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

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

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

A series of 2-alkylamino-2'-deoxyadenosine triphosphates (dATP) was prepared and found to be substrates for the Therminator DNA polymerase, which incorporated only one modified nucleotide into the primer. Using a template encoding for two consecutive adenines, conditions were found for incorporation of either one or two modified nucleotides. In all cases, addition of a mixture of natural dNTPs led to primer extension resulting in site-specific single modification of DNA in the minor groove. The allylamino-substituted DNA was used for the thiol-ene addition, whereas the propargylamino-DNA for the CuAAC click reaction was used to label the DNA with a fluorescent dye in the minor groove. The approach was used to construct FRET probes for detection of oligonucleotides.

• Klíčová slova
• DNA, fluorescent probes, nucleotides, oligonucleotides, polymerases,
• MeSH
• alylové sloučeniny chemie   MeSH
• deoxyadeninnukleotidy chemie   MeSH
• DNA chemie   MeSH
• fluorescenční barviva chemie   MeSH
• konformace nukleové kyseliny MeSH
• oligonukleotidy analýza   MeSH
• pargylin analogy a deriváty   chemie   MeSH
• propylaminy chemie   MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 2'-deoxyadenosine triphosphate MeSH Prohlížeč
• alylové sloučeniny MeSH
• deoxyadeninnukleotidy MeSH
• DNA MeSH
• fluorescenční barviva MeSH
• oligonukleotidy MeSH
• pargylin MeSH
• propargylamine MeSH Prohlížeč
• propylaminy MeSH

2'-Deoxyadenosine triphosphate (dATP) derivatives bearing diverse substituents (Cl, NH2 , CH3 , vinyl, ethynyl, and phenyl) at position 2 were prepared and tested as substrates for DNA polymerases. The 2-phenyl-dATP was not a substrate for DNA polymerases, but the dATPs bearing smaller substituents were good substrates in primer-extension experiments, producing DNA substituted in the minor groove. The vinyl-modified DNA was applied in thiol-ene addition and the ethynyl-modified DNA was applied in a CuAAC click reaction to form DNA labelled with fluorescent dyes in the minor groove.

• Klíčová slova
• DNA modification, DNA polymerase, bioconjugation, fluorescent labelling, nucleotides,
• MeSH
• denaturace nukleových kyselin MeSH
• deoxyadeninnukleotidy chemie   metabolismus   MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• sekvence nukleotidů MeSH
• substrátová specifita MeSH
• termodynamika MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2'-deoxyadenosine triphosphate MeSH Prohlížeč
• deoxyadeninnukleotidy MeSH
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH

A nucleoside bearing a solvatochromic push-pull fluorene fluorophore (dCFL ) was designed and synthesized by the Sonogashira coupling of alkyne-linked fluorene 8 with 5-iodo-2'-deoxycytidine. The fluorene building block 8 and labeled nucleoside dCFL exerted bright fluorescence with significant solvatochromic effect providing emission maxima ranging from 421 to 544 nm and high quantum yields even in highly polar solvents, including water. The solvatochromism of 8 was studied by DFT and ADC(2) calculations to show that, depending on the polarity of the solvent, emission either from the planar or the twisted conformation of the excited state can occur. The nucleoside was converted to its triphosphate variant dCFLTP which was found to be a good substrate for DNA polymerases suitable for the enzymatic synthesis of oligonucleotide or DNA probes by primer extension or PCR. The fluorene-linked DNA can be used as fluorescent probes for DNA-protein (p53) or DNA-lipid interactions, exerting significant color changes visible even to the naked eye. They also appear to be suitable for time-dependent fluorescence shift studies on DNA, yielding information on DNA hydration and dynamics.

• Publikační typ
• časopisecké články MeSH

• MeSH
• biosenzitivní techniky přístrojové vybavení   metody   MeSH
• elektrochemické techniky přístrojové vybavení   metody   MeSH
• glykomika přístrojové vybavení   metody   MeSH
• glykoproteiny analýza   metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• proteiny analýza   metabolismus   MeSH
• sacharidové sekvence MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• glykoproteiny MeSH
• proteiny MeSH

New redox labelling of DNA by an azido group which can be chemically transformed to nitrophenyltriazole or silenced to phenyltriazole was developed and applied to the electrochemical detection of DNA-protein interactions. 5-(4-Azidophenyl)-2'-deoxycytidine and 7-(4-azidophenyl)-7-deaza-2'-deoxyadenosine nucleosides were prepared by aqueous-phase Suzuki cross-coupling and converted to nucleoside triphosphates (dNTPs) which served as substrates for incorporation into DNA by DNA polymerase. The azidophenyl-modified nucleotides and azidophenyl-modified DNA gave a strong signal in voltammetric studies, at -0.9 V, due to reduction of the azido function. The Cu-catalyzed click reaction of azidophenyl-modified nucleosides or azidophenyl-modified DNA with 4-nitrophenylacetylene gave nitrophenyl-substituted triazoles, exerting a reduction peak at -0.4 V under voltammetry, whereas the click reaction with phenylacetylene gave electrochemically silent phenyltriazoles. The transformation of the azidophenyl label to nitrophenyltriazole was used for electrochemical detection of DNA-protein interactions (p53 protein) since only those azidophenyl groups in the parts of the DNA not shielded by the bound p53 protein were transformed to nitrophenyltriazoles, whereas those covered by the protein were not.

• Publikační typ
• časopisecké články MeSH