Effect of 8-oxo-1,N6-ethenoadenine derivatives on the activity of RNA polymerases of the SARS-CoV-2 virus and Escherichia coli

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Abstract

Bacterial and viral RNA polymerases are promising targets for the development of new transcription inhibitors. One of the potential blockers of RNA synthesis is 7,8-dihydro-8-oxo-1,N6-ethenoadenine (oxo-εA), a synthetic compound that is a combination of two modifications of adenine: 8-oxoadenine and 1,N6-ethenoadenine. In this study we synthesized oxo-εA triphosphate (oxo-εATP) and showed that it could be incorporated by RNA-dependent RNA polymerase of the SARS-CoV-2 virus into the synthesized RNA opposite template residues A and G in the presence of Mn2+ ions. In the case of Escherichia coli RNA polymerase, the incorporation occurred opposite A residues in the template DNA strand. If oxo-εA was present instead of adenine in the template DNA strand, transcription was completely stopped at the site of modification. At the same time, oxo-εATP did not suppress RNA synthesis by both RNA polymerases in the presence of unmodified nucleotides. Thus, oxo-εA modification significantly disrupts the template properties of the nucleotide during RNA synthesis by RNA polymerases of different classes, and the corresponding nucleotide derivatives are not potential antiviral or antibacterial transcription inhibitors.

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About the authors

I. V. Petushkov

National Research Centre “Kurchatov Institute”; Institute of Gene Biology, Russian Academy of Sciences

Author for correspondence.
Email: telomer1@rambler.ru
Russian Federation, 123182 Moscow; 119334 Moscow

A. V. Aralov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; RUDN University

Email: telomer1@rambler.ru
Russian Federation, 117997 Moscow; 117198 Moscow

I. A. Ivanov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; LLC “Organicum”

Email: telomer1@rambler.ru
Russian Federation, 117997 Moscow; 127486 Moscow

M. S. Baranov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; Pirogov Russian National Research Medical University

Email: telomer1@rambler.ru
Russian Federation, 117997 Moscow; 117997 Moscow

T. S. Zatsepin

Lomonosov Moscow State University

Email: telomer1@rambler.ru

Faculty of Chemistry

Russian Federation, 119991 Moscow

A. V. Kulbachinskiy

National Research Centre “Kurchatov Institute”; Institute of Gene Biology, Russian Academy of Sciences

Email: avkulb@yandex.ru
Russian Federation, 123182 Moscow; 119334 Moscow

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Supplementary files

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2. Fig. 1. Structure of the oxo-εA modification (a), chemical synthesis of oxo-εATP (b) and putative pairs oxo-εA:A, oxo-εA:G and oxo-A:G (c)

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3. Fig. 2. Analysis of oxo-εATP incorporation of SARS-CoV-2 RdRp. a – Schematic of the RNA substrate used in the experiments. The RNA primer is shown in red, the RNA template is shown in black, the first template base is marked “+1”. The residues incorporated by RdRp upon primer extension are shown in gray (the 7 residues incorporated upon addition of a limited set of NTPs are shown), the direction of transcription is indicated by an arrow. b – Analysis of RdRp transcription products in the presence of different sets of NTPs and oxo-εATP upon addition of Mg2+ (left panel) and Mn2+ (right panel) ions. Control samples incubated without NTPs are marked with a “−” sign. The length of RNA products in nucleotides is indicated on the right. Electropherogram of transcription products in 15% PAGE under denaturing conditions

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4. Fig. 3. Incorporation of oxo-εATP RdRp onto RNA substrates with a variable template base at the +1 position. a – Schematic of the RNA substrates used in the experiments. The RNA primer is shown in red, the RNA template is shown in black. The variable template base at the +1 position (X) is highlighted in yellow, the residue inserted opposite it (Y) and the following three residues are shown in gray. b – Analysis of RdRp transcription products in the presence of natural NTP and/or oxo-εATP upon addition of Mg2+ ions. The control sample, which was incubated without NTP, is marked with the “−” sign. Reactions were carried out with RNA templates containing residues A, G, C or U at the +1 position. c – Analysis of RdRp transcription products in the presence of natural NTP and/or oxo-εATP with the addition of Mn2+ ions by analogy with panel b. Electropherograms of transcription products in 15% PAGE under denaturing conditions. The length of RNA products in nucleotides is indicated on the right

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5. Fig. 4. Analysis of oxo-εATP incorporation of E. coli RNAP. a – Schematic of the elongation complex used in the experiments. The RNA oligonucleotide is shown in red, the template DNA strand is shown in black, and the non-template strand is shown in blue. The start point of nucleotide residue incorporation (+1) is shown, the first 4 incorporated nucleotide residues are marked in gray, and the direction of transcription is indicated by an arrow. b – Analysis of transcription products in the presence of different sets of NTP and oxo-εATP upon addition of Mg2+ (left panel) and Mn2+ (right panel) ions. Control samples incubated without NTP are marked with a “−” sign. Electropherogram of transcription products in 15% PAGE under denaturing conditions. The length of RNA products in nucleotides is marked on the right.

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6. Fig. 5. Analysis of the passage of oxo-εA residue in the template DNA strand by E. coli RNAP. a – Schematic diagram of the elongation complex used in the experiments. The RNA oligonucleotide is shown in red, the template strand in black, and the non-template strand in blue. The position of oxo-εA or control dA is marked with the letter X and highlighted in yellow. The start point of nucleotide inclusion (+1) is shown, the first 4 incorporated nucleotide residues are marked in gray, and the letter Y indicates the residue incorporated opposite to oxo-εA or dA. b – Analysis of transcription products upon RNAP passage through oxo-εA at the +2 position of the template strand (left panel) or through the control dA (right panel). The control sample, which was incubated without NTP, is marked with a “−” sign. Electropherogram of transcription products in 15% PAAG under denaturing conditions. The length of RNA products in nucleotides is indicated on the right

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7. Appendix
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