Protein-RNA interactions play important biological roles and hence reactive RNA probes for cross-linking with proteins are important tools in their identification and study. To this end, we designed and synthesized 5'-O-triphosphates bearing a reactive squaramate group attached to position 5 of cytidine or position 7 of 7-deazaadenosine and used them as substrates for polymerase synthesis of modified RNA. In vitro transcription with T7 RNA polymerase or primer extension using TGK polymerase was used for synthesis of squaramate-modified RNA probes which underwent covalent bioconjugations with amine-linked fluorophore and lysine-containing peptides and proteins including several viral RNA polymerases or HIV reverse transcriptase. Inhibition of RNA-depending RNA polymerases from Japanese Encephalitis virus was observed through formation of covalent cross-link which was partially identified by MS/MS analysis. Thus, the squaramate-linked NTP analogs are useful building blocks for the synthesis of reactive RNA probes for bioconjugations with primary amines and cross-linking with lysine residues.

• Publikační typ
• časopisecké články MeSH

Reactive RNA probes are useful for studying and identifying RNA-binding proteins. To that end, we designed and synthesized chloroacetamide-linked 7-deaza-ATP which was a good substrate for T7 RNA polymerase in in vitro transcription assay to synthesize reactive RNA probes bearing one or several reactive modifications. Modified RNA probes reacted with thiol-containing molecules as well as with cysteine- or histidine-containing peptides to form stable covalent products. They also reacted selectively with RNA-binding proteins to form cross-linked conjugates in high conversions thanks to proximity effect. Our modified nucleotide and RNA probes are promising tools for applications in RNA (bio)conjugations or RNA proteomics.

• Klíčová slova
• Bioconjugations, Cross-Linking, Modified RNA, Proteins, RNA Polymerases,
• MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• DNA metabolismus   MeSH
• nukleotidy * metabolismus   MeSH
• proteiny vázající RNA MeSH
• reagencia zkříženě vázaná MeSH
• RNA sondy MeSH
• RNA * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chloroacetamide MeSH Prohlížeč
• DNA řízené RNA-polymerasy MeSH
• DNA MeSH
• nukleotidy * MeSH
• proteiny vázající RNA MeSH
• reagencia zkříženě vázaná MeSH
• RNA sondy MeSH
• RNA * MeSH