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Mapping m6A Sites on HIV-1 RNA Using Oligonucleotide LC-MS/MS.
Baek, Alice; Rayhan, Asif; Lee, Ga-Eun; Golconda, Sarah; Yu, Hannah; Kim, Shihyoung; Limbach, Patrick A; Addepalli, Balasubrahmanyam; Kim, Sanggu.
Afiliação
  • Baek A; Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210, USA.
  • Rayhan A; Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA.
  • Lee GE; Infectious Diseases Institute, The Ohio State University, Columbus, OH 43210, USA.
  • Golconda S; Translational Data Analytics Institute, The Ohio State University, Columbus, OH 43210, USA.
  • Yu H; Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA.
  • Kim S; Center for Retrovirus Research, The Ohio State University, Columbus, OH 43210, USA.
  • Limbach PA; Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA.
  • Addepalli B; Infectious Diseases Institute, The Ohio State University, Columbus, OH 43210, USA.
  • Kim S; Translational Data Analytics Institute, The Ohio State University, Columbus, OH 43210, USA.
Methods Protoc ; 7(1)2024 Jan 10.
Article em En | MEDLINE | ID: mdl-38251200
ABSTRACT
The biological significance of chemical modifications to the ribonucleic acid (RNA) of human immunodeficiency virus type-1 (HIV-1) has been recognized. However, our understanding of the site-specific and context-dependent roles of these chemical modifications remains limited, primarily due to the absence of nucleotide-resolution mapping of modification sites. In this study, we present a method for achieving nucleotide-resolution mapping of chemical modification sites on HIV-1 RNA using liquid chromatography and tandem mass spectrometry (LC-MS/MS). LC-MS/MS, a powerful tool capable of directly analyzing native RNAs, has proven effective for mapping RNA modifications in small RNA molecules, including ribosomal RNA and transfer RNA. However, longer RNAs have posed challenges, such as the 9 Kb HIV-1 virion RNA, due to the complexity of and ambiguity in mass differences among RNase T1-cleaved RNA fragments in LC-MS/MS data. Here, we introduce a new target RNA enrichment method to isolate small local RNA fragments of HIV-1 RNA that potentially harbor site-specific N6-methyladenosine (m6A) modifications. In our initial trial, we used target-specific DNA probes only and encountered insufficient RNA fragmentation due to inefficient S1 digestion near the target site. Recognizing that inefficient S1 digestion by HIV-1 RNA is likely due to the formation of secondary structures in proximity to the target site, we designed multiple DNA probes annealing to various sites of HIV-1 RNA to better control the structures of RNA substrates for S1 digestion. The use of these non-target DNA probes significantly improved the isolation of more homogeneous target RNA fragments of approximately 50 bases in length. Oligonucleotide LC-MS/MS analysis of these isolated target RNA fragments successfully separated and detected both m6A-methylated and non-methylated oligomers at the two m6A-predicted sites. The principle of this new target enrichment strategy holds promise and should be broadly applicable to the analysis of any lengthy RNA that was previously deemed infeasible for investigation using oligonucleotide LC-MS/MS.
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Texto completo: 1 Bases de dados: MEDLINE Idioma: En Revista: Methods Protoc Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Estados Unidos

Texto completo: 1 Bases de dados: MEDLINE Idioma: En Revista: Methods Protoc Ano de publicação: 2024 Tipo de documento: Article País de afiliação: Estados Unidos