We designed and synthesized a set of six 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) bearing functional groups mimicking amino acid side chains in enzyme active sites (OH, SH, COOH, and imidazole) attached to position 5 of pyrimidines or position 7 of 7-deazapurines through different linkers. These modified dNTPs were studied as substrates in enzymatic synthesis of modified and hypermodified DNA using several DNA polymerases. In primer extension (PEX), all modified dNTPs provided DNA containing one, two, three, or, (all) four modified nucleotides each bearing a different modification, although the thiol-modified dNTPs were worse substrates compared to the others. In PCR, we observed exponential amplification for any combination of one, two, or three nonsulfur dNTPs but the thiol-modified dNTP did not work well in any combinations. Sequencing of the hypermodified DNA confirmed the good fidelity of the incorporation of all the modified nucleotides. This set of modified dNTPs extends the portfolio of building blocks for prospective use in selections of functional nucleic acids.

• Keywords
• DNA, enzymatic syntheses, nucleotides, polymerases,
• MeSH
• DNA-Directed DNA Polymerase * metabolism   chemistry   MeSH
• DNA * chemistry   chemical synthesis   MeSH
• Imidazoles * chemistry   MeSH
• Catalytic Domain MeSH
• Carboxylic Acids * chemistry   MeSH
• Polymerase Chain Reaction MeSH
• Sulfhydryl Compounds * chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 7-deazapurine MeSH Browser
• DNA-Directed DNA Polymerase * MeSH
• DNA * MeSH
• imidazole MeSH Browser
• Imidazoles * MeSH
• Carboxylic Acids * MeSH
• Purines MeSH
• Sulfhydryl Compounds * MeSH

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.

• Publication type
• Journal Article MeSH

We designed and synthesized a set of four 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) bearing cationic substituents (protonated amino, methylamino, dimethylamino and trimethylammonium groups) attached to position 5 of pyrimidines or position 7 of 7-deazapurines through hex-1-ynyl or propargyl linker. These cationic dNTPs were studied as substrates in enzymatic synthesis of modified and hypermodified DNA using KOD XL DNA polymerase. In primer extension (PEX), we successfully obtained DNA containing one, two, three, or (all) four modified nucleotides, each bearing a different cationic modification. The cationic dNTPs were somewhat worse substrates compared to previously studied dNTPs bearing hydrophobic or anionic modifications, but the polymerase was still able to synthesize sequences up to 73 modified nucleotides. We also successfully combined one cationic modification with one anionic and two hydrophobic modifications in PEX. In polymerase chain reaction (PCR), we observed exponential amplification only in the case of one cationic modification, while the combination of more cationic nucleotides gave either very low amplification or no PCR product. The hypermodified oligonucleotides prepared by PEX were successfully re-PCRed and sequenced by Sanger sequencing. Biophysical studies of hybridization, denaturation, and circular dichroism spectroscopy showed that the presence of cationic modifications increases the stability of duplexes.

• MeSH
• Deoxyribonucleotides * chemistry   chemical synthesis   MeSH
• DNA-Directed DNA Polymerase * metabolism   MeSH
• DNA * chemistry   biosynthesis   chemical synthesis   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Cations chemistry   MeSH
• Polymerase Chain Reaction MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Deoxyribonucleotides * MeSH
• DNA-Directed DNA Polymerase * MeSH
• DNA * MeSH
• Cations 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-Directed DNA Polymerase metabolism   MeSH
• DNA * chemistry   MeSH
• Nucleotides * chemistry   MeSH
• Pyrimidines MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA-Directed DNA Polymerase MeSH
• DNA * MeSH
• Nucleotides * MeSH
• Pyrimidines 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.

• Publication type
• Journal Article 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-Methylcytosine * MeSH
• Deoxyribonucleosides MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA metabolism   MeSH
• Epigenesis, Genetic MeSH
• Nucleotides metabolism   MeSH
• Pyrimidine Nucleotides * MeSH
• Pyrimidines MeSH
• DNA Restriction Enzymes metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5-Methylcytosine * MeSH
• Deoxyribonucleosides MeSH
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH
• Nucleotides MeSH
• Pyrimidine Nucleotides * MeSH
• Pyrimidines MeSH
• DNA Restriction Enzymes 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

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
• Adenine chemistry   metabolism   MeSH
• Aptamers, Nucleotide chemical synthesis   genetics   MeSH
• Cytosine chemistry   metabolism   MeSH
• Deoxyribonucleosides chemistry   genetics   metabolism   MeSH
• Dinucleoside Phosphates chemistry   genetics   metabolism   MeSH
• DNA-Directed DNA Polymerase genetics   metabolism   MeSH
• DNA chemistry   genetics   metabolism   MeSH
• Guanine chemistry   metabolism   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Base Pairing MeSH
• Polymerase Chain Reaction MeSH
• Polymers chemical synthesis   metabolism   MeSH
• DNA Replication * MeSH
• Base Sequence MeSH
• Uracil chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenine MeSH
• Aptamers, Nucleotide MeSH
• Cytosine MeSH
• Deoxyribonucleosides MeSH
• Dinucleoside Phosphates MeSH
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH
• Guanine MeSH
• Polymers 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.

• Keywords
• DNA, fluorescence, nucleotides, real-time PCR,
• MeSH
• COVID-19 * MeSH
• DNA Probes MeSH
• Humans MeSH
• Nucleotides MeSH
• DNA Replication * MeSH
• SARS-CoV-2 MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA Probes MeSH
• Nucleotides 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.

• Keywords
• DNA, DNA polymerase, bioconjugation, cross-linking reactions, proteins,
• MeSH
• DNA metabolism   MeSH
• Humans MeSH
• Lysine metabolism   MeSH
• Nucleotides metabolism   MeSH
• Peptides chemistry   MeSH
• Proteins chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Lysine MeSH
• Nucleotides MeSH
• Peptides MeSH
• Proteins MeSH