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Enhanced ac4C detection in RNA via chemical reduction and cDNA synthesis with modified dNTPs.
Relier, Sebastien; Schiffers, Sarah; Beiki, Hamid; Oberdoerffer, Shalini.
Afiliação
  • Relier S; Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
  • Schiffers S; Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
  • Beiki H; Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
  • Oberdoerffer S; Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA shalini.oberdoerffer@nih.gov.
RNA ; 30(7): 938-953, 2024 Jun 17.
Article em En | MEDLINE | ID: mdl-38697668
ABSTRACT
The functional analysis of epitranscriptomic modifications in RNA is constrained by a lack of methods that accurately capture their locations and levels. We previously demonstrated that the RNA modification N4-acetylcytidine (ac4C) can be mapped at base resolution through sodium borohydride reduction to tetrahydroacetylcytidine (tetrahydro-ac4C), followed by cDNA synthesis to misincorporate adenosine opposite reduced ac4C sites, culminating in CT mismatches at acetylated cytidines (RedaCT). However, this process is relatively inefficient, resulting in <20% CT mismatches at a fully modified ac4C site in 18S rRNA. Considering that ac4C locations in other substrates including mRNA are unlikely to reach full penetrance, this method is not ideal for comprehensive mapping. Here, we introduce "RetraCT" (reduction to tetrahydro-ac4C and reverse transcription with amino-dATP to induce CT mismatches) as a method with enhanced ability to detect ac4C in cellular RNA. In brief, RNA is reduced through NaBH4 or the closely related reagent sodium cyanoborohydride (NaCNBH3) followed by cDNA synthesis in the presence of a modified DNA nucleotide, 2-amino-dATP, that preferentially binds to tetrahydro-ac4C. Incorporation of the modified dNTP substantially improved CT mismatch rates, reaching stoichiometric detection of ac4C in 18S rRNA. Importantly, 2-amino-dATP did not result in truncated cDNA products nor increase mismatches at other locations. Thus, modified dNTPs are introduced as a new addition to the toolbox for detecting ac4C at base resolution.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: DNA Complementar / Citidina Limite: Humans Idioma: En Ano de publicação: 2024 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: DNA Complementar / Citidina Limite: Humans Idioma: En Ano de publicação: 2024 Tipo de documento: Article