A series of adenosine triphosphate (ATP) derivatives bearing chloro, fluoro, amino, methyl, vinyl, and ethynyl groups at position 2 are synthesized and tested as substrates for RNA and DNA polymerases. The modified nucleotides work well in in vitro transcription with T7 RNA polymerase and primer extension (PEX) using engineered DNA polymerases (TGK, 2M) except for the bulkier 2-vinyl- and 2-ethynyl-ATP derivatives that give truncated products. However, in single nucleotide incorporation followed by PEX, they still can be used for site-specific incorporation of reactive modifications into RNA that can be further used for postsynthetic labeling through thiol-ene or Cu-catalyzed alkyne-azide cycloadditions reactions. All modified ATPs work in polyadenylation catalyzed by poly(A) polymerase to form long 3'-polyA tails containing the modifications that also can be used for labeling.

• Klíčová slova
• DNA polymerases, RNA polymerases, click reactions, nucleosides triphosphates, nucleotides, polyA polymerase, thiol‐ene addition,
• MeSH
• adenosintrifosfát * chemická syntéza   chemie   analogy a deriváty   metabolismus   MeSH
• cykloadiční reakce MeSH
• DNA řízené RNA-polymerasy * metabolismus   chemie   MeSH
• DNA-dependentní DNA-polymerasy * metabolismus   chemie   MeSH
• RNA * chemie   metabolismus   MeSH
• virové proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát * MeSH
• bacteriophage T7 RNA polymerase MeSH Prohlížeč
• DNA řízené RNA-polymerasy * MeSH
• DNA-dependentní DNA-polymerasy * MeSH
• RNA * MeSH
• virové proteiny 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

Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).

• Klíčová slova
• DNA polymerases, bioconjugations, cross-linking reactions, nucleotides, proteins,
• MeSH
• arginin chemie   MeSH
• DNA chemická syntéza   chemie   MeSH
• histony chemie   MeSH
• ketony chemická syntéza   chemie   MeSH
• nádorový supresorový protein p53 chemie   MeSH
• peptidy chemie   MeSH
• proteiny chemie   MeSH
• reagencia zkříženě vázaná chemická syntéza   chemie   MeSH
• sérový albumin hovězí chemie   MeSH
• skot MeSH
• thiminnukleotidy chemická syntéza   chemie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• arginin MeSH
• DNA MeSH
• histony MeSH
• ketony MeSH
• nádorový supresorový protein p53 MeSH
• peptidy MeSH
• proteiny MeSH
• reagencia zkříženě vázaná MeSH
• sérový albumin hovězí MeSH
• thiminnukleotidy MeSH