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Preparation of RNAs with non-canonical 5′ ends using novel di- and trinucleotide reagents for co-transcriptional capping

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cris.lastimport.scopus2024-02-12T19:47:43Z
dc.abstract.enMany eukaryotic and some bacterial RNAs are modified at the 5′ end by the addition of cap structures. In addition to the classic 7-methylguanosine 5′ cap in eukaryotic mRNA, several non-canonical caps have recently been identified, including NAD-linked, FAD-linked, and UDP-glucose-linked RNAs. However, studies of the biochemical properties of these caps are impaired by the limited access to in vitro transcribed RNA probes of high quality, as the typical capping efficiencies with NAD or FAD dinucleotides achieved in the presence of T7 polymerase rarely exceed 50%, and pyrimidine derivatives are not incorporated because of promoter sequence limitations. To address this issue, we developed a series of di- and trinucleotide capping reagents and in vitro transcription conditions to provide straightforward access to unconventionally capped RNAs with improved 5′-end homogeneity. We show that because of the transcription start site flexibility of T7 polymerase, R1ppApG-type structures (where R1 is either nicotinamide riboside or riboflavin) are efficiently incorporated into RNA during transcription from dsDNA templates containing both φ 6.5 and φ 2.5 promoters and enable high capping efficiencies (∼90%). Moreover, uridine-initiated RNAs are accessible by transcription from templates containing the φ 6.5 promoter performed in the presence of R2ppUpG-type initiating nucleotides (where R2 is a sugar or phosphate moiety). We successfully employed this strategy to obtain several nucleotide-sugar-capped and uncapped RNAs. The capping reagents developed herein provide easy access to chemical probes to elucidate the biological roles of non-canonical RNA 5′ capping.
dc.affiliationUniwersytet Warszawski
dc.contributor.authorKowalska, Joanna
dc.contributor.authorJemielity, Jacek
dc.contributor.authorKiledjian, Megerditch
dc.contributor.authorFedorczyk, Bartlomiej
dc.contributor.authorGrudzien-Nogalska, Ewa
dc.contributor.authorDepaix, Anais
dc.date.accessioned2024-01-25T17:33:50Z
dc.date.available2024-01-25T17:33:50Z
dc.date.issued2022
dc.description.financeŚrodki finansowe, o których mowa w art. 365 pkt. 2 ustawy
dc.description.volume9
dc.identifier.doi10.3389/FMOLB.2022.854170
dc.identifier.urihttps://repozytorium.uw.edu.pl//handle/item/116965
dc.identifier.weblinkhttps://www.frontiersin.org/articles/10.3389/fmolb.2022.854170/full
dc.languageeng
dc.pbn.affiliationchemical sciences
dc.relation.ispartofFrontiers in Molecular Biosciences
dc.rightsClosedAccess
dc.sciencecloudnosend
dc.subject.enFAD
dc.subject.enIn vitro transcription
dc.subject.enNAD
dc.subject.enRNA cap
dc.subject.enUDP-glucose
dc.subject.endinucleotide
dc.subject.entrinucleotide
dc.titlePreparation of RNAs with non-canonical 5′ ends using novel di- and trinucleotide reagents for co-transcriptional capping
dc.typeJournalArticle
dspace.entity.typePublication