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

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.

• Keywords
• DNA, fluorescent probes, nucleotides, oligonucleotides, polymerases,
• MeSH
• Allyl Compounds chemistry   MeSH
• Deoxyadenine Nucleotides chemistry   MeSH
• DNA chemistry   MeSH
• Fluorescent Dyes chemistry   MeSH
• Nucleic Acid Conformation MeSH
• Oligonucleotides analysis   MeSH
• Pargyline analogs & derivatives   chemistry   MeSH
• Propylamines chemistry   MeSH
• Fluorescence Resonance Energy Transfer methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 2'-deoxyadenosine triphosphate MeSH Browser
• Allyl Compounds MeSH
• Deoxyadenine Nucleotides MeSH
• DNA MeSH
• Fluorescent Dyes MeSH
• Oligonucleotides MeSH
• Pargyline MeSH
• propargylamine MeSH Browser
• Propylamines 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.

• Keywords
• DNA modification, DNA polymerase, bioconjugation, fluorescent labelling, nucleotides,
• MeSH
• Nucleic Acid Denaturation MeSH
• Deoxyadenine Nucleotides chemistry   metabolism   MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Base Sequence MeSH
• Substrate Specificity MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2'-deoxyadenosine triphosphate MeSH Browser
• Deoxyadenine Nucleotides MeSH
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH