A series of adenosine triphosphate (ATP) derivatives bearing chloro, fluoro, amino, methyl, vinyl, and ethynyl groups at position 2 are synthesized and tested as substrates for RNA and DNA polymerases. The modified nucleotides work well in in vitro transcription with T7 RNA polymerase and primer extension (PEX) using engineered DNA polymerases (TGK, 2M) except for the bulkier 2-vinyl- and 2-ethynyl-ATP derivatives that give truncated products. However, in single nucleotide incorporation followed by PEX, they still can be used for site-specific incorporation of reactive modifications into RNA that can be further used for postsynthetic labeling through thiol-ene or Cu-catalyzed alkyne-azide cycloadditions reactions. All modified ATPs work in polyadenylation catalyzed by poly(A) polymerase to form long 3'-polyA tails containing the modifications that also can be used for labeling.

• Keywords
• DNA polymerases, RNA polymerases, click reactions, nucleosides triphosphates, nucleotides, polyA polymerase, thiol‐ene addition,
• MeSH
• Adenosine Triphosphate * chemical synthesis   chemistry   analogs & derivatives   metabolism   MeSH
• Cycloaddition Reaction MeSH
• DNA-Directed RNA Polymerases * metabolism   chemistry   MeSH
• DNA-Directed DNA Polymerase * metabolism   chemistry   MeSH
• RNA * chemistry   metabolism   MeSH
• Viral Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphate * MeSH
• bacteriophage T7 RNA polymerase MeSH Browser
• DNA-Directed RNA Polymerases * MeSH
• DNA-Directed DNA Polymerase * MeSH
• RNA * MeSH
• Viral Proteins MeSH

Innovative approaches to controlled nucleobase-modified RNA synthesis are urgently needed to support RNA biology exploration and to synthesize potential RNA therapeutics. Here we present a strategy for enzymatic construction of nucleobase-modified RNA based on primer-dependent engineered thermophilic DNA polymerases - SFM4-3 and TGK. We demonstrate introduction of one or several different base-modified nucleotides in one strand including hypermodified RNA containing all four modified nucleotides bearing four different substituents, as well as strategy for primer segment removal. We also show facile site-specific or segmented introduction of fluorophores or other functional groups at defined positions in variety of RNA molecules, including structured or long mRNA. Intriguing translation efficacy of single-site modified mRNAs underscores the necessity to study isolated modifications placed at designer positions to disentangle their biological effects and enable development of improved mRNA therapeutics. Our toolbox paves the way for more precise dissecting RNA structures and functions, as well as for construction of diverse types of base-functionalized RNA for therapeutic applications and diagnostics.

• MeSH
• DNA-Directed DNA Polymerase * genetics   MeSH
• RNA, Messenger genetics   MeSH
• Nucleotides chemistry   MeSH
• RNA * genetics   chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA-Directed DNA Polymerase * MeSH
• RNA, Messenger MeSH
• Nucleotides MeSH
• RNA * 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

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