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Incorporation of NHC-TP during T7 RNA polymerase-mediated in vitro transcription (IVT) . ( A ) Chemical structures of Molnupiravir and <t>NHC-triphosphate.</t> ( B ) Scheme of the 346 bp DNA template encoding T7 promoter sequence and a 326 bp sequence from the 5′ LTR region of HIV-1 pNL4-3 plasmid for T7 polymerase-mediated IVT reactions. ( C ) RNA copy numbers after qRT-qPCR performed with extracted RNA products from IVT reactions after DNase treatment. The T7 polymerase-mediated IVT reactions were conducted in triplicates, and each experimental replicate is analyzed by qRT-PCR in two technical replicates. Mean with SD are represented with bars. ( D ) Agarose gel electrophoresis of one representative IVT products after DNase treatment. L: RNA ladder. IVT reactions were conducted with three different nucleotide pool compositions: (1) 3 rNTPs (ATP, GTP, and UTP) without CTP (negative control), (2) four rNTPs (ATP, GTP, UTP, and CTP: positive control), and (3) the 3rNTP (ATP, GTP and UTP) and NHC-TP. The nucleotide concentrations used were their cellular concentrations previously reported ( , ).
Nhc Triphosphate, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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nhc key laboratory  (Mianyang Habio Bioengineering Co Ltd)
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Incorporation of NHC-TP during T7 RNA polymerase-mediated in vitro transcription (IVT) . ( A ) Chemical structures of Molnupiravir and <t>NHC-triphosphate.</t> ( B ) Scheme of the 346 bp DNA template encoding T7 promoter sequence and a 326 bp sequence from the 5′ LTR region of HIV-1 pNL4-3 plasmid for T7 polymerase-mediated IVT reactions. ( C ) RNA copy numbers after qRT-qPCR performed with extracted RNA products from IVT reactions after DNase treatment. The T7 polymerase-mediated IVT reactions were conducted in triplicates, and each experimental replicate is analyzed by qRT-PCR in two technical replicates. Mean with SD are represented with bars. ( D ) Agarose gel electrophoresis of one representative IVT products after DNase treatment. L: RNA ladder. IVT reactions were conducted with three different nucleotide pool compositions: (1) 3 rNTPs (ATP, GTP, and UTP) without CTP (negative control), (2) four rNTPs (ATP, GTP, UTP, and CTP: positive control), and (3) the 3rNTP (ATP, GTP and UTP) and NHC-TP. The nucleotide concentrations used were their cellular concentrations previously reported ( , ).
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Incorporation of NHC-TP during T7 RNA polymerase-mediated in vitro transcription (IVT) . ( A ) Chemical structures of Molnupiravir and <t>NHC-triphosphate.</t> ( B ) Scheme of the 346 bp DNA template encoding T7 promoter sequence and a 326 bp sequence from the 5′ LTR region of HIV-1 pNL4-3 plasmid for T7 polymerase-mediated IVT reactions. ( C ) RNA copy numbers after qRT-qPCR performed with extracted RNA products from IVT reactions after DNase treatment. The T7 polymerase-mediated IVT reactions were conducted in triplicates, and each experimental replicate is analyzed by qRT-PCR in two technical replicates. Mean with SD are represented with bars. ( D ) Agarose gel electrophoresis of one representative IVT products after DNase treatment. L: RNA ladder. IVT reactions were conducted with three different nucleotide pool compositions: (1) 3 rNTPs (ATP, GTP, and UTP) without CTP (negative control), (2) four rNTPs (ATP, GTP, UTP, and CTP: positive control), and (3) the 3rNTP (ATP, GTP and UTP) and NHC-TP. The nucleotide concentrations used were their cellular concentrations previously reported ( , ).
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Incorporation of NHC-TP during T7 RNA polymerase-mediated in vitro transcription (IVT) . ( A ) Chemical structures of Molnupiravir and <t>NHC-triphosphate.</t> ( B ) Scheme of the 346 bp DNA template encoding T7 promoter sequence and a 326 bp sequence from the 5′ LTR region of HIV-1 pNL4-3 plasmid for T7 polymerase-mediated IVT reactions. ( C ) RNA copy numbers after qRT-qPCR performed with extracted RNA products from IVT reactions after DNase treatment. The T7 polymerase-mediated IVT reactions were conducted in triplicates, and each experimental replicate is analyzed by qRT-PCR in two technical replicates. Mean with SD are represented with bars. ( D ) Agarose gel electrophoresis of one representative IVT products after DNase treatment. L: RNA ladder. IVT reactions were conducted with three different nucleotide pool compositions: (1) 3 rNTPs (ATP, GTP, and UTP) without CTP (negative control), (2) four rNTPs (ATP, GTP, UTP, and CTP: positive control), and (3) the 3rNTP (ATP, GTP and UTP) and NHC-TP. The nucleotide concentrations used were their cellular concentrations previously reported ( , ).
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Incorporation of NHC-TP during T7 RNA polymerase-mediated in vitro transcription (IVT) . ( A ) Chemical structures of Molnupiravir and NHC-triphosphate. ( B ) Scheme of the 346 bp DNA template encoding T7 promoter sequence and a 326 bp sequence from the 5′ LTR region of HIV-1 pNL4-3 plasmid for T7 polymerase-mediated IVT reactions. ( C ) RNA copy numbers after qRT-qPCR performed with extracted RNA products from IVT reactions after DNase treatment. The T7 polymerase-mediated IVT reactions were conducted in triplicates, and each experimental replicate is analyzed by qRT-PCR in two technical replicates. Mean with SD are represented with bars. ( D ) Agarose gel electrophoresis of one representative IVT products after DNase treatment. L: RNA ladder. IVT reactions were conducted with three different nucleotide pool compositions: (1) 3 rNTPs (ATP, GTP, and UTP) without CTP (negative control), (2) four rNTPs (ATP, GTP, UTP, and CTP: positive control), and (3) the 3rNTP (ATP, GTP and UTP) and NHC-TP. The nucleotide concentrations used were their cellular concentrations previously reported ( , ).

Journal: The Journal of Biological Chemistry

Article Title: DNA-dependent RNA polymerase incorporates β-D-N4-hydroxycytidine (NHC) linking Molnupiravir to host transcription-dependent mutagenesis

doi: 10.1016/j.jbc.2026.111409

Figure Lengend Snippet: Incorporation of NHC-TP during T7 RNA polymerase-mediated in vitro transcription (IVT) . ( A ) Chemical structures of Molnupiravir and NHC-triphosphate. ( B ) Scheme of the 346 bp DNA template encoding T7 promoter sequence and a 326 bp sequence from the 5′ LTR region of HIV-1 pNL4-3 plasmid for T7 polymerase-mediated IVT reactions. ( C ) RNA copy numbers after qRT-qPCR performed with extracted RNA products from IVT reactions after DNase treatment. The T7 polymerase-mediated IVT reactions were conducted in triplicates, and each experimental replicate is analyzed by qRT-PCR in two technical replicates. Mean with SD are represented with bars. ( D ) Agarose gel electrophoresis of one representative IVT products after DNase treatment. L: RNA ladder. IVT reactions were conducted with three different nucleotide pool compositions: (1) 3 rNTPs (ATP, GTP, and UTP) without CTP (negative control), (2) four rNTPs (ATP, GTP, UTP, and CTP: positive control), and (3) the 3rNTP (ATP, GTP and UTP) and NHC-TP. The nucleotide concentrations used were their cellular concentrations previously reported ( , ).

Article Snippet: The physiological condition was tested with 66 μM CTP, 859 μM GTP, 2990 μM ATP, 460 μM UTP and/or 102.8 μM of NHC-triphosphate (MedChemExpress) as described above.

Techniques: In Vitro, Sequencing, Plasmid Preparation, Quantitative RT-PCR, Agarose Gel Electrophoresis, Negative Control, Positive Control