Cell- and Polymerase-Selective Metabolic Labeling of Cellular RNA with 2′-Azidocytidine

Abstract

Metabolic labeling of cellular RNA is a powerful approach to investigate RNA biology. In addition to revealing whole transcriptome dynamics, targeted labeling strategies can be used to study individual RNA subpopulations within complex systems. Here, we describe a strategy for cell- and polymerase-selective RNA labeling with 2′-azidocytidine (2′-AzCyd), a modified nucleoside amenable to bioorthogonal labeling with SPAAC chemistry. In contrast to 2′-OH-containing pyrimidine ribonucleosides, which rely upon uridine-cytidine kinase 2 (UCK2) for activation, 2′-AzCyd is phosphorylated by deoxycytidine kinase (dCK), and we find that expression of dCK mediates cell-selective 2′-AzCyd labeling. Further, 2′-AzCyd is primarily incorporated into rRNA and displays low cytotoxicity and high labeling efficiency. We apply our system to analyze the turnover of rRNA during ribophagy induced by oxidative stress or mTOR inhibition to show that 28S and 18S rRNAs undergo accelerated degradation. Taken together, our work provides a general approach for studying dynamic RNA behavior with cell and polymerase specificity and reveals fundamental insights into nucleotide and nucleic acid metabolism.

Multiscale Invasion Assay for Probing Macrophage Response to Gram-Negative Bacteria

“…Therefore, we explored another bioorthogonal reaction called the strain promoted azide-alkyne cycloaddition (SPAAC) that does not require a cytotoxic catalyst, allowing for the possibility of live cell imaging ( Baskin et al, 2007 ). Some applications of SPAAC include labeling proteins, lipids, glycans on mammalian cells ( Neef and Schultz, 2009 ; Nikić et al, 2015 ; Debets et al, 2020 ), in living animals such as mice ( Chang et al, 2010 ), ribonucleic acid (RNA) ( Wang et al, 2020 ), and more recently, on bacteria using penicillin binding proteins ( Brown et al, 2021b ). Since we previously remodeled both E. coli and P. aeruginosa with AzNAM, we utilized a dibenzocyclooctyne (DBCO) 488 dye as our strained alkyne.…”

Chemical methods for measuring RNA expression with metabolic labeling

“…A recent manuscript from the Kleiner group demonstrates the utility of pairing 2 0 -azido-cytidine with deoxynucleotidyl kinase (dCK) as a robust strategy for cell-specific and polymerase-specific RNA labeling at high percentages (0.3% of C) (Figure 2f) (Wang et al, 2020). However, 2 0 -azido-cytidine is also incorporated into cells with no overexpression of dCK (Nainar et al, 2020;Wang et al, 2020). These results mirror our observations that 2 0 -azido-cytidine, and not 2 0 -azido-uridine, is incorporated into RNA.…”

N3‐Kethoxal‐Based Bioorthogonal Intracellular RNA Labeling

“…The 2’‐azidoadenosine (2’‐AzAd) nucleoside gave a high incorporation yield, but primarily through polyA polymerases [29] . 2’‐Azidocytidine (2’‐AzCyd) was also developed to metabolically label cellular RNA, but needed co‐expression of deoxycytidine kinase (dCK) [30] …”