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Synthesis and polymerase incorporation of 5'-amino-2',5'-dideoxy-5'-N-triphosphate nucleotides.

ABSTRACT: Owing to the markedly increased reactivity of amino functional groups versus hydroxyls, the 5'-amino-5'-deoxy nucleoside and nucleotide analogs have proven widely useful in biological, pharmaceutical and genomic applications. However, synthetic procedures leading to these analogs have not been fully explored, which may possibly have limited the scope of their utility. Here we describe the synthesis of the 5'-amino-2',5'-dideoxy analogs of adenosine, cytidine, guanosine, inosine and uridine from their respective naturally occurring nucleosides via the reduction of 5'-azido-2',5'-dideoxy intermediates using the Staudinger reaction, and the high yield conversion of these modified nucleosides and 5'-amino-5'-deoxythymidine to the corresponding 5'-N-triphosphates through reaction with trisodium trimetaphosphate in the presence of tris(hydroxymethyl)aminomethane (Tris). We also show that each of these nucleotide analogs can be efficiently incorporated into DNA by the Klenow fragment of Escherichia coli DNA polymerase I when individually substituted for its naturally occurring counterpart. Mild acid treatment of the resulting DNA generates polynucleotide fragments that arise from specific cleavage at each modified nucleotide, providing a sequence ladder for each base. Because the ladders are generated after the extension, the corresponding products may be manipulated by enzymatic and/or purification processes. The potential utility of this extension-cleavage procedure in genomic sequence analysis is discussed.

SUBMITTER: Wolfe JL

PROVIDER: S-EPMC137424 | biostudies-literature | 2002 Sep

REPOSITORIES: biostudies-literature

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Synthesis and polymerase incorporation of 5'-amino-2',5'-dideoxy-5'-N-triphosphate nucleotides.

Wolfe Jia Liu JL Kawate Tomohiko T Belenky Alexei A Stanton Vincent V

Nucleic acids research 20020901 17

Owing to the markedly increased reactivity of amino functional groups versus hydroxyls, the 5'-amino-5'-deoxy nucleoside and nucleotide analogs have proven widely useful in biological, pharmaceutical and genomic applications. However, synthetic procedures leading to these analogs have not been fully explored, which may possibly have limited the scope of their utility. Here we describe the synthesis of the 5'-amino-2',5'-dideoxy analogs of adenosine, cytidine, guanosine, inosine and uridine from ...[more]

PMID: 12202759

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Project description:The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote the synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN-RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and the design of its inhibitors. IMPORTANCE In vitro interrogations of the central replicative complex of SARS-CoV-2, RNA-dependent RNA polymerase (RdRp), by structural, biochemical, and biophysical methods yielded an unprecedented windfall of information that, in turn, instructs drug development and administration, genomic surveillance, and other aspects of the evolving pandemic response. They also illuminated the vast disparity in the methods used to produce RdRp for experimental work and the hidden impact that this has on enzyme activity and research outcomes. In this report, we elucidate the positive and negative effects of codon optimization on the activity and folding of the recombinant RdRp and detail the design of a highly sensitive in vitro assay of RdRp-dependent RNA synthesis. Using this assay, we demonstrate that RdRp is allosterically activated by nontemplating phosphorylated nucleotides, including naturally occurring alarmone ppGpp and synthetic remdesivir triphosphate.

Dataset Information

Synthesis and polymerase incorporation of 5'-amino-2',5'-dideoxy-5'-N-triphosphate nucleotides.

Publications

Synthesis and polymerase incorporation of 5'-amino-2',5'-dideoxy-5'-N-triphosphate nucleotides.

Similar Datasets

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