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.

• Publication type
• Journal Article 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.

• Keywords
• Bioconjugations, Cross-Linking, Modified RNA, Proteins, RNA Polymerases,
• MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• DNA metabolism   MeSH
• Nucleotides * metabolism   MeSH
• RNA-Binding Proteins MeSH
• Cross-Linking Reagents MeSH
• RNA Probes MeSH
• RNA * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• chloroacetamide MeSH Browser
• DNA-Directed RNA Polymerases MeSH
• DNA MeSH
• Nucleotides * MeSH
• RNA-Binding Proteins MeSH
• Cross-Linking Reagents MeSH
• RNA Probes 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).

• Keywords
• DNA polymerases, bioconjugations, cross-linking reactions, nucleotides, proteins,
• MeSH
• Arginine chemistry   MeSH
• DNA chemical synthesis   chemistry   MeSH
• Histones chemistry   MeSH
• Ketones chemical synthesis   chemistry   MeSH
• Tumor Suppressor Protein p53 chemistry   MeSH
• Peptides chemistry   MeSH
• Proteins chemistry   MeSH
• Cross-Linking Reagents chemical synthesis   chemistry   MeSH
• Serum Albumin, Bovine chemistry   MeSH
• Cattle MeSH
• Thymine Nucleotides chemical synthesis   chemistry   MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Arginine MeSH
• DNA MeSH
• Histones MeSH
• Ketones MeSH
• Tumor Suppressor Protein p53 MeSH
• Peptides MeSH
• Proteins MeSH
• Cross-Linking Reagents MeSH
• Serum Albumin, Bovine MeSH
• Thymine Nucleotides MeSH