“…[15][16][17] Recently, thiolated nucleosides, in particular 4-thiouridine (4sU), have attracted much interest in metabolic labeling-based RNA sequencing approaches (TUC-seq, SLAM-seq, TimeLapse-seq) to study cellular RNA dynamics, such as synthesis and degradation rates. [18][19][20][21][22][23] 4sU uptake into cells is mediated by two distinct families of nucleoside transporters. 24 While cell lines, such as HEK293 or HeLa cells, show robust expression of the major uridine transporters SLC29A1 and SLC29A2, and therefore readily take up 4sU, many other cell types and cell lines do not (e.g.…”

Mutually Orthogonal Bioconjugation of Vinyl Nucleosides for RNA Metabolic Labeling

“…2 RNA metabolic labeling methods accomplish this by exposing cells to chemically modified, unnatural ribonucleoside analogues that are incorporated into elongating RNA strands. 3 Applying bioconjugation chemistries to the labeled RNA allows for the affinity-based separation of labeled transcripts from unlabeled, steady-state ones. It also permits localization studies of labeled RNA in cells with microscopy.…”

“…Monitoring RNA expression as a function of time provides information about the relative synthesis and decay rates of specific transcripts . RNA metabolic labeling methods accomplish this by exposing cells to chemically modified, unnatural ribonucleoside analogues that are incorporated into elongating RNA strands . Applying bioconjugation chemistries to the labeled RNA allows for the affinity-based separation of labeled transcripts from unlabeled, steady-state ones.…”