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

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

• MeSH
• adenin chemie   metabolismus   MeSH
• aptamery nukleotidové chemická syntéza   genetika   MeSH
• cytosin chemie   metabolismus   MeSH
• deoxyribonukleosidy chemie   genetika   metabolismus   MeSH
• dinukleosidfosfáty chemie   genetika   metabolismus   MeSH
• DNA-dependentní DNA-polymerasy genetika   metabolismus   MeSH
• DNA chemie   genetika   metabolismus   MeSH
• guanin chemie   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• párování bází MeSH
• polymerázová řetězová reakce MeSH
• polymery chemická syntéza   metabolismus   MeSH
• replikace DNA * MeSH
• sekvence nukleotidů MeSH
• uracil chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenin MeSH
• aptamery nukleotidové MeSH
• cytosin MeSH
• deoxyribonukleosidy MeSH
• dinukleosidfosfáty MeSH
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• guanin MeSH
• polymery MeSH
• uracil MeSH

Squaramate-linked 2'-deoxycytidine 5'-O-triphosphate was synthesized and found to be good substrate for KOD XL DNA polymerase in primer extension or PCR synthesis of modified DNA. The resulting squaramate-linked DNA reacts with primary amines to form a stable diamide linkage. This reaction was used for bioconjugations of DNA with Cy5 and Lys-containing peptides. Squaramate-linked DNA formed covalent cross-links with histone proteins. This reactive nucleotide has potential for other bioconjugations of nucleic acids with amines, peptides or proteins without need of any external reagent.

• Klíčová slova
• DNA, DNA polymerase, bioconjugation, cross-linking reactions, proteins,
• MeSH
• DNA metabolismus   MeSH
• lidé MeSH
• lysin metabolismus   MeSH
• nukleotidy metabolismus   MeSH
• peptidy chemie   MeSH
• proteiny chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• lysin MeSH
• nukleotidy MeSH
• peptidy MeSH
• proteiny MeSH

We report proof of principle biomimetic switching of transcription in vitro through non-natural chemical reactions in the major groove of DNA templates. Photocaged DNA templates containing nitrobenzyl-protected 5-hydroxymethyluracil or - cytosine permitted no transcription with E. coli RNA polymerase (OFF state). Their irradiation with 400 nm light resulted in DNA templates containing hydroxymethylpyrimidines, which switched transcription ON with a higher yield (250-350%) compared to non-modified DNA. Phosphorylation of templates containing 5-hydroxymethyluracil (but not 5-hydroxymethylcytosine) then turned transcription OFF again. It is the first step towards artificial bioorthogonal chemical epigenetics.

• Publikační typ
• časopisecké články MeSH