Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, Nos. 12-13, pp. 2263-2273 © The Author(s) 2024. This article is an open access publication.

2263

Effect of 8-Oxo-1,N

-Ethenoadenine Derivatives

on the Activity of RNA Polymerases

from SARS-CoV-2 and Escherichia coli

Ivan V. Petushkov

1,2,a

, Andrey V. Aralov

3,4#

, Igor A. Ivanov

3,5

, Mikhail S. Baranov

3,6

Timofey S. Zatsepin

, and Andrey V. Kulbachinskiy

1,2,b

National Research Centre “Kurchatov Institute”, 123182 Moscow, Russia

Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

RUDN University, 117198 Moscow, Russia

Organicum LLC, 127486 Moscow, Russia

Pirogov Russian National Research Medical University, 117997 Moscow, Russia

Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia

e-mail: telomer1@rambler.ru

e-mail: avkulb@yandex.ru

Received October 26, 2024

Revised November 29, 2024

Accepted December 1, 2024

Abstract— Bacterial and viral RNA polymerases are promising targets for the development of new transcrip-

tion inhibitors. One of the potential blockers of RNA synthesis is 7,8-dihydro-8-oxo-1,N

-ethenoadenine (oxo-

εA), a synthetic compound that combines two adenine modifications: 8-oxoadenine and 1,N

-ethenoadenine.

In this study, we synthesized oxo-εA triphosphate (oxo-εATP) and showed that it could be incorporated by the

RNA-dependent RNA polymerase of SARS-CoV-2 into synthesized RNA opposite template residues A and G in the

presence of Mn

ions. Escherichia coli RNA polymerase incorporated oxo-εATP opposite A residues in the tem-

plate DNA strand. The presence of oxo-εA instead of adenine in the template DNA strand completely stopped

transcription at the modified nucleotide. At the same time, oxo-εATP did not suppress RNA synthesis by both

RNA polymerases in the presence of unmodified nucleotides. Therefore, the oxo-εA modification significantly

disrupts nucleotide base pairing during RNA synthesis by RNA polymerases of different classes, and the corre-

sponding nucleotide derivatives cannot be used as potential antiviral or antibacterial transcription inhibitors.

DOI: 10.1134/S0006297924120149

Keywords: modified nucleobases, RNA polymerase, transcription, transcription inhibitors, SARS-CoV-2

INTRODUCTION

Derivatives of natural nitrogenous bases, nu-

cleosides, nucleotides, and nucleic acids are widely

used in both fundamental research and development

of therapeutic agents, since modifications can lead

to the emergence of novel properties of these com-

pounds compared to their natural analogues. The re-

cent pandemic caused by the RNA virus SARS-CoV-2

has stimulated the search for new nucleoside inhibi-

tors of viral RNA-dependent RNA polymerase (RdRp)

and demonstrated the need for the development of

effective drugs in the event of emergence of resistant

strains and/or new viruses. One possible candidate

inhibitor of RdRp is 7,8-dihydro-8-oxo-1,N

-ethenoade-

nine (oxo-εA), which combines two adenine modifica-

Abbreviations: oxo-εATP, 7,8-dihydro-8-oxo-1,N

-ethenoadenosine triphosphate; oxo-εA, 7,8-dihydro-8-oxo-1,N

-ethenoade-

nine; RdRp, RNA-dependent RNA polymerase of SARS-CoV-2; RNAP, DNA-dependent RNA polymerase of Escherichia coli.

* To whom correspondence should be addressed.

These authors contributed equally to this study.

PETUSHKOV et al.2264

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

Fig. 1. Structure of oxo-εA (a), chemical synthesis of oxo-εATP(b), and geometry of oxo-εA:A, oxo-εA:G, and oxo-A:G pairs(c).

tions: 7,8-dihydro-8-oxoadenine (oxo-A) and 1,N

-ethe-

noadenine (εA) (Fig.1a). It was previously shown that

DNA containing oxo-εA can be replicated by cellular

DNA polymerases that incorporate A residues oppo-

site oxo-εA [1]. The nitrogenous base of oxo-εA in the

DNA duplex is predominantly in the syn conformation,

forming a pair with adenine that is similar in its ge-

ometry to the AT pair [1]. This allows one to expect

that oxo-εA triphosphate can potentially be incorpo-

rated into RNA.

The genome of SARS-CoV-2 is a single-stranded

RNA molecule of 29.9 thousand nucleotides, which

is replicated and transcribed by RdRp. This enzyme

has a complex structure [2] and includes the catalytic

subunit nsp12 and two auxiliary subunits (nsp7 and

nsp8). The active site of RdRp is located in nsp12 and

contains two aspartate residues, D760 and D761, in-

volved in the binding of two divalent metal ions that

play a key role in catalysis [3]. There is evidence that

SARS-CoV-2 RdRp is one of the fastest viral polymeras-

es (incorporation rate, 600 nt/s) [4], with a high error

rate (up to 10

−1

-10

−3

misincorporations per nucleotide)

[5]. Such a high error rate requires the presence of

a proofreading activity, which is carried out by the

3′-5′ exoribonuclease nsp14 regulated by the viral

protein nsp10 [6]. On the other hand, the high error

rate contributes to the adaptation of viruses under

selection conditions [7-9]. Despite a relatively low ac-

curacy, RdRp is sensitive to the structure of nucleo-

tide substrates. Thus, it strictly requires the presence

of a 2′-OH group at the 3′-ends of both RNA primer

and nucleotide substrate [10, 11]. At the same time,

RdRp is capable of incorporating some phosphorylat-

ed synthetic nucleosides into the growing RNA strand,

which has allowed the development of several nucle-

oside drugs (sofosbuvir [12,  13], remdesivir [14,  15],

and molnupiravir [16]). The incorporation of modified

residues in the synthesized RNA can block its further

elongation [13,  17,  18], while their presence in the

RNA template can inhibit subsequent synthesis of the

complementary RNA or increase the error rate [16,

19-21]. It was also shown that some natural modifi-

cations of the RNA template, such as N

-methylade-

nosine, N

-methyluridine, and 2′-O-methylguanosine,

strongly block RNA extension by RdRp [11, 22].

Unlike most viral RNA-dependent RNA polymeras-

es, bacterial, archaeal, and eukaryotic DNA-dependent

RNA polymerases (RNAPs) are multisubunit proteins

[23]. They also require Me

ions for catalysis [24, 25].

RNAPs have a lower error rate (10

−3

-10

−5

) in compar-

ison with coronaviral RdRp [26, 27] but are also able

to incorporate some modified nucleotide analogues

[28-30]. Furthermore, RNAPs sense the presence of

modified nucleotides in the DNA template. Depending

on the nature of modification, RNAPs can incorporate

an incorrect residue into the growing RNA chain op-

posite a lesion or can stop at the site of lesion, which

makes them important sensors of DNA damage [31].

The presence of oxo-A in the template DNA strand is

a serious obstacle for archaeal RNAP [32], while the

presence of εA blocks the activity of bacterial RNAP

[33]. However, it has been unknown how a combina-

tion of these two modifications can affect the activity

of RNAP.

The purpose of this study was to analyze the effect

of the oxo-εA modification in nucleotide substrates or

in the template strand on the RNA synthesis by vi-

ral and prokaryotic RNA polymerases. We aimed to

(i)establish whether RdRp is capable of incorporating

8-oxo-1,N

-ethenoadenosine triphosphate (oxo-εATP)

into the growing RNA opposite various template nu-

cleotides, and whether the incorporation of the oxo-εA

EFFECT OF 8-OXO-EA ON RNA POLYMERASES 2265

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

residue inhibits further RNA synthesis and (ii) test

whether Escherichia coli RNAP can use oxo-εATP as a

substrate, as well as to investigate the effect of oxo-εA

in the template DNA on the RNA synthesis by RNAP.

MATERIALS AND METHODS

Equipment and reagents for chemical synthe-

sis. All reagents were purchased from Sigma-Aldrich

(USA). Solvents were purchased from CHIMMED (Rus-

sia).

C, and

P NMR spectra were recorded with

a Bruker Avance III 600 spectrometer (Germany) at

600, 150, and 243 MHz, respectively. The multiplicity

of signals in the spectra was indicated using the fol-

lowing abbreviations: s (singlet), d (doublet), and m

(multiplet). The spin–spin coupling constants (J) are

given in Hz. Ion-exchange chromatography was per-

formed with an Akta Explorer 100 instrument (Cytiva,

Sweden).

Synthesis and purification of the oxo-εATP

disodium salt. Freshly distilled trimethyl phos-

phate [(CH

PO] (8.0  mL) and tributylamine (Bu

(0.95 mL) were added to oxo-εA ribonucleoside [34]

(0.62  g, 2.0  mmol) in a Schlenk flask (100  mL) un-

der inert gas atmosphere. The mixture was vigor-

ously stirred at room temperature for 30 min and

then cooled to −10°C. Phosphorus oxychloride (POCl

)

(0.33  mL, 3.6  mmol) was added under inert gas atmo-

sphere and the mixture was stirred at −10°C for 1  h.

Next, a phosphorylating mixture obtained by vigor-

ously stirring acetonitrile CH

CN (20ml), Bu

N (2.8  ml,

11.8  mmol) and bis(tributylammonium) pyrophos-

phate [(NHBu

)

] (1.2  g, 2.2  mmol) under inert

atmosphere for 20  min at −20°C, was added to the

sample. After stirring the sample for 1  h at −10°C, cold

water (65  ml) was added to the reaction mixture; the

mixture was stirred for 1  h at 0°C, then transferred

to a separatory funnel, and washed with methylene

chloride (15  mL, 5  times). The aqueous layer was col-

lected and aqueous ammonia solution was added to

pH  7.0. The resulting oxo-εATP solution was stored

in a refrigerator until purification by ion-exchange

chromatography on a 50×250  mm column packed

with HEMA-BIO 1000 DEAE 70  μm sorbent (Germa-

ny) with a gradient of 50-600  mM triethylammonium

bicarbonate (pH  7.6). Fractions containing the target

product were evaporated; the residue was re-dissolved

in water and evaporated to remove residual buffer.

The resulting product was converted to sodium salt by

re-precipitation from aqueous solution with a tenfold

volume of 3% sodium perchlorate (NaClO

) solution

in acetone. The precipitate was washed with dry ac-

etone and dried under vacuum. The yield was 0.52  g

(0.86  mmol, 43%).

H  NMR (600  MHz, D

O): δ  9.10

(s,  1H), 8.02 (d,  J  = 1.2  Hz, 1H), 7.63 (d,  J  =  1.2  Hz, 1H),

6.06 (d,  J  =  5.6 Hz, 1H), 5.36 (dd,  J  =  5.6  Hz, J  =  5.7

Hz, 1H), 4.80-4.75 (m, 1H), 4.40-4.35 (m,  2H), 4.32-4.26

(m,  1H).

C  NMR (150  MHz, D

O): δ  154.1, 135.8, 135.4,

133.8, 132.9, 112.2, 108.4, 86.1, 82.9 (d,  J  =  8.4 Hz, 1C),

70.5, 69.9, 65.3 (d,  J  =  5.2 Hz, 1C).

P  NMR (243  MHz,

O): δ  −6.10 (d,  J  = 19.7  Hz, 1P), −10.79  (d,  J  =  18.8  Hz,

1P), −21.65 (dd,  J  =  19.7 Hz, J  =  18.8  Hz, 1P) (see Online

Resource 1 for NMR spectra).

Synthesis of DNA oligonucleotides with oxo-εA

2′-deoxyribonucleotide. Synthesis of oxo-εA 2′-deoxy-

ribonucleotide 3′-phosphoramidite and modified DNA

oligonucleotides was performed as described previ-

ously [1]. Briefly, modified DNA oligonucleotides were

prepared using the solid-phase phosphoramidite meth-

od in a MerMade 12 synthesizer (Bioautomation, USA).

Protected 2′-deoxyribonucleoside 3′-phosphoramidites,

Unylinker-CPG (500  Å), and S-ethylthio-1H-tetrazole

were purchased from ChemGenes (USA). The synthe-

sis used a standard deprotection protocol by treatment

with aqueous saturated ammonia at 55°C overnight.

The solutions were evaporated, and the aliquots were

analyzed by HPLC (purity, >95%). Analysis and puri-

fication of oligonucleotides by HPLC was performed

on a 4.6×250  mm Jupiter C18 column (5  μm, Phenome-

nex, USA) with an Agilent 1260 HPLC system (USA)

equipped with an autosampler and a fraction col-

lector. Buffer  A: 0.05  M ammonium acetate (pH  7.0),

5% acetonitrile; buffer  B: 0.03 M ammonium acetate,

80%  acetonitrile (pH  7.0); gradient  B: 0→15% (1 col-

umn volume), 15→50% (10 column volumes); flow rate

1  ml/min; temperature 45°C.

Protein expression and purification. SARS-

CoV-2 RdRp was obtained by heterologous expression

in E.  coli BL-21(DE3) cells and purified by Ni-affinity

and anion-exchange chromatographies as described

previously [11]. E.  coli RNAP core enzyme was ex-

pressed in the same strain using the pVS10 vector and

purified by polyethyleneimine precipitation followed

by heparin, Ni-affinity, and anion-exchange chroma-

tography as described previously [35].

In vitro RNA synthesis by SARS-CoV-2 RdRp.

The ability of RdRp to incorporate oxo-εATP was an-

alyzed using RNA oligonucleotides (DNA Synthesis,

Russia) corresponding to the RNA primer and RNA

template. The radioactive label was introduced at the

5′-end of the primer by T4 polynucleotide kinase (New

England Biolabs, USA) using 0.8 MBq γ-[

P]ATP (Shem-

yakin–Ovchinnikov Institute of Bioorganic Chemistry)

according to the manufacturer’s protocol. The RNA

substrate was obtained by mixing the labeled primer

and the template to the final concentrations of 2 μM

and 2.2  μM, respectively, in the transcription buffer

(TB) containing 10  mM Tris-HCl, pH  7.9, 10  mM KCl,

and 0.1  mM EDTA (all reagents from Sigma-Aldrich

unless indicated otherwise). The mixture was heat-

ed at 95°C for 3  min, then cooled to 85°C in 2  min,

PETUSHKOV et al.2266

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

and gradually cooled to 25°C at 0.5°C/min. The RNA

substrate was diluted with TB and mixed with RdRp

to the final concentrations of 25 nM and 500  nM, re-

spectively, and the mixture was incubated at 30°C for

10  min. The reaction was started by adding a mix-

ture of NTPs (Illustra, UK) and MgCl

or MnCl

(Sigma-

Aldrich) to the final concentrations of 10 μM and

1.1  mM, respectively. Oxo-εATP was added to 100  μM

when indicated. Transcription was performed at 30°C

for various time intervals (from 30  s to 10  min). The

reaction was stopped by adding an equal volume of

stop solution containing formamide (Vekton, Russia)

and heparin (100  μg/ml, Sigma-Aldrich), and the sam-

ples were heated at 95°C for 3  min. RNA products

were separated by 15% PAGE (19 :  1) under denatur-

ing conditions (7.5  M urea, Roth, Germany) in TBE

buffer and detected using a Typhoon 9500 scanner

(GE Healthcare, USA).

In vitro transcription with E.  coli RNAP. The

ability of RNAP to incorporate oxo-εATP into RNA

and to pass oxo-εA in the template DNA strand was

analyzed using RNA and DNA oligonucleotides cor-

responding to the RNA transcript and DNA template

and nontemplate strands. Unmodified oligonucleotides

were synthesized by Sintol (Russia). A 5′-terminal ra-

dioactive label was introduced into RNA as described

above. Labeled RNA oligonucleotide was mixed with

the template DNA strand to the final concentrations

of 1 and 2  μM, respectively, in transcription buffer  2

(TB2) containing 40  mM Tris-HCl, pH  7.9, 40  mM KCl,

and 0.1  mM EDTA. The mixture was heated at 65°C for

3  min, and then gradually cooled to 25°C at 0.5°C/min.

The annealed duplex was diluted with TB2 to 250  nM

and E.  coli RNAP core enzyme was added to 1  μM (in

experiments with oxo-εATP) or to 2  μM (in experi-

ments with oxo-εdA DNA templates). The samples

were incubated at 37°C for 10  min. The nontemplate

strand was added to the final concentration of 2.5  μM

and the samples were incubated at 37°C for 15  min.

The reconstituted elongation complex was diluted 10-

fold with TB2. The reaction was started by adding a

mixture of NTPs (Illustra, UK) and MgCl

or MnCl

(Sigma-Aldrich) to the final concentrations of 10 μM

and 10  mM, respectively. Oxo-εATP was added to

100  μM when indicated. Transcription was performed

at 37°C for 30  s. The reaction was stopped by adding

an equal volume of stop buffer containing 8  M urea

(Roth, Germany), 30 mM EDTA (Sigma-Aldrich), and

2×  TBE. Transcription products were separated by

PAGE as described above.

RESULTS

Chemical synthesis of oxo-εATP. Oxo-εATP was

prepared starting from 7,8-dihydro-8-oxo-1,N

-ethe-

noadenosine (oxo-εA ribonucleoside) [34] and fol-

lowing the procedure described in [36] with minor

modifications (Fig. 1b). Briefly, oxo-εA ribonucleoside

was treated with POCl

in the presence of Bu

N in

(CH

PO as a solvent to produce the corresponding

nucleoside dichlorophosphoridate, whose reaction

with (NHBu

)

and subsequent hydrolysis of

the resulting cyclic intermediate produced crude oxo-

εATP, which was purified by ion exchange chroma-

tography and precipitated with NaClO

in acetone to

obtain the required product (yield, 43%).

Incorporation of oxo-εATP by RdRp into na-

scent RNA. First, we tested the ability of SARS-CoV-2

RdRp to incorporate the triphosphorylated form of

oxo-εA (oxo-εATP) into the growing RNA chain. For

this purpose, we used a model system (Fig.  2a) that

had been previously used in the studies of the bio-

chemical activity of RdRp and its inhibitors [11, 37-

39]. The RdRp enzyme used in the reactions contained

the catalytic subunit nsp12 and the accessory subunits

nsp7 and nsp8 fused to each other. The RNA substrate

consisted of two complementary RNA oligonucleotides,

an RNA primer containing a radioactive label at the

5′-end, and an RNA template strand. RdRp was first

incubated with the RNA substrate to form a complex,

then nucleotides were added, and the reaction was

carried out for 10min at 30°C. When RdRp and a full

set of unmodified NTPs were added, effective elonga-

tion of the original primer occurred in the presence

of either Mg

and Mn

ions (Fig.2b, lanes9 and18).

By adding various sets of NTPs, we determined the

efficiency of oxo-εA incorporation opposite different

template bases. The G residue was located at the

+1 position of the template (Fig. 2a). The addition of

oxo-εATP in the presence of Mg

ions does not lead

to the elongation of the RNA primer (lane2, compare

with the control sample in lane 1). In the control ex-

periment with CTP (lane 3), most of the primer was

elongated by 1-2 nucleotides (probably due to the in-

corporation of CTP opposite the first G and second A

residues, which is consistent with the published data

on a relatively low accuracy of SARS-CoV-2 RdRp[5]).

In the presence of Mn

ions, RdRp acquired the abil-

ity to incorporate two oxo-εA residues opposite G

and A in the RNA template (lane 11, compare with

the control sample in lane 20), and also incorporated

two C residues opposite G and A (lane 12).

When CTP and oxo-εATP were added together in

the presence of either Mg

and Mn

ions, they were

sequentially incorporated, as evidenced by a slower

electrophoretic mobility of the 37-nt RNA product

(lanes4 and 13) compared to the reactions performed

with CTP only (lanes3 and 12). Therefore, under these

conditions, oxo-εA was incorporated opposite the tem-

plate residue A with a greater efficiency than CTP

(which was also present in reactions in lanes4 and13).

EFFECT OF 8-OXO-EA ON RNA POLYMERASES 2267

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

Fig. 2. Incorporation of oxo-εATP by SARS-CoV-2 RdRp. a) RNA substrate used in the experiments. Red, RNA primer; black,

RNA template; the first template base is marked +1; gray, residues incorporated by RdRp upon primer extension (7 residues

incorporated upon addition of CTP, UTP, and GTP); arrow, direction of transcription. b)  Analysis of RNA extension products

synthesized in the presence of different sets of NTPs and oxo-εATP with Mg

(left panel) and Mn

(right panel). The length

of RNA products (nt) is indicated on the right. The products were separated by 15% denaturing PAGE.

When CTP and UTP were added, RNA was elongated

by 2 nucleotides with the formation of the expected

37-nt product (lanes  7 and 14). When a mixture of CTP,

UTP, and oxo-εATP was added, no further elongation

of the 37-nt product was observed (lanes  6 and 15).

Hence, oxo-εAp was not incorporated by RdRp op-

posite the template C in the next position. Addition

of CTP, UTP, and GTP in the presence of Mg

led to

the formation of a stalled complex with a 42-nt RNA

transcript (lane  7), which was not elongated when oxo-

εATP was added to the reaction (lane 8). Therefore,

RdRp did not incorporate oxo-εATP opposite the tem-

plate U in the next position. Interestingly, in the pres-

ence of Mn

, a significant portion of the elongation

complexes extended RNA beyond 42  nt, apparently,

by including non-complementary NTPs (lane 16); the

presence of oxo-εATP partially inhibited this reaction

(lane  17). When all 4 NTPs were added, no difference

in the electrophoretic mobility of RNA products was

observed in the presence or absence of oxo-εATP with

either Mg

(lanes 9 and10) or Mn

(lanes 18 and19).

Hence, no significant oxo-εATP incorporation was ob-

served in the presence of natural NTPs, and oxo-εATP

did not inhibit the activity of RdRp.

For a more detailed analysis of oxo-εATP incorpo-

ration by RdRp, we modified the test system. To test

oxo-εATP incorporation opposite each of the template

nucleotides under the same conditions, 4  identical RNA

templates with different template nucleotides at the +1

position were synthesized. In addition, a shorter RNA

primer was used for better separation of RNA prod-

ucts by PAGE (Fig.  3a). In order to reduce the incorpo-

ration of incorrect (not forming a canonical Watson–

Crick pair) nucleotides, the reaction time was reduced

to 30  s. It was shown that in the presence of Mg

ions, RdRp with a high efficiency incorporated NTPs

that formed canonical base pairs (UTP, GTP, GTP, and

ATP in the case of template A, G, C, and U residues,

respectively; Fig.  3b, lanes 2, 6, 8, and 10), but almost

did not incorporate oxo-εATP (lanes 3, 7, 9, and 11).

A different pattern of nucleotide incorporation

was observed in the presence of Mn

ions. In this

case, in the control reactions, each of the nucleotides

was incorporated not only opposite the corresponding

template residue, but also in the next position, oppo-

site the template U (Fig.  3c, lanes 2, 7, 10, and 12).

In the reactions with oxo-εATP, the modified nucleo-

tide was incorporated with a high efficiency opposite

the template residues A and G (Fig.  3c, lanes 3 and7),

but not opposite C and U (lanes 11 and13). In the case

of the A and G templates in the presence of oxo-εATP

and ATP, the RNA primer was further elongated (by a

total of 4  nt), which demonstrated the ability of RdRp

to incorporate both oxo-εATP and ATP into the syn-

thesized RNA.

Incorporation of oxo-εATP by RNAP into na-

scent RNA. To understand whether the observed abili-

ty of RdRp to incorporate oxo-εATP is a universal phe-

nomenon, we tested if cellular DNA-dependent RNA

polymerase from E.  coli, which is unrelated to RdRp,

PETUSHKOV et al.2268

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

Fig. 3. Incorporation of oxo-εATP by RdRp using RNA substrates with different template bases at the +1 position. a)  RNA

substrates used in the experiments. Red, RNA primer; black, RNA template; yellow, variable template base at +1 position

(X); gray, residue incorporated opposite it (Y) and the next three residues. b and c)  Analysis of RNA extension products

obtained in the presence of natural NTPs and/or oxo-εATP with Mg

(b) and Mn

with RNA templates containing A, G, C, or U at the +1 position. RNA products were separated by 15% denaturing PAGE.

The length of the RNA products (nt) is shown on the right.

can perform similar reactions. Unlike RdRp, RNAP

synthesizes RNA by moving along a double-stranded

DNA and melting the DNA strands during RNA elon-

gation. In this case, a synthetic elongation complex

containing the core enzyme of E.  coli RNAP, a short

RNA transcript (20nt), and DNA template and nontem-

plate strands was prepared from oligonucleotides as

described previously (Fig. 4a) [40-42]. As can be seen

in Fig.4b, oxo-εATP was not incorporated opposite dG

at position +1 in the template strand in the presence

of either Mg

or Mn

(lanes 2 and 12). Addition of

CTP or CTP and GTP resulted in RNA elongation by

1 or 3 nucleotides (to 21 or 23  nt), but these RNA

products were not extended further upon addition of

oxo-εATP (lanes 3-6 and 13-16). Therefore, oxo-εATP

also cannot be incorporated opposite dC and dT resi-

dues in the next template positions. Addition of CTP,

GTP, and ATP resulted in transcription stalling after

addition of 4 nucleotides, in accordance with the tem-

plate sequence (24-nt RNAs were synthesized; lanes 7

and 17). In the presence of oxo-εATP, additional RNA

elongation by two more nucleotides (to 26 nt) was

observed, which was more efficient in the presence

of Mn

ions (lanes 8 and 18). Probably, this involved

incorporation of oxo-εA at position 25 of RNA oppo-

site the template dA and further RNA extension due

to the incorporation of G opposite the template dC at

position 26. However, since transcription stopped af-

ter this, oxo-εATP is not a functional analogue of UTP.

Finally, when oxo-εATP and the full set of NTPs were

added together, no difference in the RNA products was

observed with the control reaction without oxo-εATP

(lanes 9, 10, 19, and 20).

Incorporation of NTPs opposite oxo-εA by

RNAP. Finally, we studied the effects of oxo-εA residue

in the template DNA strand on RNA synthesis by RNAP.

For this purpose, synthetic elongation complexes

were assembled as described above using template

DNA oligonucleotides containing a deoxyribonucle-

otide derivative of oxo-εA or control unmodified

dA at +2  position relative to the 3′-end of the RNA

primer (+1  template nucleotide in these templates

was also dA; Fig. 5a). It was found that with the

control template, RNAP added complementary NTPs

EFFECT OF 8-OXO-EA ON RNA POLYMERASES 2269

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

Fig. 4. Incorporation of oxo-εATP by E.coli RNAP. a)  Elongation complex used in the experiments. Red, RNA oligonucleotide;

black, template DNA strand; blue, non-template strand; gray, the first 4 incorporated nucleotide residues; arrow, direction of

transcription; the starting point of nucleotide incorporation (+1) is shown. b)Analysis of transcription products synthesized

in the presence of various sets of NTPs and oxo-εATP with Mg

(left panel) and Mn

(right panel) ions. RNA products were

separated by 15% denaturing PAGE. The length of RNA products (nt) is indicated on the right.

to the growing RNA chain as expected: UTP (17-nt RNA

was extended by 2 nt to 19  nt; Fig.  5b, lane  10), UTP

and GTP (RNA was extended by 4  nt to 21  nt; lane  12),

or all four NTPs (lane16). In agreement with the pre-

vious experiments, weak incorporation of oxo-εATP

opposite the template dA was also observed (lane15).

When the elongation complex contained oxo-εA at

+2 position of the template strand, only the first UTP

residue was incorporated opposite the template dA res-

idues at +1 position in all reactions, after which the

synthesis stopped and further NTP incorporation oppo-

site template oxo-εA did not occur (Fig.5c, lanes 2-8).

DISCUSSION

We found that synthetic ATP analogue oxo-εATP

can be incorporated into the nascent RNA by two

unrelated RNA polymerases. This suggests that oxo-

εATP can be accommodated in the active site of the

enzymes due to its small size and similarity to nat-

ural nucleotides. Furthermore, since Watson-Crick

interactions are blocked by the etheno modification,

oxo-εATP probably adopts the syn conformation and

pairs with the purine base of the template nucleotide

at the +1 position, forming a pair similar to oxo-εA:A

[1] or oxo-A:G [43] in the DNA context (Fig. 1c). This

explains the observed preference for oxo-εATP incor-

poration opposite purine nucleotides of the template.

In the presence of Mg

ions, SARS-CoV-2 RdRp slowly

incorporated oxo-εATP opposite the template  A (com-

pare the incorporation efficiencies at 10min in Fig.  2,

lane  4, and at 30  s in Fig.  3, lane 3). The situation

changed in the presence of Mn

ions, when incor-

poration was observed opposite both A and G. Inthe

case of E.  coli RNAP, oxo-εATP was incorporated only

opposite dA, but the efficiency of such incorporation

also increased in the presence of Mn

ions. This differ-

ence is probably due to the structural features of the

active site of cellular RNAPs, which ensures a greater

accuracy of RNA synthesis, since the oxo-εA:dA pair

is more similar to the canonical one than the oxo-

εA:dG pair (Fig.  1c). The stimulatory effect of man-

ganese ions on the oxo-εATP incorporation observed

for both RNA polymerases is likely due to the differ-

ence in the chemical properties and sizes of Mn

and Mg

cations. Previously, analysis of RNA synthe-

sis by poliovirus RdRp demonstrated that the fidelity

of NTP incorporation in the presence of Mg

is con-

trolled at two steps: reorientation of the triphosphate

PETUSHKOV et al.2270

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

Fig. 5. Incorporation of nucleotides against oxo-εA in the template DNA strand by E.  coli RNAP. a)  Elongation complex used

in the experiments. Red, RNA oligonucleotide; black, template strand; blue, non-template strand is blue; yellow, position of

oxo-εdA or control dA (X); Y, residue incorporated opposite oxo-εA or dA; gray, the first 4 incorporated nucleotide residues;

starting point of nucleotide incorporation (+1) is shown. b) Analysis of transcription products synthesized by RNAP on the

oxo-εA (left panel) or control dA (right panel) templates. RNA products were separated by 15% denaturing PAGE. The length

of the RNA products (nt) is indicated on the right.

of the incoming NTP and phosphoryl transfer [44].

In the presence of Mn

, RdRp loses its ability to use

the phosphoryl transfer step to control the fidelity be-

cause of the same incorporation rate for complementa-

ry and non-complementary NTPs [45], which results in

a decreased fidelity of RdRp in the presence of Mn

[46-48]. The observed differences during incorporation

of non-complementary and unnatural NTPs may be due

to the differences in the sizes of these cations. Mn

ion

has a smaller radius than Mg

, which frees up space

in the active site and allows the non-canonical pair to

be oriented in a position favorable for catalysis. It was

also shown for that the reactions of exo- and endonu-

cleolytic cleavage by RNA E.  coli RNAP, which are car-

ried out in the same active site of the enzyme as RNA

synthesis, proceed faster in the presence of Mn

ions

[25,  49]. This indicates the ability of Mn

to change

the nucleophilic properties of reacting molecules (in

this case, water) [49]. There is also evidence that the

binding of Mn

ion increases the flexibility of RdRp

molecule [50], which can additionally facilitate incor-

poration of NTPs forming noncanonical base pairs.

Further research is needed for better understanding

of the observed phenomenon. At the same time, the

replacement of standard Mg

cation with Mn

can

be used for enzymatic incorporation of modified NTPs

into synthesized RNA for practical purposes.

Since it was previously shown that oxo-εA can

be bypassed by DNA polymerases in  vivo [1], it could

be expected that cellular RNAP would also be able to

incorporate nucleotides opposite the template oxo-εA.

However, it was found that the presence of oxo-εA

in the template strand completely blocked the RNAP

activity even with Mn

ions. The observed difference

(oxo-εATP can be incorporated into RNA, but stalls

RNA extension when present in the DNA template) is

also probably due to the structure of the RNAP ac-

tive site. After translocation, the nucleotide residue at

+1position of the template DNA strand is fixed in the

active site [51], while the incoming NTP can be bound

in different configurations [51], i.e., it is more flexible

than the template base, which may allow it to occupy

a position more favorable for catalysis.

Although the studied RNA polymerases are able

to incorporate oxo-εATP into the synthesized RNA, we

failed to observe the inhibitory activity of oxo-εATP on

the incorporation of unmodified nucleotides. Despite

a 10-fold excess of oxo-εATP over natural NTPs, the

studied RNA polymerases did not incorporate it in the

presence of a full set of NTPs, and oxo-εATP did not

suppress incorporation of complementary nucleotides.

Therefore, oxo-εA is not a potential antiviral or anti-

bacterial transcription inhibitor; however, it may be

a prototype for obtaining more effective transcription

EFFECT OF 8-OXO-EA ON RNA POLYMERASES 2271

BIOCHEMISTRY (Moscow) Vol. 89 Nos. 12-13 2024

inhibitors by further modifications. Considering its

fluorescent properties [1], oxo-εA can also be used as

a label in studying the mechanisms of RdRp interac-

tion with RNA and nucleotide substrates. Based on our

data, it can be proposed that the simultaneous modi-

fication of the Watson–Crick and Hoogsteen edges of

purine bases leading to the redistribution of potential

hydrogen bonds and favoring the syn conformation

makes such modified nucleotides weak inhibitors of

viral and bacterial RNAPs.

Open access. This article is licensed under a Cre-

ative Commons Attribution4.0 International License,

which permits use, sharing, adaptation, distribution,

and reproduction in any medium or format, as long

as you give appropriate credit to the original au-

thor(s) and the source, provide a link to the Creative

Commons license, and indicate if changes were made.

Theimages or other third party material in this article

are included in the article’s Creative Commons license,

unless indicated otherwise in a credit line to the mate-

rial. If material is not included in the article’s Creative

Commons license and your intended use is not permit-

ted by statutory regulation or exceeds the permitted

use, you will need to obtain permission directly from

the copyright holder. To view a copy of this license,

visit https://creativecommons.org/licenses/by/4.0/.

Supplementary information. The online version

contains supplementary material available at https://

doi.org/10.1134/S0006297924120149.

Acknowledgments. The authors thank A. Makaro-

va, E. Shilkin, and E. Boldinova (Institute of Gene Bi-

ology, Russian Academy of Sciences) for valuable dis-

cussions.

Contributions. A.V.A., A.V.K., and I.V.P. planned

the study; A.V.A, I.A.I., M.S.B., and T.S.Z. synthesized

and purified oxo-εATP and modified DNA oligonucle-

otides, I.V.P performed the experiments; A.V.A., A.V.K.,

and I.V.P. discussed the results and wrote the article.

Funding. The work was carried out within the

framework of the state assignment National Research

Centre “Kurchatov Institute”.

Ethics declarations. This work does not con-

tain any studies involving human or animal subjects.

Theauthors of this work declare that they have nocon-

flicts of interest.

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