Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 9, pp. 1288-1300 © The Author(s) 2025. This article is an open access publication.

Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 9, pp. 1377-1390.

1288

Programmable DNA Cleavage

by Cyanobacterial Argonaute Proteins

Yuliya S. Zaitseva

, Ekaterina V. Kropocheva

, Andrey V. Kulbachinskiy

1,a*

and Daria M. Gelfenbein

1,b*

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

e-mail: avkulb@yandex.ru

es_dar@inbox.ru

Received August 19, 2025

Revised September 9, 2025

Accepted September 15, 2025

Abstract— Argonaute proteins are an evolutionarily conserved family of proteins capable of recognizing and

cleaving specific nucleic acid sequences using complementary guide molecules. Eukaryotic Argonautes play a

key role in RNA interference by utilizing short RNAs of various classes to recognize target mRNAs. Prokaryotic

Argonautes are much more diverse and most of them recognize DNA targets. The search for new Argonautes

that would be active under varying conditions is important for both understanding their functions and devel-

oping new tools for genetic technologies. Many previously studied Argonautes exhibit low activity at low and

moderate temperatures. To overcome this limitation, we isolated and studied two Argonaute proteins from

psychrotolerant cyanobacteria, CstAgo from Cyanobacterium stanieri and CspAgo from Calothrixsp. Both pro-

teins use short DNA guides to recognize and cleave DNA targets. CstAgo displayed no specificity for the 5′-end

structure of the guide, while CspAgo demonstrated a weak preference for the 5′-terminal nucleotide. CstAgo

was highly active and capable of cleaving single-stranded DNA at temperatures from 10 to 50°C. CspAgo was

more cold-sensitive but cleaved double-stranded plasmid DNA using specific guides. Therefore, the studied

proteins can be potentially used for DNA manipulations under a wide range of conditions.

DOI: 10.1134/S0006297925602680

Keywords: Argonaute, programmable nucleases, DNA cleavage

# These authors contributed equally to this study.

* To whom correspondence should be addressed.

INTRODUCTION

The genes for Argonaute proteins were discov-

ered in the late 20th century, shortly followed by elu-

cidation of the RNA interference (RNAi) mechanism. It

was shown that complexes of Argonautes with short

guide RNAs play a central role in recognizing target

mRNA molecules [1, 2]. Argonautes have been found

in almost all eukaryotes. The role of RNAi includes

regulation of gene expression, suppression of trans-

position of mobile elements in the germline, and de-

fense against RNA viruses [3]. RNAi studies have led

to the development of efficient tools for suppressing

expression of target genes, which are currently used

in analysis of gene functions and therapy [4].

About 12 years ago, researchers have turned their

attention to prokaryotic Argonautes (pAgos) [5, 6],

which had previously been identified by bioinfor-

matics methods [7] and used for structural studies

of RNAi mechanisms [8, 9]. pAgos are present in ap-

proximately 10% bacterial genera and 30% archaeal

genera, and they are much more diverse in structure

than their eukaryotic homologs. pAgos can be divided

into three major groups depending on the presence of

specific domains and catalytic activity: long-A pAgos

(active), long-B pAgos (inactive), and short pAgos (in-

active) [7, 10]. Inactive Argonaute proteins are often

encoded in the same operon with additional partner

proteins. Initially used as models to understand the

functions of eukaryotic proteins, pAgos are active-

ly investigated today to elucidate their biochemical,

structural, functional properties and to allow their

application in biotechnology.

Long Argonautes consist of six domains, N, L1,

PAZ, L2, MID, and PIWI. They have a two-lobe struc-

ture, with one lobe consisting of the N and PAZ do-

mains and the other consisting of the MID and PIWI

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domains, with nucleic acids positioned between them.

The PIWI domain is structurally related to RNase

H-like proteins; it contains the DEDX catalytic tet-

rad (where X is D, H, K, or N), coordinates divalent

cations, and is necessary for catalyzing the cleav-

age of the target [11]. The N-terminal domain is the

least conserved and is important for positioning the

guide-target duplex. The PAZ domain forms a pocket

for binding the 3′-end of the guide molecule in the

binary complex before the target binding. The MID

domain contains a pocket for binding the 5′-end of

the guide. Structural studies have shown that the

5′-terminal nucleotide does not form complementary

interactions with the target and can be specifically

recognized by Argonautes in the MID pocket [12].

For example, RsAgo from Rhodobacter sphaeroides

(now Cereibacter sphaeroides) and many eukaryotic

Argonautes preferentially bind guides with the 5′ ter-

minal U nucleotide, TtAgo from Thermus thermophilus

binds guides with the 5′ C nucleotide, while some pro-

teins have no specificity for the 5′-end of the guide [5,

6, 11-14]. The presence at the 5′ end of a phosphate

group, which interacts with conserved amino acid

residues of the MID pocket and a Mg

ion bound in

the pocket, is important for guide binding by most

Argonautes [11, 15, 16]. The binding of suboptimal

guides occurs with a lower affinity and may lead to

a shift in the target cleavage site due to the reduced

complex stability [15].

pAgos efficiently bind guides of 16 to 24 nucleo-

tides in vitro [17, 18] and typically use guides of the

same length in vivo [12, 16]. It has been established

that most catalytically active pAgos use single-strand-

ed DNA guides and recognize DNA targets, acting as

guide-dependent endonucleases and cleaving the tar-

get at a strictly defined position (similar to eukaryotic

proteins) between the 10th and 11th nucleotides from

the 5′ end of the guide [6, 16, 19-23]. Later, additional

groups of pAgos have been discovered that can rec-

ognize and cleave DNA targets using RNA guides [24,

25], RNA targets using DNA guides [15, 26], as well as

RNA targets using RNA guides [27]. Some Argonauts

are able to use different combinations of guides and

targets (both DNA and RNA) in  vitro, although with

lower efficiency than optimal guides and targets [23].

In the case of catalytically inactive Argonautes, addi-

tional effector nucleases encoded in the same operon

may be involved in target DNA processing [5, 7, 10,

28, 29].

Studies of first bacterial Argonautes suggested

that these proteins modulate the amount of invader

DNA in cells by affecting the processing of plasmids

and decreasing the transformation efficiency [5, 6]. Di-

rect evidence of pAgo functions in bacterial immunity

was obtained later in a heterologous E.  coli system

for clostridial Argonaute, which was shown to sup-

press viral infection [30]. The antiphage activity has

been then demonstrated for several long and short

Argonautes [18,  28, 31-33]. Some Argonautes have the

ability to process double-stranded DNA at random

sites in the absence of guide molecules (the so-called

‘chopping’ activity). It is assumed that this process

can generate new guides during initial recognition of

invader DNA in the cell [34].

Initially, most studied Argonautes were isolated

from thermophilic bacteria. Since these proteins were

easy to be crystallized, this allowed to combine the

data on the protein structure and their properties

[35]. However, a potential possibility of using Argo-

nautes for genome editing in eukaryotic cells has

stimulated the investigation of proteins from meso-

philic bacteria [16, 22]. These proteins have sever-

al advantages compared to Cas nucleases, including

smaller size, lack of requirement for a PAM motif,

and use of short DNA guides (see reviews [12,  36]).

Successful application of Argonautes in genome ed-

iting of bacterial cells has already been demon-

strated [37,  38]; optimization of Argonaute-based

tools for the use in eukaryotic cells is currently

ongoing.

pAgos have already been used for developing

tools for targeted DNA cleavage in  vitro, similar to re-

striction endonucleases [39], as well as for detection of

nucleic acids (short RNAs, rare DNA variants, nucleic

acids of pathogens, RNA and DNA modifications) [12,

36, 40-44]. Many of these methods are based on the

ability of Argonautes to bind and cleave single-strand-

ed targets in  vitro. However, the processing of dou-

ble-stranded DNA and genome editing depend on the

ability of Argonautes to cleave both DNA strands;

such activity requires the presence of two guides

and local unwinding of the two DNA strands before

their cleavage [16, 22, 23]. Genome editing in eukary-

otic cells also requires proteins with a temperature

optimum close to physiological temperatures. DNA

supercoiling, replication, transcription, and binding

of histones and regulatory proteins can significantly

alter the accessibility of target sites in the genome.

Therefore, to create optimal genomic editors, their

activity must be tested in  vitro and in  vivo under a

wide range of conditions.

The search for new catalytically active bacterial

Argonautes capable of cleaving double-stranded DNA

under physiological conditions without auxiliary pro-

teins, as well as development of methods for their

additional activation, are among the most important

tasks in the field of Argonaute research. Some pre-

viously tested Argonautes from mesophilic bacteria

have been found to cleave double-stranded DNA at

temperatures above 50°C but to have low activity at

physiological temperatures [16, 22, 23]. One way to

solve this problem is to study new representatives

ZAITSEVA et al.1290

BIOCHEMISTRY (Moscow) Vol. 90 No. 9 2025

of Argonaute proteins from psychrophilic and psy-

chrotolerant bacteria presumably capable of func-

tioning at lower temperatures.

MATERIALS AND METHODS

Cloning and expression of Argonaute genes.

The amino acid sequences of Argonaute proteins

from cyanobacteria Cyanobacterium stanieri LEGE

03274 (WP_193799343.1) and Calothrix sp. PCC 7103

(WP_019494780.1) were codon-optimized for expres-

sion in Escherichia coli cells using the IDT Codon Op-

timization Tool and obtained by chemical synthesis

(Cloning Facility, Moscow) as three fragments overlap-

ping by 20 nt. The full-length genes were cloned into

the pET28b+ vector using the Gibson assembly method

with a N-terminal His

tag and under the control of

the T7 promoter. Gene sequences are provided in the

Supplementary Materials (Online Resource 1).

Purification of Argonaute proteins. The pro-

teins were expressed in E.  coli BL21(DE3) cells trans-

formed with the corresponding vectors. To isolate

CstAgo, the cells were grown in a shaker incubator in

LB medium containing 50µg/mL kanamycin and 0.2%

glucose to OD

600

  =  0.3 at 30°C, then cooled in an ice

bath, and transferred to 18°C. Protein synthesis was

induced with 0.2 mM IPTG, and the cells were grown

for 20  h with constant shaking at 230 rpm. To obtain

CspAgo, the cells were grown in LB medium contain-

ing 50  μg/mL kanamycin to OD

600

  =  0.3 at 37°C, then

cooled in an ice bath, and transferred to 16°C. IPTG

was added to 0.2  mM, and the cells were grown for

20 h. The cells were collected by centrifugation and

stored at −20°C.

The cell pellet obtained from 2  L of culture was

thawed and resuspended in buffer  A (50mM Tris-HCl

pH7.5, 500 mM NaCl, 20mM imidazole, 5% glycerol),

passed three times through a high-pressure continu-

ous flow cell disruptor (Constant Systems, UK) at a

pressure of 30 kpsi, and 1 mM PMSF was added to

the lysate. The lysate was centrifuged twice at 30,000g

for 15 min at 4°C in a Hitachi CR22N centrifuge (ro-

tor R21A) (Japan). The supernatant was applied on a

HisTrap FF column (GE Healthcare, USA). The column

was washed with the buffer containing 20 mM and

49 mM imidazole for CspAgo or 92mM imidazole for

CstAgo, elution was performed with 260 mM imidaz-

ole. The resulting fractions were analyzed by Laemmli

electrophoresis followed by Coomassie G-250 staining.

Fractions containing expressed Argonaute proteins

were diluted 10-fold with a buffer containing 10  mM

HEPES-KOH (pH 7.0), 0.2  M  NaCl, and 5%  glycerol,

applied on a HiTrap Heparin HP column (GE Health-

care), and eluted with a stepwise NaCl gradient (200,

320, 440, 680, and 1000 mM). Fractions containing the

target proteins were concentrated using Amicon Ultra

50  kDa (Millipore, USA). HEPES-KOH (pH  7.0) and

EDTA were added to the protein samples to 20 and

0.5 mM, respectively; the samples were mixed with

glycerol at 1  :  1 ratio and stored at −20°C (for several

months) or −70°C after freezing in liquid nitrogen (for

longer periods of time). Protein concentration was de-

termined using Pierce 660 nm Protein Assay Reagent

(Thermo Fisher Scientific, USA).

Analysis of Argonaute activity in  vitro. Syn-

thetic DNA and RNA molecules (Evrogen, Russia;

DNK-Sintez, Russia) were used as guides and targets

in the cleavage reactions (see Table  S1 in the Online

Resource  1 for the sequences). RNA and DNA guides

were 5′-phosphorylated with T4 polynucleotide kinase

(New England Biolabs, USA) in a buffer solution con-

taining 20  mM HEPES-KOH (pH  7.0), 100  mM  NaCl,

10%  glycerol, and 5  mM  MgCl

. To study the effect

of divalent cations on the cleavage activity, MgCl

CoCl

, CuCl

, ZnCl

, and MnCl

at the concentrations

of 0.5 and 5  mM were used. The purified proteins (fi-

nal concentration, 500  nM) were incubated with the

guide (500  nM) at 37°C for 15  min, and the target

(100  nM) was added. The reaction was carried out at

37°C for 15  min for CstAgo and 30  min for CspAgo.

When studying the effect of temperature on the

Argonaute activity, the proteins were loaded with a

standard phosphorylated G-guide for 15  min at 37°C.

Then, aliquots of the reaction mixture were incubat-

ed at temperatures from 10 to 50°C, after which the

target was added, and the reaction was carried out

for 30  min. The reaction was stopped by adding an

equal volume of denaturing loading buffer (8  M urea,

100  mM  EDTA, 2×  TBE buffer, 0.005%  xylene cyanol,

and 0.005% bromophenol blue) after the time indi-

cated in the figure captions.

DNA or RNA molecules were separated electro-

phoretically in a denaturing 19% polyacrylamide gel

(acrylamide  :  bisacrylamide ratio, 19  :  1; 7  M urea) in

TBE buffer in a MiniPROTEAN Tetra Cell system (Bio-

Rad, USA) at 300  V for 1  h 15  min. Nucleic acids in

the gel were visualized by staining with SYBR Gold

(Invitrogen, USA), and the gel was scanned in the ap-

propriate channel on a Typhoon FLA 9500 scanner.

To determine the reaction kinetic parameters,

the targets were labeled with P

at the 5′-terminal

position using T4 polynucleotide kinase and γ-P

ATP (New England Biolabs, USA). The position of the

radioactive label in the gel was determined using a

radio-sensitive screen (GE Healthcare) and a Typhoon

FLA 9500 scanner (GE Healthcare Life Sciences). The

results were analyzed using the ImageQuant program

(GE Healthcare) and GraphPad Prism 9.

To analyze the cleavage of plasmid DNA, the

pJET1.2 plasmid with short inserts was used (see

Table  S1 in the Online Resource  1 for the sequences).

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BIOCHEMISTRY (Moscow) Vol. 90 No. 9 2025

CspAgo or CstAgo (500 nM) was mixed with 5′-phos-

phorylated guide DNA (500  nM) in the reaction buffer

and incubated at 37°C for 10 min. When 2 or 4 guides

were used in the same reaction, they were loaded into

the Argonaute protein independently and then mixed.

Next, plasmid DNA (2nM) was added and the samples

were incubated for 10 or 40 min at 37 or 50°C. The

reaction was stopped by adding 1 µL of Proteinase

K (Thermo Fisher Scientific); the samples were incu-

bated for 20 min at 25°C and then loaded on 1.2%

agarose gel containing 0.5× TBE. SYBR Gold was added

to the samples for visualization. Electrophoresis was

carried out in 0.5× TBE in a SE-2 chamber (Helicon,

Russia) at 100 V.

RESULTS

Cyanobacterial Argonautes. To search for Argo-

nautes from psychrophilic bacteria, we used a previ-

ously constructed Argonaute phylogenetic tree with

the corresponding list of strains [10]. The search

was conducted among Argonaute proteins of the

long-A group, with a predicted full active site in the

PIWI domain. The temperature optima of bacteri-

al strains in this group were manually determined

based on their description in microbiological collec-

tions. Two proteins from cyanobacteria were selected:

WP_019494780.1 (CspAgo) from Calothrix sp. PCC 7103

and WP_193799343 (CstAgo) from Cyanobacterium

stanieri LEGE 03274 (growth temperature 19°C, ac-

cording to the LEGE culture collection catalog). Since

these bacteria inhabit cold water, we assumed that

their Argonautes are adapted to functioning at lower

temperatures. However, by the time of writing this ar-

ticle, the WP_019494780.1 protein had been removed

from the NCBI database. Its closest homolog with 90%

identity is Argonaute protein RIVM261_088880 from

Rivulariasp. IAM M-261 [45] (strain isolated in Bang-

kok from a cement wall). The positions of the studied

cyanobacterial Argonautes on the phylogenetic tree is

shown in Fig. 1a.

The genes coding for CspAgo and CstAgo were

optimized for expression in E.  coli, cloned under the

control of an inducible promoter, and expressed as

described in the Materials and Methods. CstAgo was

found to be toxic under standard expression con-

ditions, so the induction conditions for this protein

were modified (see Materials and Methods). The pro-

teins were isolated from the cells disrupted under

high pressure, which allowed to preserve significant

amounts of CspAgo and CstAgo in the soluble form,

as has been previously found for other Argonautes.

Although significant amounts of CstAgo and

CspAgo were present in the cell lysate after centrifu-

gation, the proteins were also found in the precipitate,

indicating possible formation of inclusion bodies. The

proteins were purified in two steps using nickel-af-

finity and heparin chromatography to almost com-

plete absence of impurities according to staining with

Coomassie G-250, and then concentrated (see Fig. 1c

for CspAgo).

Guide specificity of CstAgo and CspAgo. To

determine the activity of the purified proteins, in

particular, whether they can cleave DNA, their spec-

ificity for guides and targets was studied in in  vitro

tests. Both Argonautes have a standard MID pocket

structure and contain conserved residues that interact

with the 5′-phosphate group of the guide, so experi-

ments were conducted with 5′-phosphorylated guides.

For this, the proteins were loaded with RNA or DNA

guides of identical length and sequence. We used

18-nt guides, as this length was found to be optimal

for most previously studied pAgos (see Introduction).

After formation of the binary complex, a 50-nt target

(RNA or DNA) with a central region complementary

to the guide was added to the reaction (Fig. 2a). The

reaction was carried out for 4 guide–target combi-

nations: DNA–DNA, DNA–RNA, RNA–RNA, RNA–DNA

(G-guides and G-targets; the letter corresponds to the

5′-terminal nucleotide of the guide, see Online Re-

source 1). The guide was incubated with the Argo-

naute at a 1  :  1 ratio at a concentration of 500  nM,

which was significantly higher than the guide binding

constants measured for related proteins (nanomolar

and subnanomolar ranges) [15, 16, 18, 46]. The reac-

tion was carried out for 15 or 30min and the reaction

products were separated by electrophoresis in dena-

turing PAAG and stained with SYBR Gold for nucleic

acid detection (Fig. 2b). It can be seen that the target

cleavage with the formation of 23 and 27 nt prod-

ucts (corresponding to the lengths of synthetic marker

oligonucleotides) occurred only in the case of DNA

guide–DNA target combination (Fig. 2b, lane 1). The

absence of activity with other guide–target combina-

tions might be explained by much less efficient bind-

ing of non-optimal guides and targets by the Argo-

naute proteins and altered position of these molecules

in the active site [6, 16, 18]. Hence, we demonstrated

that the purified proteins possess the desired activity

and can be used in further experiments to analyze

the optimal conditions for DNA cleavage.

Some Argonautes have a strict specificity for the

5′-end guide nucleotide due to the structural features

of the MID domain pocket, in which this nucleotide

is bound (see Introduction). However, the absence of

such specificity is desirable for the potential applica-

tion of Argonautes for genome editing, as it limits the

choice of guides. To check the specificity of the puri-

fied Argonaute proteins to the 5′-terminal nucleotide

of the guide, we carried out cleavage reactions with

guides containing different nucleotides at the 5′-end

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BIOCHEMISTRY (Moscow) Vol. 90 No. 9 2025

Fig. 1. Cyanobacterial Argonautes CspAgo and CstAgo. a) Phylogenetic tree of Argonautes. The branches are colored ac-

cording to three protein groups: long-A (green), long-B (black), and short (orange). The presence and absence of catalytic

activity are indicated in the outer circle by light pink and green, respectively. b) The three-dimensional model of CstAgo

with indicated domains (created by AlphaFold). c)Purification of CspAgo. Electrophoretic analysis in 10% PAAG of fractions

obtained after nickel-affinity and heparin chromatography steps: ‘in’, protein sample applied to the column; FT, flowthrough;

M, molecular weight markers.

and corresponding DNA targets: A-guide/A-target,

G-guide/G-target, C-guide/C-target, and T-guide/T-tar-

get. In all cases, the guides were phosphorylated at

the 5′-end and fully complementary to the target.

The cleavage of the target was carried out for 3

and 10min to observe a possible effect of the 5′-termi-

nal nucleotide in the guide on the rate of target cleav-

age. CstAgo did not display any preference for the

5′-terminal nucleotide of the guide. CspAgo cleaved

the targets in the presence of A, T, or G at the 5′-end

of the guide with similar efficiencies, but the presence

of C at the 5′-end of the guide somewhat reduced the

efficiency of cleavage (Fig. 2c).

To determine the preference of CstAgo and CspA-

go for catalytic metal ions, the cleavage reaction was

carried out with various divalent metal cations tak-

en at two concentrations, 0.5  mM and 5  mM. The re-

sults of the experiment (Fig. 3a) showed that CstAgo

was active with both concentrations of Mg

(0.5 and

5  mM), as well as in the presence of 5  mM  Mn

In contrast, CspAgo exhibited activity at both concen-

trations of Mn

and only at 5 mM Mg

. The pattern

of target cleavage by both proteins in the presence

of both cations was the same. Cu

, Zn

, Co

did not

act as catalytic cations. Therefore, both studied pro-

teins were active at the physiological concentration

of magnesium ions.

Temperature dependence of Argonaute activity

and the rate of target cleavage. To check wheth-

er CstAgo and CspAgo can function at physiological

temperatures, we studied the temperature depen-

dence of target cleavage by these proteins (Fig. 3b).

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Fig. 2. Specificity of CstAgo and CspAgo toward guides and targets. a)The reaction scheme showing the Argonaute protein,

guide, target and the reaction products. b)Determination of the specificity of CstAgo and CspAgo toward DNA (D) and RNA

(R) guides (G) and targets (T). P, oligonucleotides with the lengths corresponding to the expected lengths of products of

the target cleavage between the 10th and 11th nucleotides of the guide. The cleavage reaction was carried out for 30 min.

c) Dependence of the CstAgo and CspAgo nuclease activity on the 5′-terminal nucleotide in guide DNA. Reactions were

carried out at 37°C with 5′-phosphorylated guides with different 5′-terminal nucleotides and complementary DNA targets.

CstAgo was active within the temperature range from

10 to 50°C, and the ratio of the reaction products to

the unreacted target increased with the tempera-

ture increase. CspAgo had a different profile of tem-

perature dependence. It was active only above 20°C,

and even at 30°C the amount of cleaved target re-

mained low; the highest activity was observed at 50°C.

Such temperature dependence is similar to that found

for previously studied Argonautes from mesophilic

bacteria [47, 48]. At the same time, both CstAgo and

CspAgo are active at 37°C and are therefore promising

candidates for use in cells at moderate temperatures.

The ability of CstAgo to exhibit activity at 10°C makes

it the most cold-resistant Argonaute protein among

a large number of previously studied representatives

of this group [16, 22, 23, 47, 48].

Another important characteristic of potential ge-

nome editors is the rate of target cleavage. For a more

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BIOCHEMISTRY (Moscow) Vol. 90 No. 9 2025

Fig. 3. Effect of reaction conditions on the activity of CstAgo and CspAgo. a) Dependence of the Argonaute nuclease activ-

ity on divalent cations. The reaction was carried out for 15 min with CstAgo and 30 min with CspAgo; T, target; G, guide;

P, products. b) Dependence of the Argonaute activity on reaction temperature. The reaction was carried out for 30 min

at the indicated temperatures. c) Kinetics of DNA target cleavage by CstAgo and CspAgo. The reaction was carried out at

37°C. For each time point, the ratio of the shortened reaction product to the full-length target was determined. To calculate

the observed rate constants(k

obs

), the reaction kinetics were approximated by a first-order kinetics equation. The data are

shown as means ± standard deviations for three independent experiments.

accurate measurement of this parameter, we used a

DNA target radioactively labeled at the 5′-end. The re-

action was carried out at 37°C, and the ratio of the 5′-la-

beled cleavage products to the total amount of labeled

DNA was measured at different time intervals (Fig. 3c;

observed rate constants k

obs

are shown on the right).

CstAgo cleaved the target approximately 10 times faster

than CspAgo; CstAgo cleaved half of the target within

several minutes vs. CspAgo that required tens of min-

utes (Fig. 3c). Therefore, CstAgo has one of the highest

rates of target cleavage among the studied Argonautes,

comparable to the previously described KmAgo [23].

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Fig. 4. Cleavage of plasmid DNA by Argonautes. a)Scheme of plasmid DNA cleavage using two pairs of DNA guides. b)Se-

quences of guides and targets. The arrows show the cleavage sites. c)Cleavage of plasmid DNA by CspAgo. Electrophoresis

of cleavage products in native agarose gel stained with SYBR Gold. Plasmid treated with XhoI restriction endonuclease

(lane1, p+XhoI) and RNase H2 (lane2, p+RH2) was used as a control. The reaction was carried out with one or two pairs of

guides for the indicated time at 37°C or 50°C. The products of DNA hydrolysis at the indicated sites (1956bp and 1318bp)

are shown. d) Cleavage of plasmid DNA by CstAgo. The reaction was carried out at 50°C for 10 and 40 min.

Plasmid DNA cleavage. Next, we tested the activ-

ity of CspAgo and CstAgo Argonautes toward the plas-

mid DNA, since the ability to cleave double-stranded

DNA may be important for genome editing in  vivo. The

hydrolysis of plasmid DNA by Argonautes was studied

using a previously developed pJET vector containing

an insert of the target DNA. Two pairs of guides were

selected for the sites located 1.3 kb apart: G-guide (+)

used in the experiments on single-stranded DNA and

its complementary G-guide (−) in the DNA insert re-

gion, and the Ori-guide (+) and its complementary

Ori-guide (−) in the plasmid replication origin region.

Complementary guides were offset relatively to each

other by 5 nucleotides so that cleavage of DNA by two

Argonautes at the same locus would lead to the for-

mation of 5 nt sticky ends (Fig. 4a and b). Successful

cleavage of plasmid DNA by Argonautes in this system

would led to the formation of two linear products

1956 and 1318 bp long (Fig. 4a).

CstAgo and CspAgo were independently loaded

with four guides and incubated with plasmid DNA in

the reaction buffer at 37°C or 50°C for 10 or 40 min.

The plasmid cleaved at a unique XhoI site (linear DNA)

and plasmid treated with RNase H2 (relaxed circular

form) were used as controls. The relative positions

of all plasmid forms and the products of the control

reaction with CbAgo (which specifically cleaves plas-

mid DNA [16, 22]) are shown in the supplementary

data (Online Resource  2). It was found that in the

absence of guides (Fig. 4c, lanes 3-6), CspAgo did not

affect the distribution of plasmid DNA forms at both

temperatures, indicating the absence of non-specific

(chopping) activity under these conditions. When one

pair of the guides was added (Fig.4c, lanes 7-10), one

of the bands corresponding to the supercoiled plas-

mid disappeared (the lower band on the gel), which

could be seen after 40 min of incubation at 37°C and

at both time points at 50°C.

ZAITSEVA et al.1296

BIOCHEMISTRY (Moscow) Vol. 90 No. 9 2025

When two pairs of guides were used, two linear

cleavage products of the expected length were ob-

served after 40 min of incubation at 37°C (lane 12)

and at both time points at 50°C (lanes 13 and 14).

Therefore, the cleavage of the plasmid DNA by

CspAgo occurred more efficiently at 50°C than at 37°C;

however, this efficiency did not reach 100% under

the tested conditions. At the same time, CstAgo was

unable to cleave the plasmid DNA in the presence of

two pairs of guides either at 37°C or at 50°C (Fig. 4d;

results obtained at 50°C are shown). Hence, of the two

studied Argonautes, only CspAgo cleaved plasmid DNA

using specific guides.

DISCUSSION

Argonautes are programmable nucleases that

recognize target nucleic acids using short guide mol-

ecules; hence they can be potentially used in bio-

technological applications along with CRISPR-Cas nu-

cleases [12, 36]. Studied pAgos have diverse catalytic

properties and can use DNA or RNA guides and recog-

nize DNA or RNA targets. However, an important lim-

itation of the previously studied Argonautes is their

low activity at physiological temperatures, especially

toward double-stranded DNA. Many Argonautes have

been isolated from thermophilic bacteria and archaea,

so they have a high thermostability but are unable

to cleave DNA at low or moderate temperatures [6,

19, 34, 35]. In order to expand the range of available

Argonautes, we purified and investigated the prop-

erties of two Argonautes, CstAgo and CspAgo, from

psychrotolerant cyanobacteria, whose temperature

optimum for growth is lower than that of most me-

sophilic bacteria previously used as a source of Ar-

gonaute proteins [47, 48]. Due to the difficulties in

obtaining specific bacterial strains, the genes for the

studied proteins were constructed from synthetic DNA

fragments, cloned into expression vectors and used

for isolation of highly purified preparations of the

expressed proteins.

The main goal of this work was to study the

efficiency of DNA cleavage in vitro by the new cya-

nobacterial Argonautes to assess their activity under

physiological conditions. The experiments showed

that both CstAgo and CspAgo are DNA guide-depen-

dent DNA nucleases that have no preference for the

5′-terminal nucleotide of the guide and are active in

the presence of magnesium and manganese cations.

At the same time, CspAgo exhibited a preference for

manganese ions, similar to the previously studied

cyanobacterial Argonautes LroAgo and SynAgo [16,

21]. This might be related to the physiology of cy-

anobacteria and increased content of manganese in

cells, since it is a component of manganese cluster

of the oxygen-evolving complex (OEC) of photosys-

tem II (PSII) [49].

Measuring the temperature dependence and the

rate of single-stranded DNA cleavage showed that

CstAgo was active within the temperature range from

10 to 50°C, while the activity of CspAgo was observed

only at 20°C and above. Moreover, CstAgo cleaved tar-

gets an order of magnitude faster than CspAgo. Hence,

CstAgo has the highest activity at low temperatures

among the known pAgos. Unfortunately, the removal

of this protein from the original database makes it

impossible to unambiguously determine its host spe-

cies. Nevertheless, it can be assumed that the high

activity of the studied proteins at low temperatures

is associated with the adaptation of cyanobacteria to

low environmental temperatures.

The data obtained suggest that the studied Ar-

gonautes can be potential tools for DNA manipula-

tion, as they retained activity in a wide temperature

range in the presence of standard concentrations of

divalent cations, and did not depend on the 5′-ter-

minal nucleotide of the guide (with the exception of

reduced activity of CspAgo in the presence of cytosine

at the 5′-end). Although CspAgo demonstrated lower

activity toward single-stranded DNA, it specifically

cleaved double-stranded plasmid DNA at physiologi-

cal temperature.

The cleavage of double-stranded DNA by Argo-

nautes requires the use of two guides complementary

to the two DNA strands at the target locus, as each

protein introduces a single-strand break. It was pre-

viously shown that some Argonautes from thermo-

philic and mesophilic bacteria can specifically cleave

double-stranded DNA at high temperatures and excise

target DNA fragments from plasmids [16, 23]. Thus,

a technology based on the thermophilic Argonaute

TtAgo has been created, which allows manipulations

of plasmid DNA invitro [39]. Several Argonautes can

cleave plasmid DNA at 37°C, but their cleavage effi-

ciency is low and strongly depends on the GC con-

tent of the cleavage site [47,  48]. The efficiency of

double-stranded DNA cleavage can be increased by

heating the reaction mixture [23,  47] or adding UvrD

helicase, RecBC (which lacks nuclease activity but un-

winds DNA), and SSB proteins [50,  51]. An alternative

approach to the targeted cleavage of plasmids/dou-

ble-stranded DNA in  vitro using Argonautes is based

on the application of catalytically inactive proteins

directed to specific loci using guides and fused with

FokI nuclease. To facilitate the binding of Argonautes

to the target DNA, this technology uses modified oligo-

nucleotides (peptide nucleic acids, PNA) complemen-

tary to both strands of the target and stabilizing the

melted DNA region. This allowed a pair of chimeric

proteins bound to the adjacent sites to form an ac-

tive FokI dimer and introduce a double-strand break

CYANOBACTERIAL ARGONAUTE PROTEINS 1297

BIOCHEMISTRY (Moscow) Vol. 90 No. 9 2025

even at medium and low temperatures, albeit with

low efficiency [52].

In summary, the existing approaches involving

Argonautes do not yet allow an efficient cleavage of

target double-stranded DNA at physiological tempera-

tures in the absence of additional factors. Significant

activity of CstAgo and CspAgo proteins at moderate

temperatures, as well as the ability of CspAgo to

cleave double-stranded DNA at 37°C (albeit with low

efficiency), makes these proteins promising candidates

for further development of genome editors. The next

steps in this direction may be optimization of proper-

ties of the studied proteins using artificial intelligence

tools (as has been recently done for KmAgo [53]),

characterization of new Argonautes among hyperpsy-

chrophiles, and search for auxiliary factors capable

of stimulating the cleavage of double-stranded DNA

by Argonautes.

Supplementary information

The online version contains supplementary material

available at https://doi.org/10.1134/S0006297925602680.

Contributions

D.M.G. and A.V.K. developed the concept and super-

vised the study; Yu.S.Z., E.V.K., and D.M.G. performed

the experiments and collected the data; E.V.K., D.M.G.,

and A.V.K. analyzed the data and prepared the man-

uscript. All authors agree with the publication of the

final version of the manuscript.

Funding

This work was supported by the Ministry of Science

and Higher Education of the Russian Federation (proj-

ect no.075-15-2024-539).

Acknowledgments

The equipment used in this study was provided by

the Institute of Gene Biology, Russian Academy of Sci-

ences.

Ethics approval and consent to participate

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest

The authors of this work declare that they have

noconflicts of interest.

Open access

This article is licensed under a Creative Commons At-

tribution4.0 International License, which permits use,

sharing, adaptation, distribution, and reproduction

in any medium or format, as long as you give appro-

priate credit to the original author(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 material. If materi-

al is not included in the article’s Creative Commons

license and your intended use is not permitted by

statutory regulation or exceeds the permitted use,

you will need to obtain permission directly from the

http://creativecommons.org/licenses/by/4.0/.

REFERENCES

1. Bohmert, K., Camus, I., Bellini, C., Bouchez, D.,

Caboche, M., and Benning, C. (1998) AGO1 defines a

novel locus of Arabidopsis controlling leaf develop-

ment, EMBO J., 17, 170-180, https://doi.org/10.1093/

emboj/17.1.170.

2. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A.,

Driver, S. E., and Mello, C. C. (1998) Potent and spe-

cific genetic interference by double-stranded RNA in

Caenorhabditis elegans, Nature, 391, 806-811, https://

doi.org/10.1038/35888.

3. Olina, A. V., Kulbachinskiy, A. V., Aravin, A. A., and

Esyunina, D.M. (2018) Argonaute proteins and mech-

anisms of RNA interference in eukaryotes and pro-

karyotes, Biochemistry (Moscow), 83, 483-497, https://

doi.org/10.1134/S0006297918050024.

4. Jadhav, V., Vaishnaw, A., Fitzgerald, K., and Maier,

M. A. (2024) RNA interference in the era of nucle-

ic acid therapeutics, Nat. Biotechnol., 42, 394-405,

https://doi.org/10.1038/s41587-023-02105-y.

5. Olovnikov, I., Chan, K., Sachidanandam, R., Newman,

D. K., and Aravin, A. A. (2013) Bacterial argonaute

samples the transcriptome to identify foreign DNA,

Mol. Cell, 51, 594-605, https://doi.org/10.1016/j.molcel.

2013.08.014.

6. Swarts, D.C., Jore, M.M., Westra, E.R., Zhu,Y., Janssen,

J.H., Snijders, A.P., Wang,Y., Patel, D.J., Berenguer,J.,

Brouns, S.J.J., and van der Oost,J. (2014) DNA-guided

DNA interference by a prokaryotic Argonaute, Nature,

507, 258-261, https://doi.org/10.1038/nature12971.

7. Makarova, K.S., Wolf, Y.I., van der Oost,J., and Koonin,

E. V. (2009) Prokaryotic homologs of Argonaute pro-

teins are predicted to function as key components

of a novel system of defense against mobile genetic

elements, Biol. Direct, 4, 29, https://doi.org/10.1186/

1745-6150-4-29.

8. Parker, J. S., Roe, S. M., and Barford, D. (2004) Crys-

tal structure of a PIWI protein suggests mechanisms

for siRNA recognition and slicer activity, EMBOJ., 23,

4727-4737, https://doi.org/10.1038/sj.emboj.7600488.

9. Yuan, Y.R., Pei,Y., Ma, J.B., Kuryavyi,V., Zhadina,M.,

Meister, G., Chen, H. Y., Dauter, Z., Tuschl, T., and

Patel, D. J. (2005) Crystal structure of A. aeolicus

ZAITSEVA et al.1298

BIOCHEMISTRY (Moscow) Vol. 90 No. 9 2025

argonaute, a site-specific DNA-guided endoribonucle-

ase, provides insights into RISC-mediated mRNA cleav-

age, Mol. Cell, 19, 405-419, https://doi.org/10.1016/j.

molcel.2005.07.011.

10. Ryazansky, S., Kulbachinskiy, A., and Aravin,

A. A. (2018) The expanded universe of prokary-

otic argonaute proteins, mBio, 9, e0193518, https://

doi.org/10.1128/mBio.01935-18.

11. Lisitskaya, L., Aravin, A. A., and Kulbachinskiy, A.

(2018) DNA interference and beyond: structure and

functions of prokaryotic Argonaute proteins, Nat.

Commun., 9, 5165, https://doi.org/10.1038/s41467-

018-07449-7.

12. Kropocheva, E. V., Lisitskaya, L. A., Agapov, A. A.,

Musabirov, A. A., Kulbachinskiy, A. V., and Esyunina,

D.M. (2022) Prokaryotic argonaute proteins as a tool

for biotechnology, Mol. Biol., 56, 854-873, https://

doi.org/10.1134/S0026893322060103.

13. Miyoshi, T., Ito, K., Murakami, R., and Uchiumi, T.

(2016) Structural basis for the recognition of guide

RNA and target DNA heteroduplex by Argonaute,

Nat. Commun., 7, 11846, https://doi.org/10.1038/

ncomms11846.

14. Matsumoto, N., Nishimasu, H., Sakakibara, K.,

Nishida, K.M., Hirano,T., Ishitani,R., Siomi,H., Siomi,

M. C., and Nureki, O. (2016) Crystal structure of silk-

worm PIWI-clade Argonaute Siwi bound to piRNA,

Cell, 167, 484-497 e489, https://doi.org/10.1016/j.cell.

2016.09.002.

15. Lisitskaya, L., Shin, Y., Agapov, A., Olina, A., Kro-

pocheva,E., Ryazansky,S., Aravin, A.A., Esyunina,D.,

Murakami, K. S., and Kulbachinskiy, A. (2022) Pro-

grammable RNA targeting by bacterial Argonaute

nucleases with unconventional guide binding and

cleavage specificity, Nat. Commun., 13, 4624, https://

doi.org/10.1038/s41467-022-32079-5.

16. Kuzmenko, A., Yudin, D., Ryazansky, S., Kulbachin-

skiy,A., and Aravin, A.A. (2019) Programmable DNA

cleavage by Ago nucleases from mesophilic bacteria

Clostridium butyricum and Limnothrix rosea, Nucleic

Acids Res., 47, 5822-5836, https://doi.org/10.1093/nar/

gkz379.

17. Olina, A., Agapov, A., Yudin, D., Sutormin, D., Gali-

vondzhyan, A., Kuzmenko, A., Severinov, K., Aravin,

A. A., and Kulbachinskiy, A. (2023) Bacterial argo-

naute proteins aid cell division in the presence of

topoisomerase inhibitors in Escherichia coli, Micro-

biol. Spectr, 11, e0414622, https://doi.org/10.1128/

spectrum.04146-22.

18. Lisitskaya,L., Kropocheva,E., Agapov,A., Prostova,M.,

Panteleev,V., Yudin,D., Ryazansky, S., Kuzmenko, A.,

Aravin, A. A., Esyunina, D., and Kulbachinskiy, A.

(2023) Bacterial Argonaute nucleases reveal different

modes of DNA targeting in vitro and in vivo, Nucleic

Acids Res., 51, 5106-5124, https://doi.org/10.1093/nar/

gkad290.

19. Zander, A., Willkomm, S., Ofer, S., van Wolferen, M.,

Egert, L., Buchmeier, S., Stockl, S., Tinnefeld, P.,

Schneider, S., Klingl, A., Albers, S. V., Werner, F., and

Grohmann, D. (2017) Guide-independent DNA cleav-

age by archaeal Argonaute from Methanocaldo-

coccus jannaschii, Nat. Microbiol., 2, 17034, https://

doi.org/10.1038/nmicrobiol.2017.34.

20. Swarts, D. C., Hegge, J. W., Hinojo, I., Shiimori, M.,

Ellis, M. A., Dumrongkulraksa, J., Terns, R. M.,

Terns, M. P., and van der Oost, J. (2015) Argonaute

of the archaeon Pyrococcus furiosus is a DNA-guid-

ed nuclease that targets cognate DNA, Nucleic Ac-

ids Res., 43, 5120-5129, https://doi.org/10.1093/nar/

gkv415.

21. Olina, A., Kuzmenko, A., Ninova, M., Aravin, A. A.,

Kulbachinskiy, A., and Esyunina, D. (2020) Genome-

wide DNA sampling by Ago nuclease from the cya-

nobacterium Synechococcus elongatus, RNA Biol.,

17, 677-688, https://doi.org/10.1080/15476286.2020.

1724716.

22. Hegge, J.W., Swarts, D.C., Chandradoss, S.D., Cui, T.J.,

Kneppers, J., Jinek, M., Joo, C., and van der Oost, J.

(2019) DNA-guided DNA cleavage at moderate tem-

peratures by Clostridium butyricum Argonaute, Nu-

cleic Acids Res., 47, 5809-5821, https://doi.org/10.1093/

nar/gkz306.

23. Kropocheva, E., Kuzmenko, A., Aravin, A. A., Esyuni-

na,D., and Kulbachinskiy,A. (2021) A programmable

pAgo nuclease with universal guide and target spec-

ificity from the mesophilic bacterium Kurthia mas-

siliensis, Nucleic Acids Res., 49, 4054-4065, https://

doi.org/10.1093/nar/gkab182.

24. Agapov, A., Panteleev, V., Kropocheva, E., Kanevska-

ya,A., Esyunina,D., and Kulbachinskiy,A. (2024) Pro-

karyotic Argonaute nuclease cooperates with co-en-

coded RNase to acquire guide RNAs and target in-

vader DNA, Nucleic Acids Res., 52, 5895-5911, https://

doi.org/10.1093/nar/gkae345.

25. Kaya, E., Doxzen, K. W., Knoll, K. R., Wilson, R. C.,

Strutt, S.C., Kranzusch, P.J., and Doudna, J. A. (2016)

A bacterial Argonaute with noncanonical guide RNA

specificity, Proc. Natl. Acad. Sci. USA, 113, 4057-4062,

https://doi.org/10.1073/pnas.1524385113.

26. Li, W., Liu, Y., He, R., Wang, L., Wang, Y., Zeng, W.,

Zhang,Z., Wang,F., and Ma, L. (2022) A programma-

ble pAgo nuclease with RNA target preference from

the psychrotolerant bacterium Mucilaginibacter

paludis, Nucleic Acids Res., 50, 5226-5238, https://

doi.org/10.1093/nar/gkac315.

27. Bastiaanssen, C., Bobadilla Ugarte, P., Kim, K.,

Finocchio,G., Feng,Y., Anzelon, T.A., Köstlbacher, S.,

Tamarit, D., Ettema, T. J. G., Jinek, M., MacRae, I. J.,

Joo, C., Swarts, D. C., and Wu, F. (2024) RNA-guided

RNA silencing by an Asgard archaeal Argonaute, Nat.

Commun., 15, 5499, https://doi.org/10.1038/s41467-

024-49452-1.

CYANOBACTERIAL ARGONAUTE PROTEINS 1299

BIOCHEMISTRY (Moscow) Vol. 90 No. 9 2025

28. Prostova, M., Kanevskaya, A., Panteleev, V., Lisits-

kaya, L., Perfilova Tugaeva, K. V., Sluchanko, N. N.,

Esyunina, D., and Kulbachinskiy, A. (2024) DNA-tar-

geting short Argonautes complex with effector pro-

teins for collateral nuclease activity and bacterial

population immunity, Nat. Microbiol., 9, 1368-1381,

https://doi.org/10.1038/s41564-024-01654-5.

29. Song, X., Lei, S., Liu, S., Liu, Y., Fu, P., Zeng, Z.,

Yang,K., Chen, Y., Li, M., She, Q., and Han, W. (2023)

Catalytically inactive long prokaryotic Argonaute

systems employ distinct effectors to confer immu-

nity via abortive infection, Nat. Commun., 14, 6970,

https://doi.org/10.1038/s41467-023-42793-3.

30. Kuzmenko, A., Oguienko, A., Esyunina, D., Yudin, D.,

Petrova, M., Kudinova, A., Maslova, O., Ninova, M.,

Ryazansky, S., Leach, D., Aravin, A. A., and Kulba-

chinskiy,A. (2020) DNA targeting and interference by

a bacterial Argonaute nuclease, Nature, 587, 632-637,

https://doi.org/10.1038/s41586-020-2605-1.

31. Koopal, B., Potocnik, A., Mutte, S. K., Aparicio-

Maldonado,C., Lindhoud,S., Vervoort, J.J.M., Brouns,

S. J. J., and Swarts, D. C. (2022) Short prokaryotic

Argonaute systems trigger cell death upon detection

of invading DNA, Cell, 185, 1471-1486.e19, https://

doi.org/10.1016/j.cell.2022.03.012.

32. Zaremba, M., Dakineviciene, D., Golovinas, E.,

Zagorskaite, E., Stankunas, E., Lopatina, A.,

Sorek,R., Manakova,E., Ruksenaite,A., Silanskas, A.,

Asmontas, S., Grybauskas, A., Tylenyte, U., Jurge-

laitis, E., Grigaitis, R., Timinskas, K., Venclovas, C.,

and Siksnys, V. (2022) Short prokaryotic Argonautes

provide defence against incoming mobile genetic el-

ements through NAD

depletion, Nat. Microbiol., 7,

1857-1869, https://doi.org/10.1038/s41564-022-01239-0.

33. Bobadilla Ugarte, P., Barendse, P., Swarts D.C. (2023)

Argonaute proteins confer immunity in all domains

of life, Curr. Opin. Microbiol., 74, 102313, https://

doi.org/10.1016/j.mib.2023.102313.

34. Swarts, D. C., Szczepaniak, M., Sheng, G., Chandra-

doss, S.D., Zhu,Y., Timmers, E.M., Zhang,Y., Zhao,H.,

Lou, J., Wang, Y., Joo, C., and van der Oost, J. (2017)

Autonomous Generation and Loading of DNA Guides

by Bacterial Argonaute, Mol Cell., 65, 985-998, https://

doi.org/10.1016/j.molcel.2017.01.033.

35. Wang,Y., Sheng, G., Juranek,S., Tuschl,T., and Patel,

D. J. (2008) Structure of the guide-strand-containing

argonaute silencing complex, Nature, 456, 209-213,

https://doi.org/10.1038/nature07315.

36. Graver, B. A., Chakravarty, N., and Solomon, K. V.

(2024) Prokaryotic Argonautes for in vivo biotechnol-

ogy and molecular diagnostics, Trends Biotechnol., 42,

61-73, https://doi.org/10.1016/j.tibtech.2023.06.010.

37. Huang, S., Wang, K., and Mayo, S. L. (2023) Ge-

nome manipulation by guide-directed Argonaute

cleavage, Nucleic Acids Res., 51, 4078-4085, https://

doi.org/10.1093/nar/gkad188.

38. Esyunina, D., Okhtienko, A., Olina, A., Panteleev, V.,

Prostova, M., Aravin, A. A., and Kulbachinskiy, A.

(2023) Specific targeting of plasmids with Argonaute

enables genome editing, Nucleic Acids Res., 51, 4086-

4099, https://doi.org/10.1093/nar/gkad191.

39. Enghiad, B., Xue, P., Singh, N., Boob, A. G., Shi, C.,

Petrov, V. A., Liu, R., Peri, S. S., Lane, S. T., Gaither,

E.D., and Zhao,H. (2022) PlasmidMaker is a versatile,

automated, and high throughput end-to-end platform

for plasmid construction, Nat. Commun., 13, 2697,

https://doi.org/10.1038/s41467-022-30355-y.

40. He, R., Wang, L., Wang, F., Li, W., Liu, Y., Li, A.,

Wang,Y., Mao,W., Zhai,C., and Ma,L. (2019) Pyrococ-

cus furiosus Argonaute-mediated nucleic acid detec-

tion, Chem. Commun. (Camb), 55, 13219-13222, https://

doi.org/10.1039/c9cc07339f.

41. Shin, S., Jung, Y., Uhm, H., Song, M., Son, S., Goo, J.,

Jeong, C., Song, J. J., Kim, V. N., and Hohng, S. (2020)

Quantification of purified endogenous miRNAs with

high sensitivity and specificity, Nat. Commun., 11,

6033, https://doi.org/10.1038/s41467-020-19865-9.

42. Song, J., Hegge, J.W., Mauk, M. G., Chen, J., Till, J. E.,

Bhagwat, N., Azink, L. T., Peng, J., Sen, M., Mays, J.,

Carpenter, E.L., van der Oost,J., and Bau, H.H. (2020)

Highly specific enrichment of rare nucleic acid frac-

tions using Thermus thermophilus argonaute with

applications in cancer diagnostics, Nucleic Acids Res.,

48, e19, https://doi.org/10.1093/nar/gkz1165.

43. Wu, Z., Yu, L., Shi, W., and Ma, J. (2023) Argonaute

protein-based nucleic acid detection technology,

Front. Microbiol., 14, 1255716, https://doi.org/10.3389/

fmicb.2023.1255716.

44. Xiao,G., Fu, X., Zhang,J., Liu,S., Wang,Z., Ye,T., and

Zhang, G. (2021) Rapid and cost-effective screening

of CRISPR/Cas9-induced mutants by DNA-guided Ar-

gonaute nuclease, Biotechnol. Lett., 43, 2105-2110,

https://doi.org/10.1007/s10529-021-03177-z.

45. Hirose,Y., and Katayama,M. (2021) Draft genome se-

quence of the phototropic cyanobacterium Rivularia

sp. strain IAM M-261, Microbiol. Resour. Announc., 10,

e0079021, https://doi.org/10.1128/MRA.00790-21.

46. Liu, Y., Esyunina, D., Olovnikov, I., Teplova, M., Kul-

bachinskiy, A., Aravin, A. A., and Patel, D. J. (2018)

Accommodation of helical imperfections in Rhodo-

bacter sphaeroides Argonaute ternary complexes

with guide RNA and target DNA, Cell Rep., 24, 453-462,

https://doi.org/10.1016/j.celrep.2018.06.021.

47. Xu, X., Yang, H., Dong, H., Li, X., Liu,Q., and Feng,Y.

(2024) Characterization of argonaute nucleases from

mesophilic bacteria Pseudobutyrivibrio ruminis,

Bioresour. Bioprocess, 11, 94, https://doi.org/10.1186/

s40643-024-00797-x.

48. Cao,Y., Sun,W., Wang,J., Sheng,G., Xiang,G., Zhang,T.,

Shi,W., Li,C., Wang,Y., Zhao,F., and Wang,H. (2019)

Argonaute proteins from human gastrointestinal

bacteria catalyze DNA-guided cleavage of single- and

ZAITSEVA et al.1300

BIOCHEMISTRY (Moscow) Vol. 90 No. 9 2025

double-stranded DNA at 37 °C, Cell Discov., 5, 38,

https://doi.org/10.1038/s41421-019-0105-y.

49. Stengel,A., Gugel, I.L., Hilger,D., Rengstl,B., Jung,H.,

and Nickelsen, J. (2012) Initial steps of photosystem

II denovo assembly and preloading with manganese

take place in biogenesis centers in Synechocystis,

Plant Cell, 24, 660-675, https://doi.org/10.1105/tpc.

111.093914.

50. Hunt, E. A., Evans, T. C., Jr., and Tanner, N. A. (2018)

Single-stranded binding proteins and helicase en-

hance the activity of prokaryotic argonautes in vi-

tro, PLoS One, 13, e0203073, https://doi.org/10.1371/

journal.pone.0203073.

51. Vaiskunaite, R., Vainauskas, J., Morris, J. J. L., Pota-

pov, V., and Bitinaite, J. (2022) Programmable cleav-

age of linear double-stranded DNA by combined ac-

tion of Argonaute CbAgo from Clostridium butyricum

and nuclease deficient RecBC helicase from E. coli,

Nucleic Acids Res., 50, 4616-4629, https://

doi.org/10.1093/nar/gkac229.

52. Wang, Q., Rao, G. S., Marsic, T., Aman, R., and

Mahfouz, M. (2024) Fusion of FokI and catalytically

inactive prokaryotic Argonautes enables site-specif-

ic programmable DNA cleavage, J. Biol. Chem., 300,

107720, https://doi.org/10.1016/j.jbc.2024.107720.

53. Zhou, B., Zheng, L., Wu, B., Yi, K., Zhong, B., Tan, Y.,

Liu,Q., Lio,P., and Hong,L. (2024) A conditional pro-

tein diffusion model generates artificial programma-

ble endonuclease sequences with enhanced activity,

Cell Discov., 10, 95, https://doi.org/10.1038/s41421-

024-00728-2.

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