Eric Kool, Postdoctoral Faculty Sponsor
Acylation probing of "generic" RNA libraries reveals critical influence of loop constraints on reactivity.
Cell chemical biology
The reactivity of RNA 2'-OH acylation is broadly useful both in probing structure and in preparing conjugates. To date, this reactivity has been analyzed in limited sets of biological RNA sequences, leaving open questions of how reactivity varies inherently without regard to sequence in structured contexts. We constructed and probed "generic" structured RNA libraries using homogeneous loop sequences, employing deep sequencing to carry out a systematic survey of reactivity. We find a wide range of RNA reactivities among single-stranded sequences, with nearest neighbors playing substantial roles. Remarkably, certain small loops are found to be far more reactive on average (up to 4,000-fold) than single-stranded RNAs, due to conformational constraints that enhance reactivity. Among loops, we observe large variations in reactivity based on size, type, and position. The results lend insights into RNA designs for achieving high-efficiency local conjugation and provide new opportunities to refine structure analysis.
View details for DOI 10.1016/j.chembiol.2022.05.005
View details for PubMedID 35662395
Control of RNA with quinone methide reversible acylating reagents.
Organic & biomolecular chemistry
Caging RNA by polyacylation (cloaking) has been developed recently as a simple and rapid method to control the function of RNAs. Previous approaches for chemical reversal of acylation (uncloaking) made use of azide reduction followed by amine cyclization, requiring 2-4 h for the completion of cyclization. In new studies aimed at improving reversal rates and yields, we have designed novel acylating reagents that utilize quinone methide (QM) elimination for reversal. The QM de-acylation reactions were tested with two bioorthogonally cleavable motifs, azide and vinyl ether, and their acylation and reversal efficiencies were assessed with NMR and mass spectrometry on model small-molecule substrates as well as on RNAs. Successful reversal both with phosphines and strained alkenes was documented. Among the compounds tested, the azido-QM compound A-3 displayed excellent de-acylation efficiency, with t1/2 for de-acylation of less than an hour using a phosphine trigger. To test its function in RNA caging, A-3 was successfully applied to control EGFP mRNA translation in vitro and in HeLa cells. We expect that this molecular caging strategy can serve as a valuable tool for biological investigation and control of RNAs both in vitro and in cells.
View details for DOI 10.1039/d1ob01713f
View details for PubMedID 34528657
DNA tiling enables precise acylation-based labeling and control of mRNA.
Angewandte Chemie (International ed. in English)
Methods for site-selective labeling of long, native RNAs are needed for studying mRNA biology and future therapies. Current approaches involve engineering RNA sequences, which may alter folding, or are limited to specific sequences or bases. Here, we describe a versatile strategy for mRNA conjugation via a novel DNA tiling approach. The method, TRAIL, exploits a pool of "protector" oligodeoxynucleotides to hybridize and block the mRNA, combined with an "inducer" DNA that extrudes a reactive RNA loop for acylation at a predetermined site. Using TRAIL, an azido-acylimidazole reagent was employed for labeling and controlling RNA for multiple applications in vitro and in cells, including analysis of RNA-binding proteins, imaging mRNA in cells, and analysis and control of translation. The TRAIL approach offers an efficient and accessible way to label and manipulate RNAs of virtually any length or origin without altering native sequence.
View details for DOI 10.1002/anie.202112106
View details for PubMedID 34624169
Site-Selective RNA Functionalization via DNA-Induced Structure.
Journal of the American Chemical Society
2020; 142 (38): 16357–63
Methods for RNA functionalization at specific sites are in high demand but remain a challenge, particularly for RNAs produced by transcription rather than by total synthesis. Recent studies have described acylimidazole reagents that react in high yields at 2'-OH groups stochastically at nonbase-paired regions, covering much of the RNA in scattered acyl esters. Localized reactions, if possible, could prove useful in many applications, providing functional handles at specific sites and sequences of the biopolymer. Here, we describe a DNA-directed strategy for in vitro functionalization of RNA at site-localized 2'-OH groups. The method, RNA Acylation at Induced Loops (RAIL), utilizes complementary helper DNA oligonucleotides that expose gaps or loops at selected positions while protecting the remainder in DNA-RNA duplexes. Reaction with an acylimidazole reagent is then carried out, providing high yields of 2'-OH conjugation at predetermined sites. Experiments reveal optimal helper oligodeoxynucleotide designs and conditions for the reaction, and tests of the approach are carried out to control localized ribozyme activities and to label RNAs with dual-color fluorescent dyes. The RAIL approach offers a simple and novel strategy for site-selective labeling and control of RNAs, potentially of any length and origin.
View details for DOI 10.1021/jacs.0c06824
View details for PubMedID 32865995