Article

ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 3, pp. 405-431 © Pleiades Publishing, Ltd., 2026.

405

REVIEW

Circular RNAs Fifty Years After Their Discovery

Ivan B. Filippenkov

1,a

*, Ekaterina V. Tsareva

, Ivan V. Mozgovoy

Olga Yu. Sudarkina

, Lyudmila V. Dergunova

, and Svetlana A. Limborska

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

e-mail: filippenkov_IB@nrcki.ru, filippenkov-ib.img@yandex.ru

Received October 8, 2025

Revised February 2, 2026

Accepted February 2, 2026

Abstract— Circular RNAs (circRNAs) are a unique class of covalently closed molecules formed through non-ca-

nonical splicing and characterized by a markedly greater stability compared to linear RNAs. Although the

first circRNA was discovered half a century ago in 1976 in a viroid, they had remained largely overlooked

for several decades. Over the past ten years, the however, interest in circRNAs has grown substantially, even

as their biological functions and overall significance continue to be debated. It is now well established that

circRNAs constitute a large and diverse group of molecules with varied origins and properties. They have

been identified across a wide range of organisms, from prokaryotes to plants and mammals, where they

participate in the regulation of numerous cellular processes. The unique properties of circRNAs are begin-

ning to be exploited for practical applications, including their use as disease biomarkers and platforms for

the development of novel therapeutic strategies. This review summarizes the knowledge accumulated on

circRNAs since their discovery and highlights recent advances in understanding their biology and potential

applications.

DOI: 10.1134/S0006297925603594

Keywords: circular RNAs, structure, biogenesis, and degradation of circRNAs, functions of circRNAs, compet-

itive endogenous RNAs, disease biomarkers, therapy

* To whom correspondence should be addressed.

INTRODUCTION

Circular RNAs (circRNAs) are covalently closed,

circular long non-coding RNA (lncRNA) molecules.

They were first described in 1976 by Heinz Ludwig

Sänger and colleagues, who deciphered the structure

of the potato spindle tuber viroid (PSTVd), originally

discovered five years earlier by Theodor Otto Diener,

and introduced the term “circular RNA” (circRNA)

[1,  2]. By the late 1970s, circRNAs had been found

in several satellite viruses, including hepatitis delta

virus (HDV). The HDV genome was the first circRNA

isolated from a human organism  [3-5].

Over the past half-century, the accumulation of

knowledge about circRNAs has been highly uneven.

During the 1980s-1990s and into first decade of the

21st century, less than 200 articles had been pub-

lished on this topic (PubMed), most of which were

focused on isolated examples of individual circRNAs

in specific eukaryotic organisms, e.g., the freshwater

ciliate Tetrahymena, slime mold, Chinese hamster

ovary cells, monkey CV-1 cells, and human HeLa cells

[6-9]. During the first 35 years following their discov-

ery, up to 2011, circRNAs had remained largely out-

side the mainstream of molecular biology research.

This situation has changed dramatically over the past

15 years, primarily due to the advent of whole-tran-

scriptome sequencing technologies. These approach-

es have uncovered an unexpected abundance and

diversity of circRNAs, particularly in human cells.

As a result, more than 25,000 articles published

during this period (PubMed) have substantially ex-

panded our understanding of circRNA biogenesis,

structure, and biological functions, firmly establishing

circRNAs as biologically important molecules.

CircRNAs originate from a wide range of genomic

sources. They can be generated from different func-

tional DNA regions, including exons (exonic circRNAs)

[10,  11], introns (intronic circRNAs)  [12], and com-

bined exon–intron sequences (exon–intron circRNAs,

FILIPPENKOV et al.406

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

EIciRNAs)  [13]. In addition, circRNAs may arise from

genomic regions involved in gene or chromosom-

al translocations, giving rise to fusion circRNAs [14,

15], mitochondrial DNA (mitochondrial circRNAs, or

mecciRNAs)  [16], and tRNA introns, producing circu-

lar tRNA introns (tricRNAs)  [17]. CircRNAs have been

identified across a broad spectrum of organisms, in-

cluding humans, rodents, and other mammals [11,

18-20], birds  [21], fruit flies (Drosophila spp.) [22,  23],

amphibians  [24], as well as plants [2,  25] and even

archaea  [18].

CircRNAs have attracted considerable attention

due to their unique and biologically valuable prop-

erties. First, due to the lack of free 5′ and 3′ ends,

circRNAs are more metabolically stable compared

to linear RNAs [12,  26]. Second, although circRNAs

frequently originate from protein-coding genes, they

generally do not encode proteins and are regulated by

expression mechanisms distinct from those governing

messenger RNAs (mRNAs) [22, 27, 28]. Third, circRNAs

are particularly abundant in brain tissue [11,  20,

29,  30]. Fourth, numerous studies have demonstrated

that circRNAs are actively expressed under a wide

range of pathological conditions [28, 31-34]. In recent

years, substantial progress has been made in under-

standing the functional roles of circRNAs. One of the

best-characterized functions is their ability to inter-

act with microRNAs (miRNAs), sequester them, and

thereby alleviate miRNA-mediated repression of pro-

tein-coding transcripts [35-38]. Through the involve-

ment of specific competitive circRNA–miRNA–mRNA

regulatory axes, circRNAs can modulate key cellular

processes, including cell differentiation, proliferation,

invasion, and metastasis in cancer [39-41], as well

as viral infections, including COVID-19, and antiviral

immune responses  [42]. Extensive research has also

been focused on the role of circRNAs in cardiovas-

cular diseases, particularly in regulating the blood–

brain barrier permeability, limiting ischemic injury,

and promoting neuroprotection [36, 37, 43-45]. The

substantial regulatory potential of circRNAs, which

has been long underestimated, provides grounds for

their potential use in biomedical applications. Emerg-

ing studies describe the use of circRNAs as disease

biomarkers, development of strategies for targeted

circRNA delivery as therapeutic agents, creation of

novel circRNA-based vaccines, and other advanced

therapeutic approaches  [46-48].

Currently, two terms are used in Russian-lan-

guage literature to denote covalently closed RNA

molecules: “циклические РНК” and “кольцевые

РНК” (ring-like RNAs). The term “кольцевые РНК”

was used on the Biomolecula website in2018 (https://

biomolecula.ru/articles/vlast-kolets-vsemogushchie-

koltsevye-rnk [in Russian]), as well as in the article by

Duk and Samsonova in 2021  [49] and in the review

by Baulina et al. in  2024 [50]. In contrast, the term

“циклические РНК” was first introduced by our

group in 2016. This term was chosen as the most

linguistically and conceptually consistent with the

English-language term “circular RNAs” and has re-

mained in active use since then [51-56].

According to PubMed, about 4000 review arti-

cles on circRNAs have been published worldwide,

providing comprehensive overviews of these mole-

cules, their functions, and their potential roles in the

pathogenesis of various diseases. In contrast, in the

Russian-language scientific literature, circRNAs have

been described in only a limited number of publi-

cations. In the present review, focused primarily

on the studies of Russian researchers, we provide a

comprehensive overview of the structural organiza-

tion of circRNAs, their expression features, functional

roles, and prevalence. For the first time, information

on prokaryotic circRNAs is presented, including vi-

roid-like RNAs known as obelisks. The review discuss-

es recent advances and current trends in the develop-

ment of practical applications of circRNAs, including

their use as disease biomarkers and therapeutic

agents, as well as potential applications in forensic

science and genome editing technologies. In addition,

we present the most significant results of our own

studies on circRNAs in brain cells. These include the

discovery and characterization of the structural and

functional organization of circRNAs derived from the

human sphingomyelin synthase  1 gene (SGMS1) and

its animal homolog (Sgms1), as well as the analysis

of circRNA expression patterns in cerebral ischemia.

We believe that this review will be of interest to spe-

cialists in biochemistry and researchers working in

related fields of science.

CircRNAs IN CELLS

CircRNA formation in  vivo. The main stages of

circRNA biogenesis have been described in numerous

reviews [19, 33, 46, 50, 57, 58]. A key event in circRNA

formation is alternative splicing, during which pre-

cursor RNAs are processed into either linear or circu-

lar RNA molecules. CircRNAs are frequently derived

from protein-coding genes and are often generat-

ed concomitantly with mRNA production. CircRNA

biogenesis occurs through a noncanonical splicing

mechanism known as backsplicing. This process in-

volves the joining of a downstream 5′ splice donor

site to an upstream 3′ splice acceptor site, which is

enabled by the formation of looped RNA structures.

Such loops are typically formed by intronic sequenc-

es flanking the circularized exon(s). Loop formation

can be mediated by base pairing between inverted

repeat elements, such as Alu elements in primates

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

or B1 andB2 elements in rodents, which are common-

ly present within introns [11, 59-61]. In the absence

of inverted repeat elements, circRNA formation can

be facilitated by RNA-binding proteins. For example,

proteins such as QKI  [62] and FUS  [63] can dimerize

after binding to specific intronic motifs flanking the

circRNA-forming region and bring the backsplicing

sites into close proximity, thereby promoting circu-

larization. Studies in Drosophila have demonstrated

that the efficiency of backsplicing is influenced by the

activity of canonical pre-mRNA splicing. Thus, deple-

tion of spliceosome components leads to a substantial

increase in steady-state circRNA levels. This phenom-

enon is thought to arise because circRNA formation

requires fewer splicing factors than linear splicing.

Similarly, circRNA production is enhanced when

3′-end processing of pre-mRNA is inhibited. Reduced

polyadenylation activity can result in the transcrip-

tional read-through across adjacent genes, producing

unusually long transcripts that impose increased de-

mand on the splicing machinery. Under these condi-

tions of limited splicing factor availability, circRNAs

gain an advantage and become the predominant

products of pre-mRNA splicing  [22,  27].

The formation of circRNAs is still debated, with

some studies interpreting them as “splicing noise”

and associating their presence with background

mRNA isoforms  [64]. Because the structures of many

circRNAs have been inferred primarily through bio-

informatic analysis of short reads from high-through-

put RNA sequencing, their actual existence in cells

has been questioned. Technical limitations at multiple

stages, including circRNA enrichment, RNA-Seq library

preparation, and bioinformatic analysis, can lead to

the identification of artifactual circRNAs  [65]. Increas-

ing the reliability of circRNA detection requires the

use of complementary experimental approaches, such

as PCR and Northern blotting  [66]. Using these meth-

ods, we detected and confirmed several circRNAs de-

rived from the SGMS1 gene, predominantly composed

of exons from the 5′  untranslated region (5′  UTR)  [11].

Moreover, SGMS1 circRNAs exhibited evolutionary

conservatism and tissue-specific expression, support-

ing the view that their formation is a non-random

process  [20,  67].

It is believed that circRNAs do not require their

own promoters for synthesis. However, in the study of

circRNAs derived from the rat Sgms1 gene, we demon-

strated that the use of alternative gene promoters can

regulate circRNA accumulation. The Sgms1 gene is

transcribed from at least three promoters. Atan early

stage of rat embryonic development (7  days in  utero),

the majority of transcripts in the embryonic brain

originated from the internal promoter. These tran-

scripts retained an intact open reading frame and all

elements required for mRNA translation, enabling the

production of sphingomyelin synthase  1 protein. How-

ever, they lacked the extended 5′  UTR, whose exons

were included in the Sgms1 circRNAs. Consequently,

transcription from the internal promoter did not sup-

port circRNA formation. At later stages of embryonic

development (11-21  days in  utero) and in 2-month-old

adult rats, mRNA expression predominantly originat-

ed from distal promoters. This shift was paralleled

by the increase in the circRNA level, suggesting that

circRNA biogenesis can be regulated through the use

of alternative gene promoters.

Diversity of circRNAs. As a result of alterna-

tive splicing, polyadenylation, and use of alternative

promoters, several classes of circRNAs are generated,

including exonic, intronic, and EIciRNAs (Fig.  1). Each

class exhibits distinct structural and functional char-

acteristics. Thus, exonic circRNAs are true circular

molecules formed by canonical 3′-5′ phosphodiester

bonds. In addition to circRNAs derived from exons

of a single gene, exonic circRNAs can also arise as

chimeric molecules composed of exons from different

genes. Such fusion circRNAs may result from chro-

mosomal translocations or read-through transcription

of downstream genes [14, 15, 22, 27]. The presence

of such chimeric circRNAs is often indicative of on-

going oncogenic processes. Intronic circRNAs are

typically lariat-like structures containing an atypical

2′-5′ phosphodiester bond. These molecules can be

experimentally distinguished from true circRNAs by

sequential treatment with the RNA lariat debranch-

ing enzyme (DBR) and RNase  R. DBR cleaves the

2′-5′ bond, converting the lariat into a linear RNA,

which is subsequently degraded by RNaseR, whereas

true circRNAs remain resistant to both enzymes [12,

26]. Intronic circRNAs may also be generated through

the self-splicing of group II introns with intrinsic ri-

bozyme activity  [68]. EIciRNAs contain both exon se-

quences and intronic regions  [13]. In some instances,

circRNAs do not encompass entire introns but instead

include specific intronic segments referred to as in-

ternal recursive exons. These elements participate in

the stepwise splicing of exceptionally large introns,

sometimes spanning tens of thousands of nucleotides,

and can be retained within circRNA molecules  [69].

Although recursive exons may be preserved between

canonical exons, transcripts harboring them are fre-

quently targeted for degradation through the non-

sense-mediated decay pathway  [70,  71]. Previously,

we identified six circRNAs derived from the human

SGMS1 gene that contain recursive exons. Interest-

ingly, these recursive exons were not only positioned

between canonical exons in an order consistent with

linear mRNA but also directly participated in back-

splicing  [69]. Based on these observations, we pro-

posed a model of recursive backsplicing in which

circularization occurs between a canonical exon

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

Fig.  1. CircRNA origin: circRNAs are synthesized in different cellular compartments and are derived from exons of one or

more genes, introns, tRNA precursors, and obelisks.

and a recursive exon, followed by excision of the in-

tron from the circular precursor [69].

Another conserved mechanism of RNA circular-

ization has been identified in archaea and eukary-

otes. tRNA intron lyase (also known as tRNA splicing

endonuclease, TSEN) cleaves intron-containing pre-

cursor tRNAs at the characteristic bulge–helix–bulge

(BHB) motif. Following the cleavage, the exon ends

are ligated by specific ligase complexes to generate

mature tRNA. In animals, the intron ends can also

be ligated to form circular RNAs derived from tRNA

introns, known as tRNA intronic circular RNAs (tric-

RNAs). However, this process is not typical in yeast

and plants [17,  72] (Fig.  1). It has been proposed that

maintaining tricRNAs in plants and yeast would incur

high energy costs, whereas degradation of tRNA in-

trons may help conserve both energy and nucleotide

resources  [72].

Recently, circRNAs encoded by mitochondrial and

chloroplast genomes have been discovered (Fig.  1).

Hundreds of mitochondrial genome-encoded circRNAs

have been found across various cells and tissues in

humans, mice, and other species. These molecules,

termed mitochondrial-encoded circRNAs (mecciRNAs),

are synthesized from mitochondrial DNA templates

through the activity of nuclear-encoded splicing fac-

tors imported into mitochondria from the nucleus.

Notably, mecciRNAs can facilitate the transport of

certain proteins into mitochondria, as they are ca-

pable of passing through the mitochondrial pores

[16]. In plants, chloroplast genome-encoded circRNAs

(cp-circRNAs) have been identified and characterized

in Arabidopsis thaliana. Sequence analysis suggests

that cp-circRNAs may participate not only in plant

development, but also in responses to environmen-

tal stimuli  [73]. Although the precise mechanism of

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

cp-circRNA biogenesis remains unclear, it is thought

to be associated with splicing. Plastid genomes, in-

cluding chloroplast DNA, are known to contain in-

trons, and gene expression in plastids is subject to

extensive post-transcriptional regulation  [74,  75].

Unlike in eukaryotes, circRNA formation in bacte-

ria occurs independently of splicing. A study in Bacil-

lus altitudinis demonstrated that specific 3′-terminal

sequences of linear RNA precursors play a crucial role

in the circularization of the DuCS (dual-conformation

small) non-coding RNA that regulates oxidative stress

resistance in this bacterium. Mutations within these

terminal sequences abolished the circRNA formation

[76]. It was proposed that DuCS circularization may

involve ribonucleases, including 5′-3′ exoribonucle-

ases and/or site-specific endoribonucleases, together

with bacterial RNA ligases. However, the precise mo-

lecular mechanism of circRNA biogenesis in bacteria

remains to be elucidated  [76].

In 2024, Zheludev et  al.  [77] discovered a new

class of viroid-like RNAs, named obelisks, in meta-

transcriptomes of human gut microorganisms and

oral microbiota (Fig.  1). These elements are approx-

imately 1000 nucleotides in length and adopt a

rod-like secondary structure reminiscent of viroids.

However, unlike classical viroids, obelisks encode

proteins of the Oblin family (Oblin-1, Oblin-2, Oblin-

SS, etc.), which places them closer to viruses. Oblin-1

contains an RNA-binding domain and is structurally

conserved across diverse obelisks, whereas Oblin-2

has a propensity to form protein polycondensates.

Some obelisks also exhibit type  III hammerhead ri-

bozyme self-cleavage activity  [77]. Subsequent stud-

ies confirmed that obelisks are rather common. In

Streptococcus sanguinis SK36, the content of obelisk

RNAs can exceed that of cellular mRNAs  [78]. In  2025,

López-Simón et  al.  [79] identified dozens of additional

obelisks through the analysis of marine metagenom-

ic and metatranscriptomic datasets. The authors pro-

posed that obelisks represent an evolutionary inter-

mediate between viroids and viruses, combining the

features of both types of organisms. Studying them

may provide new insights into RNA-mediated regula-

tion in microbiomes and shed light on the evolution

of pre-cellular life forms.

Several databases (e.g., circAtlas, circBank, and

circBase) provide comprehensive information on

circRNAs in different species. In Russia, a user-friend-

ly automated Unix/Linux pipeline called CircParser

was developed for the annotation of circRNAs from

both local and public databases, such as the National

Center for Biotechnology Information (NCBI). This tool

integrates outputs from widely used in  silico circRNA

prediction programs (CIRI, CIRI2, CircExplorer2,

find_circ, and circFinder) and classifies circRNAs ac-

cording to their structural features (exonic, intronic,

exon-intron, or intergenic) based on genome anno-

tation files [80]. Application of CircParser enabled

the identification of myogenic circRNAs in Nile tila-

pia [81].

Additionally, a web-based platform, CircPrime,

was developed to facilitate the design of PCR primers

and optimization of thermocycling conditions for the

reliable identification of circRNAs using conventional

PCR techniques. The platform is universally applicable

for studying multiple biological species whose genome

assemblies are available in the NCBI database  [82].

CircRNA degradation. The cellular levels of

circRNAs, like those of other transcripts, are deter-

mined by the balance between their biogenesis and

degradation. CircRNAs are often more stable than lin-

ear RNAs, which allows them to accumulate in the

cells. Nonetheless, several mechanisms exist to reduce

their abundance  [83]. In the nucleus, circRNAs can be

cleaved by RNase H when they form R-loops through

interactions with single-stranded DNA  [84]. In the cy-

toplasm, multiple degradation pathways have been

identified. One such pathway involves RNase  L, an

enzyme that participates in the primary immune re-

sponse by cleaving viral and certain cellular RNAs

[85,  86]. CircRNAs frequently form 16 to 26-nucleo-

tide intramolecular RNA duplexes that act as endog-

enous inhibitors of double-stranded RNA-dependent

protein kinase (PKR), and RNase L-mediated circRNA

degradation is thought to be required for PKR acti-

vation during viral infection  [85]. Another degrada-

tion pathway targets m

A-modified circRNAs via an

RNase P-dependent mechanism  [87]. Structure-medi-

ated degradation has also been described, in which

highly structured hairpin motifs in circRNAs are rec-

ognized by UPF1 and G3BP1, triggering endoribonu-

clease-mediated cleavage  [88]. Additionally, Hansen

et  al.  [35] reported an AGO2-dependent pathway,

where circRNAs are cleaved following recognition by

miRNA–AGO complexes. Beyond enzymatic degrada-

tion, circRNAs can be removed from cells through

the export in exosomes, an alternative pathway for

circRNA clearance [47, 89-91].

FUNCTIONAL SIGNIFICANCE OF circRNAs

CircRNA interactions with miRNAs. In 2013, it

was demonstrated that the circRNA Cdr1as can bind

complementarily to the miRNA miR-7 and inhibit its

activity in mammalian cells [35]. Because of this func-

tion, Cdr1as was also termed Cirs-7 (circRNA sponge

for miR-7). The identification of Cirs-7 sparked exten-

sive research into the role of circRNAs as endogenous

miRNA sponges.

miRNAs are a class of short (18-21  nt) non-cod-

ing RNAs found in various organisms [92, 93] and

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

Fig. 2. Biological functions of circRNAs.

involved in post-transcriptional control of gene ex-

pression through interaction with mRNAs. In animal

cells, this interaction typically occurs via an miRNA

sequence complementary to the corresponding mRNA

target  [94]. The formation of the mRNA–miRNA

duplex usually leads to the repression of mRNA

translation or its degradation  [92]. However, if a

circRNA containing a sequence complementary to the

miRNA is present, the miRNA can alternatively bind

to the circRNA. This sequestration prevents miRNA

from interacting with its target mRNA, thereby in-

creasing the levels of both the mRNA and the protein

it encodes.

The sponging of miRNAs is considered the pri-

mary and most well-established function of circRNAs

(Fig.  2). Numerous competitive circRNA–miRNA–

mRNA axes have been identified that regulate both

normal physiological and pathological processes. Ex-

amples include prostate cancer (circDHRS3/miR-421/

MEIS2), ischemic stroke (circHECTD1/miR-27a-3p/

FSTL1; circHECTD1/miR-133b/TRAF3; circMap2k1/miR-

135b-5p/Pidd1), and Alzheimer’s disease (circHDAC9/

miR-138/Sirtuin-1) [36, 37, 41, 43-45, 95]. It is believed

that circRNAs (predominantly exonic) can be trans-

ported to the cytoplasm, where they act as competi-

tive endogenous RNAs. Recent studies have elucidated

the transport mechanism, which involves Ran–GTP,

exportin-2, and IGF2BP1  [96].

It is important to note that circRNAs can compete

with mRNAs and lncRNAs in regulating miRNA levels

through the target RNA-directed miRNA degradation

(TDMD) on mRNA or lncRNA templates, as demon-

strated for Cdr1as  [38,  97]. The TDMD mechanism has

recently attracted significant research interest, and

its underlying details, including the contribution of

circRNAs, are currently the subject of active investi-

gation. Recent studies indicate that efficient miRNA

binding to circRNA sequences depends on the pres-

ence of multiple corresponding binding sites and a

high level of circRNA expression  [49]. Establishing

and validating the role of circRNAs as sponges for

miRNAs requires a multifaceted approach, combining

bioinformatics analyses with experimental methods,

including dual-luciferase reporter assays, RNA precip-

itation techniques such as RNA pull-down (RPD) and

RNA immunoprecipitation (RIP), as well as circRNA

overexpression and knockdown  [98]. Several data-

bases provide information on miRNA–circRNA inter-

actions. For example, CircFunBase offers data derived

both from bioinformatic predictions and experimental

validation  [99]. CircInteractome focuses on predicted

binding sites and includes tools for designing prim-

ers that selectively amplify circRNAs in PCR, avoid-

ing linear RNAs  [100]. ENCORI (formerly starBase)

provides interaction data obtained through immu-

noprecipitation methods and also allows prediction

CIRCULAR RNAs: FIFTY YEARS AFTER DISCOVERY 411

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

of whether an miRNA may undergo degradation

via the TDMD mechanism upon interacting with

a circRNA  [101].

It should be noted that many reported circRNA–

miRNA interactions are derived from single studies

and are not always supported by a robust evidence

base, which may raise questions about their biolog-

ical significance. Moreover, circRNAs exhibit diverse

functional roles beyond acting as sponges for miRNAs,

thus emphasizing the complexity of these molecules

as research subjects.

Functions of circRNAs in the nucleus. In ad-

dition to their cytoplasmic roles, circRNAs exhibit

important nuclear functions. For instance, circular

intronic RNAs (ciRNAs) have been shown to regu-

late transcription by interacting with the RNA poly-

merase  II complex [12, 102, 103]. Similarly, EIciRNAs

can associate with RNA polymerase  II and small nu-

clear RNAs (snRNAs) due to the presence of snRNA-

binding sites within their retained introns  [13]. These

examples highlight that many circRNA functions are

predominantly nuclear in nature. The interest in

nuclear circRNAs has grown significantly in recent

years [104,  105]. One notable function involves the

prevention of R-loop formation  [84]. R-loops occur

when a strand of genomic DNA hybridizes with its

nascent RNA transcript, which can interfere with the

pre-mRNA post-transcriptional processing. Cells typi-

cally mitigate R-loop accumulation through the activ-

ity of helicases such as DHX9 and DDX5  [106] or the

enzyme RNase  H  [107]. While RNase  H can resolve

R-loops by cleaving RNA, this often results in the

degradation of pre-mRNA and consequent decrease

in gene expression. It was shown that circRNAs can

compete with the synthesized RNA for the binding

to the single-stranded DNA in the R-loop. RNase  H

recognizes the DNA–circRNA hybrid and cleaves the

circRNA, eliminating the R-loop without damaging

the pre-mRNA, thereby preserving gene expression

[84,  108].

The ability of circRNAs to interact with DNA

had formed the basis for the 2022 hypothesis of

eco-crossover, also termed circRNA-regulated meta-

bolic crossover, that was suggested by the renowned

theoretical biology scientist A.  M.  Olovnikov  [109].

According to this hypothesis, various stress factors

can alter the expression of specific genes and their

associated circRNAs. As the levels of these circRNAs

increase, they may influence chromatin architecture,

thereby modulating the transmission of hereditary

information. Several intrinsic properties of circRNAs

support this idea: their enhanced metabolic stability,

stress-responsive expression, presence in germline tis-

sues [110,  111], and capacity for intercellular transfer

via exosomes or other extracellular vesicles [47, 89,

90]. Based on these features, Olovnikov proposed that

circRNAs could function as adapters targeting specif-

ic genomic sequences, facilitating recombination and

potentially guiding non-random mutagenesis under

environmentally regulated conditions [109].

CircRNAs interactions with proteins. Various

interactions between circRNAs and proteins have

been described. The resulting circular ribonucleopro-

tein complexes (circRNPs) perform diverse function-

al roles in cellular processes [112,  113] (Fig.  2). For

instance, circRNAs were found to directly interact

with transcription factors in many forms of cancer.

A  well-characterized example is Cdr1as, which in-

teracts with the tumor suppressor and transcription

factor p53 at a region critical for p53 binding to the

MDM2 protein. Normally, MDM2 represses p53 activ-

ity, thus facilitating cancer cell proliferation. By bind-

ing to p53, Cdr1as prevents the formation of the p53–

MDM2 complex, thereby stabilizing p53 function and

enhancing its tumor-suppressive activity. Conversely,

inactivation of Cdr1as contributes significantly to

tumorigenesis, highlighting its anti-oncogenic role

[114]. In contrast, circRHOT1 exhibits a pro-oncogen-

ic function. It binds transcription factors and facili-

tates their recruitment to the promoter of the NR2F6

gene. Expression of NR2F6 in T  cells suppresses the

anti-tumor immune response. Elevated expression of

circRHOT1 has been identified as a negative prognos-

tic factor in hepatocellular carcinoma  [115].

There is also evidence that circRNAs are present

in RNP complexes involved in the epigenetic regu-

lation of gene expression. For instance, circFECR1

has been identified in a complex that demethylates

the promoter of the leukemia virus FLI1 gene  [116].

In contrast, circLRP6 promotes DNA methylation and

functions as an oncogene. This circRNA interacts

with LSD1 and EZH2 proteins, thereby enhancing

methylation and reducing expression of tumor sup-

pressor genes such as KLF2 and APC that normally

act to slow osteosarcoma progression  [117]. In all the

above cases, the interactions between circRNAs and

their associated proteins have been experimentally

validated.

Another notable property of certain circRNAs is

regulation of the primary immune response, in which

the cell detects viral infection and activates a system

to degrade foreign nucleic acids through a network of

receptors and nucleases. During this process, specific

circRNAs, e.g., circPOLR2AP, are produced that bind

to NF90 and NF110 proteins (co-factors in immune

gene transcription) and sequester them in the cyto-

plasm. In the absence of infection, these proteins re-

main bound to circRNAs and are inactive. Upon viral

infection, circRNAs are rapidly degraded by RNase  L,

releasing NF90 and NF110 to interact with viral

RNA and trigger the immune response. Additionally,

under normal conditions, these proteins promote the

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

formation of corresponding circRNAs in the nucleus,

creating a reservoir that is ready for rapid deploy-

ment upon subsequent infections [118-120].

Functional interactions with proteins have also

been reported for mecciRNAs in humans, mice, and

other species. These circRNAs can be transported

from mitochondria to the cytoplasm, where they

participate in the transport and processing of nucle-

ar proteins, such as RPA (replication protein  A) and

hnRNPA (heterogeneous nuclear ribonucleoprotein  A),

which are critical for the mitochondrial genome rep-

lication and transcription  [16]. MecciRNAs have also

been implicated in cancer progression and resistance

to anticancer therapies  [121].

A recent study revealed that circRNAs play a

key role in modulating cellular responses to heavy

metal-induced stress. Central to this response is the

RNA-binding protein gawky, which translocates to the

nucleus and functions as a chromatin-associated fac-

tor, activating the transcription of numerous stress-re-

sponsive genes. In the case of copper-induced stress,

gawky has been shown to interact with circRNAs

containing metal-sensitive elements. Overexpression

of these circRNAs suppresses stress-induced transcrip-

tion by promoting the dissociation of gawky from the

chromatin  [122]. Therefore, circRNAs can act as neg-

ative regulators of stress-responsive gene expression,

impairing the chromatin-dependent activity of the

stress-activated gawky protein.

Translation of circRNAs. For a long time, cir-

cRNAs had been classified as lncRNAs. However,

emerging studies indicate that some endogenous cir-

cRNAs have a potential for translational, which can

be initiated by N6-methyladenosine (m

A), the most

common RNA base modification, the presence of in-

ternal ribosome entry sites (IRESs), or AU-rich motifs

(Fig.  2). CircRNAs have been shown to produce both

full-length proteins [123-125] and small peptides [126-

128]. Certain RNA-binding proteins facilitate cap-in-

dependent translation by recognizing motifs in the

circRNA sequence that resemble IRES, exhibiting

varying specificity and affinity for these sites  [129].

Moreover, recent evidence suggests that most eukary-

otic initiation factors (eIFs) are also essential for cir-

cRNA translation  [130]. Several bioinformatics tools,

such as TransCirc, RiboCirc, CircAtlas, and CRAFT,

can predict circRNAs with the coding potential  [131].

A collaborative effort involving Russian researchers

has led to the creation of the AthRiboNC database,

which catalogs non-coding RNAs, including circRNAs

with the coding potential, in A.  thaliana. This data-

base is based on ribosome profiling data (Ribo-Seq)

and contains information on 1871 circRNAs. Its inter-

face allows alignment with nucleotide or amino acid

sequences, as well as search and filtering by genomic

location, expression level, and other criteria  [132].

CircRNAs as participants in low-affinity nu-

cleic acid interactions. Molecular recognition be-

tween nucleic acids is traditionally thought to rely on

complementary base-pairing. However, recent study

by Nikitin  [133] has described the phenomenon of

strand commutation – a reversible, low-affinity bind-

ing of essentially non-complementary strands. Nikitin

demonstrated in  vitro that the role of such low-com-

plementary interactions can be similar to that of

classical complementary interactions. Specifically, the

addition of a single-stranded DNA with a limited com-

plementarity to the target DNA significantly altered

the efficiency of DNA cleavage by RNase  H  [133]. This

effect can be explained by the competition between

low-complementary nucleic acids, consistent with the

principles of reversible reactions. These findings are

particularly relevant for understanding the functions

of non-coding RNA, including miRNAs and circRNAs.

CircRNAs are highly stable compared to linear RNAs

due to their resistance to nucleases, suggesting they

may play a substantial regulatory role even in low-af-

finity interactions. For instance, miRNAs are approx-

imately 20 nucleotides long but typically recognize

RNA targets through short 6-8 nucleotide sequenc-

es. CircRNAs may influence miRNA target selection,

thereby modulating their regulatory impact. Moreover,

circRNAs could interact directly with DNA, potentially

preventing R-loop formation and thereby influencing

processes such as RNaseH-mediated cleavage (Fig.  2).

Competitive interactions involving circRNAs, includ-

ing low-affinity binding, may therefore significantly

affect the outcome of nucleic acid–nucleic acid inter-

actions within R-loops.

CircRNA EXPRESSION IN NORMAL

AND PATHOLOGICAL CONDITIONS

Tissue-specific expression of circRNAs. Nu-

merous studies have demonstrated tissue-specific

expression of circRNAs. Elevated levels of circRNAs

are observed in the heart, liver, and other tissues

[134-137]. In addition, circRNAs show developmental

stage-specific expression [135,  138] and accumulate

in anucleate platelets  [139] and exosomes  [140-142].

Thus, brain tissues have a high circRNA level [19,

20,  30], with genes involved in neurotransmission,

neuronal differentiation, and synaptogenesis express-

ing the greatest number of circRNAs [11, 20, 29, 30].

Our previous study on circRNAs of the SGMS1 gene

in humans, rats, and mice revealed an increased

abundance of these molecules in brain cells  [11,  69].

You et  al.  [143] reported that circRNAs predominant-

ly localize to neuronal regions, including neuropils,

axons, and dendrites, with their levels dependent on

the synapse development and homeostatic plasticity.

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

Due to a higher synaptic density, human brains often

contain more circRNAs than rodent brains [20,  144].

Expression of circRNAs in the brain can also be cell

type-specific. For example, Dong et  al.  [145] identified

1526 circRNAs in dopaminergic neurons and 3308

circRNAs in pyramidal neurons. Some circRNAs have

clear functional roles; for instance, circTulp4 regu-

lates neurotransmitter release at excitatory synapses

and enhances behavioral responses to aversive and

anxiogenic stimuli in mice  [146].

Moreover, an age-related increase in the circRNA

expression has been reported across various organ-

isms [147-151]. In tissues with a high proportion of

post-mitotic cells, such as the brain, circRNAs are

likely to accumulate over time due to their inher-

ent resistance to degradation. Such elevated circRNA

abundance in the brain may result from both en-

hanced activity of factors that promote circRNA bio-

genesis, such as QKI (Quaking, KH domain containing

RNA binding protein), and reduced activity of factors

that inhibit their formation, e.g., ADAR1 (adenosine

deaminase acting on RNA  1) [20, 62,  152].

Involvement of circRNAs in disease patho-

genesis. In recent years, research on circRNAs has

expanded significantly, revealing their crucial role

in the pathogenesis of various diseases. Numerous

circRNAs have been implicated in cardiovascular

conditions, including cardiomyopathy, chronic heart

failure, hypertension, ischemic heart disease, and

atherosclerosis [98, 153-155]. One of the most studied

circRNAs in vascular pathology is circANRIL. Its role,

however, appears to be context-dependent. In vascu-

lar smooth muscle cells and macrophages, high cir-

cANRIL expression is associated with a reduced se-

verity of coronary artery disease  [31]. Conversely, in

endothelial cells, circANRIL overexpression correlates

with an increased atherogenic index, elevated serum

lipid levels, and higher expression of inflammato-

ry cytokines (IL-1, IL-6), matrix metalloproteinase-9

(MMP-9), and C-reactive protein (CRP)  [156]. Other

circRNAs, including circLrp6, circDiaph3, circCHFR,

and circDcbld1, contribute to vascular pathologies

by regulating key cellular processes, such as pro-

liferation, migration, and differentiation of vascu-

lar smooth muscle cells [157-160]. CircRNA_000203

(circMyo9a) promotes the expression of myocardial

fibrosis-related genes through the interaction with

miR-26b-5p  [161]. Similarly, circRNA_010567 exerts the

profibrotic effects via the circRNA_010567/miR-141/

TGF-β1 regulatory axis.

The increased level of circRNAs in brain cells

[11, 20, 29, 30] has highlighted their role as key reg-

ulators in various neuroinflammatory and neurode-

generative diseases  [162,  163]. Moreover, circRNAs

have been implicated in the pathogenesis of glioma,

schizophrenia, and autism spectrum disorders [30,

164,  165]. Specific circRNAs, including circMyst4,

circKlhl2, circAagab, and circHomer1, may contrib-

ute to neuroplasticity and synaptogenesis  [166]. Sev-

eral studies have also emphasized the critical role of

circRNAs in cellular responses to cerebral ischemia.

For instance, Zuo et  al.  [167] reported that elevated

levels of circFUNDC1, circPDS5B, and circCDC14A

positively correlate with cerebral infarct volume.

Conversely, Bai et  al.  [37] demonstrated that upreg-

ulation of circDLGAP4 significantly mitigates neuro-

logical deficits and protects against infarct expansion

and blood-brain barrier damage in a mouse stroke

model. Han et  al.  [36] showed that circHectd1 regu-

lates regenerative mechanisms in brain cells during

ischemia, notably reducing infarct size in ischemic

mice. CircMap2k1 has been identified as a potential

contributor to the pathogenesis of cerebral ischemic

stroke  [45].

Using the transient middle cerebral artery oc-

clusion (tMCAO) model, we characterized the whole-

genome array of circRNAs potentially involved in the

rat brain response to ischemia in the affected hemi-

sphere [28,  51,  168]. A differential circRNA expres-

sion was observed across brain regions with varying

degrees of injury, including the striatum and frontal

cortex, suggesting involvement of these molecules in

regulating cellular responses, including those critical

for the functional recovery after cerebral ischemia.

Furthermore, we predicted mRNA–miRNA–circRNA

interaction networks that may modulate genome ac-

tivity during ischemia, both within the ischemic focus

and in the surrounding penumbral regions containing

recoverable cells [28,  51,  169].

The studies on the role of circRNAs in various

pathologies can expand our understanding of the

molecular processes underlying tissue damage and

recovery.

DEVELOPMENT

OF circRNA-BASED TECHNOLOGIES

Synthesis of circRNAs in  vitro. CircRNAs hold

considerable promise for biomedical applications,

and their potential use in the development of thera-

peutics, vaccines, genome editing systems, and tools

for studying diverse metabolic processes has been

actively explored  [170]. These prospects highlight

the importance of efficient synthetic approaches

for circRNA production. CircRNAs can be generated

invitro using either chemical or enzymatic methods.

Chemical strategies typically rely on the modification

of RNA molecule ends to promote circularization. For

example, RNA molecules bearing a 3′-amino group

and a 5′-phosphate can be circularized using the

phosphate-activating reagent 1-ethyl-3-(3-dimethylami-

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

nopropyl)carbodiimide (EDC). This reaction produces

a non-natural phosphoramidate (P–N) bond at the

ligation site. Although this linkage does not natural-

ly occur in cells, circRNAs synthesized in this man-

ner are recognized by the cellular translational ma-

chinery and can serve as templates for polypeptide

synthesis  [171]. An alternative chemical approach

involves modification of the phosphorylated 5′-end

of RNA molecule with a nitrile group, which subse-

quently reacts with the 3′-hydroxyl group to achieve

circularization. However, this strategy frequently

generates undesired by-products, including circRNAs

containing 2′-5′  phosphodiester linkages. To improve

the ligation efficiency and reduce side reactions,

single-stranded DNA oligonucleotides complementa-

ry to the splice junction can be used to bring the 3′

and 5′  ends into close proximity, thereby facilitating

intramolecular ligation. Among chemical approach-

es, significant attention has recently been directed

toward click chemistry strategies and bioorthogo-

nal reactions. These reactions are characterized by

rapid kinetics, high yields, and minimal byproducts

and involve functional groups that are not natural-

ly present in biological systems and therefore do not

interfere with endogenous biomolecules. A  common-

ly employed strategy is azide–alkyne cycloaddition,

in which the 3′ and 5′  ends of the RNA are modified

with azide and alkyne groups, respectively. Although

this method can provide high yields of circular prod-

ucts, it also introduces a non-natural linkage at the

ligation site. Chemical ligation strategies have been

comprehensively reviewed in [172-176]; however, the

advantages and limitations of chemical approach-

es for circRNA synthesis require further systematic

investigation.

Enzymatic approaches for generating circRNAs

in  vitro are primarily based on ligating the 5′ and

3′  ends of linear RNA molecules using RNA ligases.

In this method, a 5′-monophosphorylated linear RNA

is first produced by in  vitro transcription of a PCR

product fused with the T7 phage promoter, and the

DNA template is then removed by treatment with

DNase. The 5′ and 3′  ends of the linear RNA are li-

gated using T4 RNA ligase, and residual linear RNA

is subsequently eliminated by digestion with RNase  R.

Importantly, the choice of ligase may depend on spe-

cific nucleotides (A, U, G, or C) present at the ligation

junction. To further enhance the ligation efficiency,

complementary DNA splints are commonly employed

to bring the reactive RNA ends into close proximi-

ty, thereby facilitating intramolecular circularization

[173, 174, 176].

CircRNA formation can be driven by the catalytic

activity of group  I introns, which undergo self-splic-

ing. To utilize this capability, an artificial construct is

designed in which the sequence intended for circular-

ization (exon) is positioned between two ribozyme-ac-

tive intron fragments. Specifically, the 3′  half of the

intron along with the 3′  splice site is placed upstream

of the exon, while the 5′  splice site and the 5′  half

of the intron are positioned downstream. During the

splicing reaction, group  I intron catalyzes excision

of the internal exon as a covalently closed circular

RNA. This system can be applied both in  vivo during

transcription and in  vitro to generate target circRNAs

[174, 177, 178].

CircRNA overexpression is commonly used in

research practice. This approach involves generation

of an expression construct in which the circRNA se-

quence is flanked by artificially designed inverted

repeats and placed under control of a strong pro-

moter, such as the cytomegalovirus (CMV) promoter,

in a plasmid vector [35,  127]. High levels of circRNA

overexpression can also be achieved by cloning the

sequence of interest into a tRNA gene driven by a

strong promoter. In this strategy, the native tRNA in-

tron is replaced with the target circRNA sequence.

During pre-tRNA processing in animal cells, this mod-

ified transcript is circularized, leading to the forma-

tion of the desired circRNA instead of a tricRNA  [17].

The choice of the method for generating circRNAs

depends on specific research objectives and intend-

ed applications. For example, circRNAs produced

using ribozyme-mediated circularization have been

shown to support efficient translation in eukaryotic

cells [179, 180]. Such constructs have been used in

the development of vaccines against SARS-CoV-2 and

its variants, where they induced higher proportions

of neutralizing antibodies compared to convention-

al mRNA vaccines [181,  182]. However, ribozyme-de-

rived circRNAs may contain additional sequences,

commonly referred to as splicing scars, which can

trigger an undesired innate immune response. Toad-

dress this limitation, a scar-free strategy was devel-

oped in which the target sequence is rearranged so

that its ends mimic exon sequences required for the

intron-mediated splicing [170,  183]. Subsequently, an

alternative scar-free approach based on group  II in-

trons was introduced that involves modification of

the exon-binding site within the D1 domain [170,

184]. Another strategy for the scar-free circRNA syn-

thesis employs trans-splicing ribozymes [170,  185].

Inaddition, circRNAs with a minimal immunogenicity

can be generated enzymatically using T4  RNA ligase

[186]. When circRNAs are considered as templates

for protein synthesis, overexpression systems can be

employed to evaluate the translation efficiency and

activity of IRESs. However, it is essential to ensure

that no trans-splicing occurs during circRNA forma-

tion from the expression construct. Otherwise, linear

mRNA species may be generated, leading to the pro-

tein production independently of circRNA  [187].

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

Fig. 3. CircRNA applications.

CircRNAs in genome editing. Genome editing

technologies are rapidly advancing, with CRISPR–

Cas systems currently representing the most widely

used platforms. Guide RNAs (gRNAs) are essential

components of these systems, as they direct Cas nu-

cleases to specific DNA or RNA targets for precise

modification. However, linear gRNAs exhibit a rela-

tively short half-life compared to Cas proteins, which

can substantially limit the overall editing efficien-

cy  [188]. Due to their enhanced stability, circRNAs

have recently emerged as promising alternatives to

linear gRNAs in genome editing applications [189,

190] (Fig.  3). Their covalently closed structure con-

fers resistance to exonuclease-mediated degradation,

potentially prolonging guide activity and improving

editing outcomes. Recent studies have demonstrated

that engineered circRNAs capable of recruiting ADAR

enzymes significantly enhance both the efficiency

and specificity of RNA editing [189,  190]. In 2023,

a research group from China reported the develop-

ment of a guide circRNAs compatible with the Cas12a

and Cas13d systems  [191]. CRISPR platforms based

on Cas12a and Cas13 offer potential therapeutic ad-

vantages over Cas9, including reduced off-target ac-

tivity and improved suitability for multiplex gene

editing [192-194]. Furthermore, in 2025, Zhang et  al.

[195] demonstrated that guide circRNAs can also be

adapted for the use with the compact Cas12f (Cas14)

nuclease, which may be particularly advantageous

for precise genome editing and biomedical appli-

cations.

Recently, circRNAs have been explored for the

use in genome editing via the prime editing approach

[196-198]. The prime editing system typically consists

of a Cas9 nickase fused to a reverse transcriptase and

a specialized gRNA known as a prime editing gRNA

(pegRNA). This pegRNA not only directs the nickase to

the target DNA site but also serves as a template for

the synthesis of a new DNA segment during reverse

transcription [199,  200]. CircRNAs have demonstrated

applicability in CRISPR–Cas12a-based systems  [198],

in configurations employing separate nickase and re-

verse transcriptase components  [197], and in so-called

reverse editing systems that enable modifications at

the sites located closer to the 5′  end relative to the

nickase cleavage site  [196]. Further development of

these approaches is expected to substantially broaden

the scope of nucleic acid editing, enhancing both the

efficiency and translational potential of these technol-

ogies in biomedicine.

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

CircRNAs AS BIOMARKERS

It has been well established that circRNA levels

can vary depending on specific pathological condi-

tions, making circRNAs promising biomarkers for var-

ious diseases (Fig.  3). The first proposals for develop-

ing the panels of circRNA biomarkers, primarily for

cancer samples, have already been reported [201-203].

Earlier, we identified circRNAs originating from the

HDLBP and TNFRSF1A genes in RNA extracted from

human peripheral blood mononuclear cells. Using

bioinformatics analysis, we predicted regulatory net-

works of competitive interactions between miRNAs

and mRNAs or circRNAs for genes involved in lip-

id metabolism  [204]. Upregulation of circSPARC and

circTMEM181 has been observed in peripheral blood

mononuclear cells of patients with ischemic heart dis-

ease compared to individuals without atherosclerosis,

supporting the pro-atherogenic role of these molecules

[205]. Recent studies indicate that circRNAs can also

serve as markers of age and life experience, reflect-

ing the organism’s ability to “remember” stress. For

instance, exposure of Drosophila flies to low (18°C)

or high (29°C) temperatures for 10  days induced ex-

pression of specific subgroups of circRNAs, whose el-

evated levels persisted for several weeks even after

returning to standard conditions  [151].

Establishing regulatory networks involving

circRNAs enables the development of test systems that

account for the mutual interactions of different RNA

types, thus facilitating the selection of RNA panels for

more accurate diagnostics with reduced false results.

The primary approach relies on the well-established

functions of circRNAs, particularly their ability to

bind miRNAs. By leveraging individual competitive

circRNA–microRNA–mRNA axes, it is possible to mod-

ulate global cellular processes, including differentia-

tion, proliferation, invasion, and metastasis in cancer

[39-41], as well as coordination of progression of vi-

ral infection, such as COVID-19, and antiviral immune

responses [42,  206]. Consequently, potential diagnos-

tic systems could be based on the expression levels

of circRNAs and their associated miRNAs. Such sys-

tems might be highly efficient because circRNAs and

miRNAs are stable, exhibit tissue- and disease-spe-

cific expression, and circulate in extracellular fluids,

making them convenient analytes. For example, the

circRNA-to-miRNA ratio (circR-284/miR-221) has been

proposed as a predictive marker for carotid artery

disease and stroke [207].

Several databases summarize experimentally

validated data on the circRNA expression in vari-

ous diseases. In  2017, Yao et  al.  [208] developed the

Circ2Disease database, which provided 274 experi-

mentally confirmed associations between the circRNA–

miRNA axes and human diseases; however, this da-

tabase is no longer updated. The circRNADisease

database (updated in 2023) contains approximately

7000 entries documenting experimentally confirmed

associations of circRNAs with a wide range of hu-

man and animal diseases  [209]. For instance, the use

of “brain ischemia” prompt for searching this data-

base provides information on circRNAs derived from

the CDR1, DLGAP4, OGDH, FUNDC1, and other genes,

detailing their differential expression during ischemia

and miRNAs with which they can interact. The data-

base also catalogs over 7 million associations between

circRNAs and mutations across 30 cancer types, with

the highest number of circRNA mutations observed

in endometrial cancer, skin melanoma, and colorec-

tal adenocarcinoma. Additional resources, including

MiOncoCirc  [210], CircNet  2.0  [211], and CSCD2  [212],

provide information on circRNA expression and func-

tions in various cancers. At the end of 2025, Yuan

et  al.  [213] launched the BloodCircR database, which

compiles data from RNA sequencing of 5430 human

peripheral blood samples associated with 58 diseases.

This resource holds significant promise for identify-

ing circRNA biomarkers and circRNAs with a poten-

tial therapeutic relevance.

However, circRNA-based test systems may exhib-

it significant variability. Factors such as the cellular

concentrations of proteins interacting with circRNAs

or R-loops generated during transcription and repli-

cation, should be taken in consideration, as circRNAs

have been shown to participate in resolving R-loops

[84]. Evidence also suggests the involvement of these

mechanisms in various pathological processes. Clear-

ly, in such contexts, different strategies for the quan-

titative detection of analytes in the corresponding test

systems will be necessary. At present, however, these

possibilities remain largely in the realm of scientific

prospects.

The potential of circRNAs in forensic medicine

has been increasingly recognized (Fig.  3). Because

of their metabolic stability, circRNAs are promising

biomarkers for estimating the postmortem interval

(PMI). For instance, the level of circRnf169 in liver

tissue has been suggested as a potential marker for

early PMI assessment. However, as noted by the au-

thors, its utility for late PMI estimation is limited due

to the dynamic degradation of circRNAs in the liver

[214]. To address this limitation, the authors inves-

tigated circRNA markers in brain tissue and found

that circFat3 exhibits tissue-specific expression in the

mouse brain and demonstrates a strong correlation

with PMI over a wide temperature range  [215].

Although increasing evidence highlights the po-

tential of circRNAs as biomarkers, reproducibility

across different studies remains low. Therefore, fur-

ther experimental validation is essential to establish

circRNAs as reliable components of diagnostic panels.

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

CircRNAs AS THERAPEUTIC TARGETS

Advances in deciphering the mechanisms by

which circRNAs regulate gene expression have opened

new avenues for therapeutic interventions. Targeting

circRNAs or associated regulatory networks to modu-

late their activity is a promising strategy for disease

treatment. Emerging evidence demonstrates that al-

tering circRNA activity can profoundly reprogram

cellular metabolism and function. Such modulation

has been shown to affect the blood–brain barrier

permeability and infarct size  [37], as well as key

oncogenic processes, including cell differentiation,

proliferation, invasion, and metastasis [39,  216]. Fur-

thermore, circRNAs are involved in pathogenesis of

viral infections, including COVID-19, where they con-

tribute to the viral replication dynamics and mod-

ulation of antiviral immune responses  [42] (Fig.  3).

A  notable example of circRNA-targeted therapy in-

volves triple-negative breast cancer (TNBC). Approxi-

mately one-third of TNBC patients express circHER2,

a circular RNA encoding the oncogenic protein HER2-

103. The therapy of HER2-amplified breast cancer

widely uses the monoclonal antibody pertuzumab,

which has been shown to significantly reduce the

tumorigenicity of cells expressing circHER2/HER2-103

in  vivo  [217]. In another study, the authors demon-

strated that the circRNA hsa_circ_0003220 mediated

resistance of non-small cell lung cancer (NSCLCs) to

chemotherapeutic drugs through regulation of the

miR-489-3p/IGF1 axis. Suppression of this circRNA

reduced the expression of the pro-oncogenic protein

IGF1 and restored the sensitivity of tumor cells to ther-

apy  [218]. Similarly, the circ_ZFR/miR-195-5p/KPNA4

regulatory axis has been identified as a key determi-

nant of paclitaxel sensitivity in NSCLCs, representing

a potential therapeutic target  [219]. Paclitaxel exerts

its antitumor effects by disrupting the microtubule

dynamics and mitosis progression, and circRNA-me-

diated regulation significantly influences cellular

responsiveness to this drug. The hsa_circ_0074027/

miR-379-5p/IGF1 axis has been shown to contribute

to the molecular mechanisms underlying chemore-

sistance in NSCLCs  [220]. Therefore, assessment of

circRNAs as therapeutic targets is extremely import-

ant, as targeted modulation of specific circRNAs or

circRNA-dependent signaling pathways may signifi-

cantly enhance the treatment efficacy [221,  222].

CircRNA-BASED DRUGS

Currently, circRNA-based therapeutics are being

actively developed. Particular attention is focused

on their potential application in glioma treatment.

Accumulating evidence indicates that circRNAs con-

tribute to glioma resistance, primarily by mediating

resistance to radiotherapy and chemotherapy. Mech-

anistically, circRNAs exert these effects by function-

ing as competitive endogenous RNAs (ceRNAs), regu-

lating miRNA activity within specific signaling axes,

including circATIC/miR-520d-5p/Notch2-Hey1  [223],

circ-0008344/miR-433-3p/RNF2  [224], and circASAP1/

miR-502-5p/NRAS  [225]. Exosome- or lipid nanoparti-

cle-encapsulated circPRKD3 has been shown to stim-

ulate CXCL10 secretion through the reprogramming

of tumor-associated macrophages, thereby enhancing

recruitment and tumor infiltration of CD8

  T  cell.

Recently engineered circRNA encoding interleukin-2

(IL-2) demonstrated significant tumor-suppressive

effects in a glioma model  [226]. To facilitate its de-

livery, a specialized lipid nanoparticle system based

on ursodeoxycholic acid was developed for efficient

circRNA transport.

CircRNAs can be delivered via extracellular vesi-

cles (e.g., exosomes) to target cells, where they exert

their protective effects. It was shown that circRNAs

are naturally transported in exosomes [91,  140]. Ac-

cordingly, one of the most actively developing re-

search areas focuses on characterizing the circRNA

cargo of exosomes derived from various sources and

elucidating the functional role of these molecules.

Yang et  al.  [47] showed that extracellular vesicle-me-

diated delivery of circSCMH1 promotes its interac-

tion with MeCP2, thereby relieving repression of tar-

get genes regulated by this protein. This results in

a significantly enhanced neuroplasticity, along with

reduced glial reactivity and decreased infiltration of

peripheral immune cells in rodent and primate mod-

els of ischemia. Similarly, exosome-mediated delivery

of circDYM alleviated chronic unpredictable stress-

induced depressive-like behavior in mice  [89]. Collec-

tively, these findings highlight the therapeutic poten-

tial of exosome-based circRNA delivery as a promising

strategy for neuroprotection and treatment of neuro-

psychiatric disorders (Fig.  3). However, clinical trans-

lation remains limited by insufficient understanding

of exosome biology, as well as concerns regarding

the efficiency and safety of developed technologies.

Despite these challenges, combining exosome-based

delivery systems with bioactive molecules such as

circRNAs may open new avenues for the treatment

of complex multifactorial diseases.

CircRNA-based vaccines. The onset of the

COVID-19 pandemic has significantly accelerated re-

search into novel RNA-based vaccine platforms. Due

to their enhanced metabolic stability and recently

demonstrated capacity for efficient and specific trans-

lation, circRNAs have emerged as a promising alter-

native to linear mRNAs in vaccine development  [48]

(Fig. 3). Recent studies have successfully generated ar-

tificial circRNAs capable of robust protein expression,

FILIPPENKOV et al.418

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

including constructs that elicit antiviral immune re-

sponses [180-182], and used them in the development

of prototype vaccines against SARS-CoV-2  [182]. Nota-

bly, circRNAs have been shown to sustain protein ex-

pression for longer periods compared to their linear

mRNA counterparts. In particular, circRNA constructs

encoding the VFLIP-X spike protein (modified VFLIP

containing six amino acid substitutions) were devel-

oped. A vaccine prototype based on the circRNA-me-

diated expression of VFLIP-X induced neutralizing an-

tibodies in mice that persisted for up to seven weeks

following booster immunization, demonstrating effica-

cy against SARS-CoV-2 and its variants  [182].

At the same time, circRNAs themselves can mod-

ulate immune system activity [86,  227]. They are ca-

pable of activating the innate immune sensor RIG-I

and, under certain conditions, may even trigger auto-

immune responses. The immunostimulatory potential

of circRNAs depends on factors such as their biogen-

esis, structural features, and method of production.

Thus, circRNAs of different origins can elicit distinct

cellular immune responses. RNA-binding proteins

play a crucial role in this process by recognizing spe-

cific secondary structure motifs within circRNAs and

mediating their discrimination as “self” or “non-self.”

Therefore, when developing safe circRNA-based vac-

cines, it is essential to carefully consider the immuno-

modulatory properties of circRNAs and their potential

contribution to the overall immune response.

Recently, the first data on circRNA-based onco-

lytic vaccines have appeared  [228]. The authors re-

ported that a vaccine incorporating the tumor-specific

circRNA circFAM53B and its non-canonically encoded

cryptic peptides induced a robust anti-tumor immune

response in mouse models of melanoma and breast

cancer  [229]. In addition, a recent study described

the development of a circRNA encoding chimeric

antigen receptor (CAR) proteins, the key tools for

the T  cell-mediated tumor eradication. The authors

demonstrated that the circRNA CAR suppressed tumor

growth and reshaped the tumor microenvironment

in mice  [230].

Although circRNA-based vaccines have a poten-

tial to enhance therapeutic efficacy, several challeng-

es remain unresolved. These include the synthesis of

target circRNAs with minimal impurities, optimiza-

tion of delivery systems, and mitigation of unwanted

immune responses. Intensive research efforts are un-

derway to address these issues, including the applica-

tion of bioinformatics approaches and artificial intel-

ligence-based methods [231-233]. Another important

concern is the potential for off-target effects arising

from the extensive interactions of circRNAs with bio-

active molecules, such as nucleic acids and proteins.

Growing evidence suggests that the development of

safe and effective circRNA vaccines requires a com-

prehensive understanding of circRNA interaction net-

works and their regulatory roles at a genome-wide

level.

CONCLUSION

In this review, we examined the key features

of circRNAs, including their structure, biogenesis,

functions, and practical applications. CircRNAs play

diverse roles in the regulation of gene expression:

they can act as molecular sponges for miRNAs and

proteins, modulate alternative splicing and chromatin

organization, regulate R-loop formation, and serve as

templates for translation. Due to their high stability

and tissue-specific expression patterns, circRNAs are

regarded as promising biomarkers in the diagnostics

of a wide range of diseases and in forensic applica-

tions. In addition, they can function as therapeutic

targets and even as standalone therapeutic agents.

Particular attention is given to their potential in the

diagnostics and treatment of neurological disorders,

including neurodegenerative diseases and ischemic

stroke. The use of circRNAs in genome editing tech-

nologies to enhance the efficiency of CRISPR–Cas

systems is very promising. Furthermore, the develop-

ment of next-generation vaccines based on circRNAs,

including antiviral and oncolytic vaccines with im-

proved stability and immunogenicity, represents a

rapidly evolving field.

Despite substantial advances in circRNA re-

search, many important questions remain unresolved.

CircRNAs are still sometimes regarded as byproducts

of aberrant splicing – molecules that exist in cells but

lack functional significance. Moreover, although nu-

merous circRNAs and their potential roles have been

predicted using bioinformatic approaches, many of

these predictions have not yet been validated exper-

imentally, increasing the risk of overinterpretation

or mischaracterization of their biological relevance.

Key challenges include elucidating circRNA-centered

regulatory networks, particularly their roles in mod-

ulating low-affinity molecular interactions; develop-

ing reliable methods for the preparative isolation of

pure circRNAs; overcoming obstacles related to their

efficient and targeted delivery in  vivo; and addressing

a potential ethnic specificity of circRNA-based thera-

peutic strategies. CircRNA research remains a rapidly

evolving field, significant not only for advancing fun-

damental biological knowledge but also for enabling

the development of innovative technologies in mod-

ern medicine and biotechnology.

Abbreviations

circRNA circular  RNA

EIciRNA exon–intron circular  RNA

CIRCULAR RNAs: FIFTY YEARS AFTER DISCOVERY 419

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 3 2026

IRES internal ribosome entry site

mecciRNA mitochondrial-encoded circular RNA

miRNA microRNA

mRNA messenger RNA

cp-circRNA chloroplast genome-encoded circRNA

TDMD target RNA-directed miRNA degrada-

tion

tricRNA tRNA intronic circular RNA

Contributions

I.B.F., O.Yu.S., S.A.L., and L.V.D. developed the concept

and supervised the study; I.B.F., I.V.M., and E.V.Ts.

wrote and edited the manuscript; I.B.F. and E.V.Ts. pre-

pared the figures; S.A.L. and I.B.F. acquisition funding.

Funding

This work was carried out within the state assign-

ment of NRC “Kurchatov Institute” (circRNA-based

technologies) and with the financial support of the

Russian Science Foundation (project no. 25-14-00047;

structure, expression, and functions of circRNAs).

Ethics approval and consent to participate

This work does not contain any studies involving hu-

man or animal subjects.

Conflict of interest

The authors of this work declare that they have no

conflicts of interest.

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