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Expedient production of site specifically nucleobase-labelled or hypermodified RNA with engineered thermophilic DNA polymerases.
Brunderová, Mária; Havlícek, Vojtech; Matyasovský, Ján; Pohl, Radek; Postová Slavetínská, Lenka; Krömer, Matous; Hocek, Michal.
Afiliação
  • Brunderová M; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16000, Prague, 6, Czech Republic.
  • Havlícek V; Department of Organic Chemistry, Faculty of Science, Charles University, Hlavova 8, CZ-12843, Prague, 2, Czech Republic.
  • Matyasovský J; MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, UK.
  • Pohl R; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16000, Prague, 6, Czech Republic.
  • Postová Slavetínská L; Department of Organic Chemistry, Faculty of Science, Charles University, Hlavova 8, CZ-12843, Prague, 2, Czech Republic.
  • Krömer M; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16000, Prague, 6, Czech Republic.
  • Hocek M; Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, CZ-16000, Prague, 6, Czech Republic.
Nat Commun ; 15(1): 3054, 2024 Apr 09.
Article em En | MEDLINE | ID: mdl-38594306
ABSTRACT
Innovative approaches to controlled nucleobase-modified RNA synthesis are urgently needed to support RNA biology exploration and to synthesize potential RNA therapeutics. Here we present a strategy for enzymatic construction of nucleobase-modified RNA based on primer-dependent engineered thermophilic DNA polymerases - SFM4-3 and TGK. We demonstrate introduction of one or several different base-modified nucleotides in one strand including hypermodified RNA containing all four modified nucleotides bearing four different substituents, as well as strategy for primer segment removal. We also show facile site-specific or segmented introduction of fluorophores or other functional groups at defined positions in variety of RNA molecules, including structured or long mRNA. Intriguing translation efficacy of single-site modified mRNAs underscores the necessity to study isolated modifications placed at designer positions to disentangle their biological effects and enable development of improved mRNA therapeutics. Our toolbox paves the way for more precise dissecting RNA structures and functions, as well as for construction of diverse types of base-functionalized RNA for therapeutic applications and diagnostics.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: RNA / DNA Polimerase Dirigida por DNA Idioma: En Revista: Nat Commun Ano de publicação: 2024 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: RNA / DNA Polimerase Dirigida por DNA Idioma: En Revista: Nat Commun Ano de publicação: 2024 Tipo de documento: Article