A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA

Abstract

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.

Enzymatic synthesis of base-modified RNA by T7 RNA polymerase. A systematic study and comparison of 5-substituted pyrimidine and 7-substituted 7-deazapurine nucleoside triphosphates as substrates

“…[38][39][40][41] Our current work for the first time compares the same substituents directly linked (without any flexible View Article Online tether) to different NTPs. This not only confirms the previous findings [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] that the pyrimidine NTPs bearing even bulkier groups at position 5 are good substrates for T7 RNA polymerase, but also shows that the corresponding 7-substituted 7-deazapurine NTPs (with smaller or mid-sized groups up to benzofuryl) can be reasonably efficiently incorporated into RNA. The substituents here were not selected for a specific function but to study the influence of the size of the group on the enzymatic incorporation.…”

“…Most of the previous studies were performed only with 5-modified UTPs [25][26][27][28][29][30][31][32][33][34][35][36][37] and with just a few examples of 5-modified CTPs. [38][39][40][41] Our current work for the first time compares the same substituents directly linked (without any flexible View Article Online tether) to different NTPs.…”

“…1, Table S1 in the ESI †). The transcription has been performed in analogy to literature procedures [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39]54 using [α-32 P]-GTP or [α-32 P]-ATP and the RNA products were analyzed by PAGE and quantified using QuantityOne software (Fig. 2, means from 2-3 independent experiments).…”

“…22 The known examples of polymerase synthesis of modified RNAs include the incorporation of useful functional groups, i.e. biotin for affinity probes, 23,24 alkyne or azido groups for click reactions, 25,26 5-vinylU for further chemical modifications, 27 5-iodoU for posttranscriptional cross-coupling modification, 28 amino acid-like side chains for selection of aptamers, [29][30][31] diazirine for crosslinking 32 or fluorophores. [33][34][35][36][37] In the vast majority of cases, the modification was attached at position 5 of uridine tripho-sphate (UTP) because of easy synthetic access and because modification at this position does not much perturb the structures of RNA duplexes.…”

In vitro labeling strategies for in cellulo fluorescence microscopy of single ribonucleoprotein machines

“…Herein, we exploited T7 RNA polymerase and yeast poly(A) polymerase to integrate modified nucleotides into one of the three segments of an mRNA: randomly into the body (which includes the 5′ UTR, coding region and 3′ UTR), randomly into the poly(A) tail, or specifically between the body and tail (BBT). Coupling transcription and tailing with modification and labeling methods has the potential to save time and cost, as well as to increase the yield.…”