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Chemical methods for measuring RNA expression with metabolic labeling

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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
Chemical probes for metabolic labeling of RNA. (a) Schematic of metabolic labeling experiments. (b) Schematic of pulse and chase experiments: two classes of experiments are traditionally performed. First a pulse experiment where RNA is labeled and collected at desired times and enriched for newly transcribed RNA. A second type of experiment is one in which RNA is pulse labeled for longer time point and the metabolic label is taken away and RNA isolated to characterize RNA decay. (c) Chemical structures and derivatives of adenosine and uridine. (d) Bioorthogonal chemical approaches to label metabolically labeled RNA and append biotin groups for enrichment
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Biological applications for cell‐specific metabolic labeling of RNA. (a,b) Application of 4TU‐UPRT (TU‐tagging) system for profiling differential RNA expression in neural development and human cytomegalovirus latency. (c) Utilization of 5EC‐incorporating CD‐UPRT fusion system to study signaling behavior and characterize transcriptomes of rare cell populations in Drosophila melanogaster
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Nucleoside recoding to track nascent RNA synthesis. (a) Schematic of step‐wide approach of nucleoside recoding for tracking nascent RNA synthesis. (b) Different methods for conversion of 4SU to cytidine or alkyl cytidine catalyzed by NaIO4 or OsO4, respectively. (c) Iodoacetamide mediated conversion of 4SU to 4‐thiol(SH)‐linked alkyl uridine. This modification is identified as cytidine after reverse transcription
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Chemical probes for cell‐specific metabolic labeling of RNA. (a) Schematic of cell‐specific metabolic labeling experiments. (b) Example reaction between uracil or uracil analogs reacting with uracil phosphoribosyltransferase (UPRT). (c) “Caged” cytidine analogs can be “uncaged” by penicillin G amidase to liberate 5‐ethynyl‐cytidine (5EC) for subsequent incorporation into RNA. (d) 2′‐azido‐uridine can be phosphorylated by uridine/cytidine kinase 2 (UCK2) for RNA metabolic labeling. (e) Co‐crystal structure of UCK2 bound to 5′‐phosphorylated 2′‐azido‐uridine (the kinase product. PDB ID for this structure is 6N53). (f) Chemical reaction of 2′‐deoxy‐2′azido‐cytidine with deoxynucleotidyl kinase (dCK) for cell‐specific metabolic incorporation into RNA
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RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry
RNA Evolution and Genomics > Ribonomics
RNA Methods > RNA Analyses in Cells

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