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-dependentní DNA-polymerasy * genetika   MeSH
• messenger RNA genetika   MeSH
• nukleotidy chemie   MeSH
• RNA * genetika   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA-dependentní DNA-polymerasy * MeSH
• messenger RNA MeSH
• nukleotidy 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.

• 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

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