DNA in eukaryotes usually exists in a double helix structure,and two deoxynucleotide chains are connected by hydrogen bonds according to the principle of complementary base pairing,forming a stable B-type double helix structure(two complementary double helix chains are antiparallel,with the same central longitudinal axis,spiraling from the right to the top).However,during replication,transcription,and DNA repair,double-stranded DNA may be temporarily unwound into two Single-stranded DNA(ssDNA).single-stranded DNA is related to some non-B-type structures,such as R-loop,G-quadruplex,H-DNA,Slipped-strand,hairpin structure,cruciform,etc^{[1][2][3][4][5][6]}。 These single-stranded DNAs and non-B-type structures containing single-stranded DNAs in cells regulate many important cellular processes.Therefore,high-throughput sequencing of intracellular ssDNA is an important means to understand the location of ssDNA in the genome and to carry out functional studies。

ssDNA can arise from normal biological processes.In the process of DNA replication,transcription and repair,dsDNA is partially melted.If the temporarily formed ssDNA has a special sequence,it may form a non-B-type structure and produce a relatively stable ssDNA structure.For example,the R-loop is formed by the combination of the new RNA and the template DNA strand during transcription,thus replacing the non-template DNA strand.the local three-stranded structure is composed of the replaced single-stranded DNA and an RNA-DNA hybrid(Fig.1A).the RNA-DNA hybrid structure is often more stable than the double-stranded DNA,so the R-loop can exist stably^[7,8]。 G-quadruplex is usually folded by DNA sequences rich in tandem repeats.G-quartet is the basic structural unit of G-quadruplex,which is a square planar structure composed of four guanines,and each guanine is connected with two adjacent guanines through Hoogsteen hydrogen bond interaction(Fig.1B).Hoogsteen hydrogen bond is a special type of hydrogen bond,which is different from the common Watson-Crick hydrogen bond in the relative position,orientation and special pairing between bases.It connects the bases in a non-classical way and helps to maintain the stability of DNA with specific structure^[9]。 A stack of multiple G-tetrad planes forms a G-quadruplex,and the sequences between them are squeezed into a single-stranded loop^[10]; At that same time,when the G-quadruplex is compose of one strand of dsDNA,the complementary strand cannot pair with it to form a single strand(Fig.1B).H-DNA is an intramolecular triplex,with the third strand folded inward by the rotation of the mirror repeat in one strand of the double-stranded DNA molecule,resulting in a triplex structure,and the other strand unpaired,single-stranded DNA(fig.1C)^[11]。 in the triplex structure of H-DNA,purines In the double strand can form Hoogsteen hydrogen bonds with the third strand bases through rotation around the glycosidic bond and base flipping,thus maintaining the stability of the structure^[12]。 A slip-strand structure is formed when the direct repeat base pairs with the complementary strand in a misaligned manner(Fig.1D),and after DNA unwinding,a hairpin or exocyclic base may be generated^[13]。 the inverted repeats can form hairpin structures(Fig.1E)and cruciform structures(Fig.1F).In this structure,sequences equidistant from the center of symmetry are complementary to each other,thus forming an intrachain hairpin,with the nonrepetitive sequence at the center of the structure protruding to form a single-stranded loop.the hairpin structure is formed when the single-stranded DNA inverted repeat sequence folds back on itself to form an intrastrand pair^[14]。 Similarly,inverted repeats in double-stranded DNA may trigger two intrastrand hairpins,forming a cruciform structure^[15]。

single-stranded DNA is produced in many cellular processes,and the structure containing Single-stranded DNA will in turn affect some important cellular physiological processes,including the transcription of genetic information,DNA replication,homologous recombination repair,meiosis and so on^[16⇓~18]。

R-loops can play a role in transcriptional activation and termination in mammals.R-loops of different lengths formed under different conditions help recruit specific chromatin remodeling factors to regulate transcription.Many ncRNAs may also assist transcriptional control by changing chromatin status through R-loops^[19]。 When R-loops accumulate during transcription,SOSS-INTAC exerts RNA endonuclease action to induce termination of transcription near the promoter,possibly through SSB1-mediated single-stranded DNA recognition,thereby preventing genomic instability induced by R-loop accumulation^[20]。 In addition,G-quadruplex structures may be formed during transcription,which may either promote or inhibit transcription at or near the promoter region.If the G-quadruplex motif is located on the template strand,it blocks the transcription mechanism and thus inhibits transcription^[21]。 If the G-quadruplex motif is located on the non-template strand,it may prevent annealing of the transcription template to the complementary strand to maintain the single-stranded conformation of the transcribed strand to facilitate transcription^[10]。 Negative supercoiling generated by the transcription process may induce the formation of H-DNA,and in some cases,nascent purine-rich RNA binding to single-stranded DNA in the H-DNA structure can further stabilize the formation of H-DNA,which may inhibit or block the transcription process when it is formed^[11]。 Hairpin or cruciform structures affect the helical state of DNA and may promote or prevent DNA-protein interactions,thereby affecting the transcription process.cruciform overhang reduces local supercoiling of DNA,and local changes in supercoiling density can affect promoter activity,so cruciform overhang of promoter regions may reduce its activity^[22]。

For DNA replication,in the initial stage of replication,RNA will act as a primer to participate in complementary pairing with template DNA as an origin of replication to form DNA:RNA hybrids,but the length of DNA:RNA hybrids produced by DNA replication is shorter,which is different from the more stable R-loop^[23]。 G-quadruplexes are easily produced in the lagging strand of DNA replication,and when replication is slowed down,G-quadruplexes are more easily formed,and replication will not continue after the formation of G-quadruplex,and it is likely that helicases are needed to unwind the G-quadruplex structure^[10]。 in addition,simultaneous DNA replication and gene transcription in eukaryotic and prokaryotic cells may occur"conflict",that is,when DNA replication and gene transcription occur in opposite directions or in the same direction,R-loops formed during transcription may affect DNA replication^[24]。 Because R-loops block DNA replication forks,they may cause DNA damage,such as DNA replication fork breaks,DNA double-strand breaks and single-strand DNA gaps(ssDNA gaps)^[25,26]。 When these DNA damages occur,repair mechanisms such as homologous recombination are generally activated to repair DNA damage in time to avoid adverse effects on genome stability such as gene mutation^[17,27]。 R-loops can trigger homologous recombination,and the occurrence of this type of homologous recombination associated with gene transcription(Transcription associated recombination,TAR)may prevent cells from Transcription-induced genomic instability^[24,28]。 in studies of S.cerevisiae and C.elegans,it was found that R-loops can form in meiosis and negatively affect meiotic replication and genome stability,while THO and THSC/TREX-2,which can prevent the formation of R-loops in meiosis,play an important role in the reproductive inheritance of organisms^[29]。

In addition to affecting cellular physiological processes,single-stranded DNA is vulnerable to nucleases,chemical agents,and inappropriate protein binding,affecting genome stability^[18]。 single-stranded DNA may also be a target for the action of specific mutagens or enzymes,such as AID/APOBEC cytosine deaminase,AID(activation-induced deaminase)expressed predominantly in B cells,Its main function is to convert cytosine(C)in Single-stranded DNA to uracil(U)by deamination,and to mediate somatic hypermutation and immunoglobulin class recombination switch with APOBEC(polypeptide-like apolipoprotein B that catalyzes mRNA editing)^[30,31]。 ssDNA is more susceptible to mutagenic DNA damage and mutation than dsDNA,and therefore,single-stranded DNA regions need to be adequately protected to avoid loss of genetic information^[32]。 When ssDNA is generated,it is usually rapidly wrapped by single-stranded DNA-binding proteins such as Replication protein A(RPA)complex.This not only protects ssDNA from nuclease degradation,but also coordinates the activation of DNA damage checkpoint response and DNA repair,while RPA also prevents the formation of DNA secondary structure^[33][34][35]。 However,because RPA binding to ssDNA is dynamic,RPA is unable to fully protect the bases of ssDNA from chemical damage^[36]。 Nucleases such as Yen1/GEN1 and SLX1/SLX4 can recognize and cleave cruciform structures,and are also recruited to cut fragile sites containing repetitive sequences,resulting in double-strand breaks and genomic instability^[37]。 the slip-strand structure may produce mismatch repair in The process of DNA damage repair,leading to genome instability^[38]。

Single-stranded DNA is also associated with many human diseases and cancers.the H-DNA formed in the human c-MYC gene promoter coincides with a breakpoint associated with c-MYC-induced leukemia,and the H-DNA in the human PKD1 gene may increase the mutation rate of the gene and lead to the genetic disease Autosomal dominant polycystic kidney disease(ADPKD)^{[39,40][41,42]}。 Transcription of repeats such as CTG and GAA may lead to the formation of R-loops and promote the instability of the repeats themselves,resulting in their expansion or contraction^[43][44,45]。 nervous system diseases such as Friedreich ataxia(FRDA)and fragile X syndrome are closely related to the genetic instability of trinucleotide repeat expansion,so R-loop may be a potential factor inducing these Nervous system diseases^[43][46][47]。 the clearance of intracellular R-loops depends on RNase H,which specifically recognizes the DNA-RNA hybrid in the R-loop and excises the RNA strand in it.When the activity of RNase H1 is reduced or RNase H1 and RNase H2 are knocked out,the instability of CAG and other repetitive sequences will increase.in addition to RNase H,R-loops can also be unwound by DNA-RNA helicases such as AQR and SETX,which are mutated in two neurodegenerative diseases and are closely related to the accumulation of R-loops^[44][48,49]。

genomic instability and replication stress are symptomatic hallmarks of tumor cells,and R-loop accumulation can lead to Genomic instability and replication stress,so R-loops are potential drivers of cancer^[50]。 R-loops are linked to cancer through the DNA Double strand break(DSB)repair genes BRCA1 and BRCA2.When cells knock out BRCA1 or BRCA2,R-loop accumulation and DSB increase,which is partially reduced by overexpression of RNase H1^[51,52]。 Upon R-loop dysregulation,a portion of the nuclear R-loop that cannot be resolved is processed by XPG,resulting in cytoplasmic accumulation of DNA-RNA hybrids that are subsequently recognized by cytoplasmic cGAS and TLR3,activating IRF3-mediated immune signaling and apoptosis^[53]。 Burkitt lymphoma is caused by the translocation between the MYC proto-oncogene and the immunoglobulin S region.in mice,AID is important for the generation of DNA double-strand breaks In the GC-rich S region and the transcribed Myc region,and this process may form R-loops,so R-loops may be the cause of Burkitt lymphoma^[19]。

G-quadruplexes have been implicated in human leukemia.In some leukemia patients,the expression of Aven protein is increased.MLL1 and MLL4 are two important genes related to leukemia.In the coding region of mRNA,Aven protein can bind to the G-quadruplex structure of RGG/RG region,promote the expression of MLL1 and MLL4,and induce the transcription of leukemia genes.Loss of Aven protein results in decreased synthesis of MLL1 and MLL4 proteins,leading to decreased proliferation of leukemic cells^[54]。 G-quadruplex is also closely related to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia.the formation of G-quadruplex structure will block the movement of RNA polymerase II during transcription and stop transcription.This not only leads to the loss of normal protein products,but also to incomplete transcript fragments,which can fold into G-quadruplex structures by themselves,which may hinder RNA-binding proteins and lead to nucleolar stress response to damage cells.It may also escape from the nucleus and bind to ribosomal complexes to be translated into dipeptide repeat proteins to destroy cells^[55]。

Among the existing single-stranded DNA sequencing technologies,only some methods can be used to sequence ssDNA structures,such as ssDNA-seq,KAS-seq,etc.,and most of the methods are used to sequence and locate specific non-B-type structures(mainly R-loops and G-quadruplexes)containing single-stranded DNA,such as DRIP-seq,R-ChIP,MapR,SMRF-seq^[56]。

ssDNA exists widely in organisms and plays an important role in maintaining the normal function of cells and the transmission of genetic information.with the deepening of research,its importance has become increasingly prominent.in this paper,the types of intracellular ssDNA,the causes of its formation,its biological functions,its relationship With diseases,and its high-throughput analysis techniques are described.However,due to the limitation of ssDNA high-throughput analysis technology,the distribution and function of ssDNA in the genome still have problems and challenges.Accurate analysis of the distribution of ssDNA across the genome is critical to understanding highly dynamic transcriptional events,DNA replication and repair,and other processes。

in terms of methodology,the following aspects can be improved and attempted in future studies:establishing a new mild ssDNA labeling method;Reduce the damage of ssDNA structure caused by DNA treatment;Improve method resolution.Methodological advances will also further reveal the biological functions of the above ssDNA and help people understand the role of ssDNA in disease。