{"title":"Aqueous Activation of RNA 2′-OH for Conjugation with Amines and Thiols","authors":"Ryuta Shioi, Lu Xiao and Eric T. Kool*, ","doi":"10.1021/acs.bioconjchem.3c00370","DOIUrl":null,"url":null,"abstract":"<p >Strategies for covalent modification of RNA are important for enabling biological studies of the biopolymer and for enhancing properties of therapeutic RNAs. While a number of electrophiles have been observed to react with RNA, few methods exist for reaction with nucleophiles. Here, we describe new reagents that enable efficient conjugation of amines and other nucleophiles to unmodified RNA postsynthetically via transient activation of 2′-OH groups. Reaction of single-stranded RNA in aqueous solution with phenolic imidazolecarbamates at room temperature results in stoichiometric and superstoichiometric yields of imidazolecarbonyl group adducts, and control experiments with DNA confirm the site of reaction in RNA as 2′-OH. Subsequent incubation of imidazolecarbonyl-activated RNAs with primary or selected secondary amines results in rapid, high-yield conversion to carbamate conjugates. The activation and subsequent nucleophile reaction can be carried out either stepwise or in a one-pot reaction. Thiols and phenol species react to yield (thio)carbonate adducts, and amino acid sidechains also react, suggesting possible future utility for protein conjugates and analysis of protein–RNA interactions. The activation method is found to be selective to unpaired regions of RNA, and can be directed to a specific location in a strand by use of a loop-inducing helper DNA. The results establish novel and efficient reagents and methods for modifying RNA postsynthetically with nucleophiles.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry Bioconjugate","volume":null,"pages":null},"PeriodicalIF":4.0000,"publicationDate":"2023-12-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bioconjugate Chemistry Bioconjugate","FirstCategoryId":"1","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acs.bioconjchem.3c00370","RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Strategies for covalent modification of RNA are important for enabling biological studies of the biopolymer and for enhancing properties of therapeutic RNAs. While a number of electrophiles have been observed to react with RNA, few methods exist for reaction with nucleophiles. Here, we describe new reagents that enable efficient conjugation of amines and other nucleophiles to unmodified RNA postsynthetically via transient activation of 2′-OH groups. Reaction of single-stranded RNA in aqueous solution with phenolic imidazolecarbamates at room temperature results in stoichiometric and superstoichiometric yields of imidazolecarbonyl group adducts, and control experiments with DNA confirm the site of reaction in RNA as 2′-OH. Subsequent incubation of imidazolecarbonyl-activated RNAs with primary or selected secondary amines results in rapid, high-yield conversion to carbamate conjugates. The activation and subsequent nucleophile reaction can be carried out either stepwise or in a one-pot reaction. Thiols and phenol species react to yield (thio)carbonate adducts, and amino acid sidechains also react, suggesting possible future utility for protein conjugates and analysis of protein–RNA interactions. The activation method is found to be selective to unpaired regions of RNA, and can be directed to a specific location in a strand by use of a loop-inducing helper DNA. The results establish novel and efficient reagents and methods for modifying RNA postsynthetically with nucleophiles.
期刊介绍:
Bioconjugate Chemistry invites original contributions on all research at the interface between man-made and biological materials. The mission of the journal is to communicate to advances in fields including therapeutic delivery, imaging, bionanotechnology, and synthetic biology. Bioconjugate Chemistry is intended to provide a forum for presentation of research relevant to all aspects of bioconjugates, including the preparation, properties and applications of biomolecular conjugates.