Nejvíce citovaný článek - PubMed ID 31181911
Enzymatic Synthesis of Base-Functionalized Nucleic Acids for Sensing, Cross-linking, and Modulation of Protein-DNA Binding and Transcription
The LutR protein represses the transcription of genes encoding enzymes for the utilization of l-lactate in Bacillus subtilis through binding to a specific DNA region. In this study, we employed oligonucleotide probes modified by viscosity-sensitive tetramethylated thiophene-BODIPY fluorophores to investigate the impact of selected metabolites on the LutR-DNA complex. Our goal was to identify the effector molecule whose binding alters the protein-DNA affinity, thereby enabling gene transcription. The designed DNA probes exhibited distinctive responses to the binding and release of the protein, characterized by significant alterations in fluorescence lifetime. Through this method, we have identified l-lactate as the sole metabolite exerting a substantial modulating effect on the protein-DNA interaction and thus confirmed its role as an effector molecule. Moreover, we showed that our approach was able to follow conformation changes affecting affinity, which were not captured by other methods commonly used to study the protein-DNA interaction, such as electro-mobility shift assays and florescence anisotropy binding studies. This work underlines the potential of environment-sensitive fluorophore-linked nucleotide modifications, i.e. dCTBdp, for studying the dynamics and subtle changes of protein-DNA interactions.
- Publikační typ
- časopisecké články MeSH
We designed and synthesized a set of four 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) derived from 5-substituted pyrimidines and 7-substituted 7-deazapurines bearing anionic substituents (carboxylate, sulfonate, phosphonate, and phosphate). The anion-linked dNTPs were used for enzymatic synthesis of modified and hypermodified DNA using KOD XL DNA polymerase containing one, two, three, or four modified nucleotides. The polymerase was able to synthesize even long sequences of >100 modified nucleotides in a row by primer extension (PEX). We also successfully combined two anionic and two hydrophobic dNTPs bearing phenyl and indole moieties. In PCR, the combinations of one or two modified dNTPs gave exponential amplification, while most of the combinations of three or four modified dNTPs gave only linear amplification in asymmetric PCR. The hypermodified ONs were successfully re-PCRed and sequenced by Sanger sequencing. Biophysical studies including hybridization, denaturation, CD spectroscopy and molecular modelling and dynamics suggest that the presence of anionic modifications in one strand decreases the stability of duplexes while still preserving the B-DNA conformation, whilst the DNA hypermodified in both strands adopts a different secondary structure.
- MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- DNA * chemie MeSH
- nukleotidy * chemie MeSH
- pyrimidiny MeSH
- sekvence nukleotidů MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA-dependentní DNA-polymerasy MeSH
- DNA * MeSH
- nukleotidy * MeSH
- pyrimidiny MeSH
Nucleic acids aptamers often fail to efficiently target some proteins because of the hydrophilic character of the natural nucleotides. Here we present hydrophobic 7-phenylbutyl-7-deaadenine-modified DNA aptamers against the Heat Shock Protein 70 that were selected via PEX and magnetic bead-based SELEX. After 9 rounds of selection, the pool was sequenced and a number of candidates were identified. Following initial screening, two modified aptamers were chemically synthesised in-house and their binding affinity analysed by two methods, bio-layer interferometry and fluorescent-plate-based binding assay. The binding affinities of the modified aptamers were compared with that of their natural counterparts. The resulting modified aptamers bound with higher affinity (low nanomolar range) to the Hsp70 than their natural sequence (>5 µM) and hence have potential for applications and further development towards Hsp70 diagnostics or even therapeutics.
- Publikační typ
- časopisecké články MeSH
Five 2'-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorporation of these substrates was evaluated by a restriction endonucleases cleavage-based assay and by a kinetic study of single nucleotide extension. All of the modified pyrimidine dNTPs were good substrates for the studied DNA polymerases that incorporated a significant percentage of the modified nucleotides into DNA even in the presence of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2'-deoxyuridine triphosphate was not the best substrate for SPO1 DNA polymerase, which naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage. The results shed light onto the possibility of gene silencing through recycling and random incorporation of epigenetic nucleotides and into the replication of modified bacteriophage genomes.
- MeSH
- 5-methylcytosin * MeSH
- deoxyribonukleosidy MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- DNA metabolismus MeSH
- epigeneze genetická MeSH
- nukleotidy metabolismus MeSH
- pyrimidinové nukleotidy * MeSH
- pyrimidiny MeSH
- restrikční enzymy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 5-methylcytosin * MeSH
- deoxyribonukleosidy MeSH
- DNA-dependentní DNA-polymerasy MeSH
- DNA MeSH
- nukleotidy MeSH
- pyrimidinové nukleotidy * MeSH
- pyrimidiny MeSH
- restrikční enzymy 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
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
Thymidine triphosphate bearing benzylidene-tetrahydroxanthylium near-IR fluorophore linked to the 5-methyl group via triazole was synthesized through the CuAAC reaction and was used for polymerase synthesis of labelled DNA probes. The fluorophore lights up upon incorporation to DNA (up to 348-times) presumably due to interactions in major groove and the fluorescence further increases in the single-stranded oligonucleotide. The labelled dsDNA senses binding of small molecules and proteins by a strong decrease of fluorescence. The nucleotide was used as a light-up building block in real-time PCR for detection of SARS-CoV-2 virus.
- Klíčová slova
- DNA, fluorescence, nucleotides, real-time PCR,
- MeSH
- COVID-19 * MeSH
- DNA sondy MeSH
- lidé MeSH
- nukleotidy MeSH
- replikace DNA * MeSH
- SARS-CoV-2 MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA sondy MeSH
- nukleotidy 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
