This perspective highlights the emerging significance of noncanonical nucleic acid structures-such as G-quadruplexes, Z-DNA/Z-RNA, and cruciforms-in viral genomes. Once considered structural oddities, these motifs are now recognized as critical regulators of viral replication, transcription, genome stability, and host-pathogen interactions. Despite mounting evidence of their functional relevance and therapeutic potential, these structures remain largely overlooked in virology and antiviral drug development. Their unique conformations offer highly specific molecular targets, with several small molecules already demonstrating the ability to modulate viral gene expression by stabilizing or destabilizing these motifs. The persistent underestimation of non-B DNA/RNA structures represents a missed opportunity in the fight against viral diseases. By synthesizing recent discoveries and emphasizing their biological and pharmacological promise, we aim to elevate awareness and catalyze interdisciplinary research. Harnessing the structural diversity of viral genomes could unlock novel antiviral strategies with high specificity and minimal off-target effects.

Department of Biology and Ecology Faculty of Science University of Ostrava Ostrava Czech Republic

Institute of Biophysics of the Czech Academy of Sciences Brno Czech Republic

School of Biological Sciences University of East Anglia Norwich United Kingdom

Zobrazit více v PubMed

Bartošová-Sojková P, Butenko A, Richtová J, Fiala I, Oborník M, Lukeš J. 2024. Inside the host: understanding the evolutionary trajectories of intracellular parasitism. Annu Rev Microbiol 78:39–59. doi: 10.1146/annurev-micro-041222-025305 PubMed DOI

Yella VR, Vanaja A. 2023. Computational analysis on the dissemination of non-B DNA structural motifs in promoter regions of 1180 cellular genomes. Biochimie 214:101–111. doi: 10.1016/j.biochi.2023.06.002 PubMed DOI

Georgakopoulos-Soares I, Victorino J, Parada GE, Agarwal V, Zhao J, Wong HY, Umar MI, Elor O, Muhwezi A, An J-Y, Sanders SJ, Kwok CK, Inoue F, Hemberg M, Ahituv N. 2022. High-throughput characterization of the role of non-B DNA motifs on promoter function. Cell Genom 2:100111. doi: 10.1016/j.xgen.2022.100111 PubMed DOI PMC

Makova KD, Weissensteiner MH. 2023. Noncanonical DNA structures are drivers of genome evolution. Trends Genet 39:109–124. doi: 10.1016/j.tig.2022.11.005 PubMed DOI PMC

Chiang DC, Li Y, Ng SK. 2021. The role of the Z-DNA binding domain in innate immunity and stress granules. Front Immunol 11. doi: 10.3389/fimmu.2020.625504 PubMed DOI PMC

Schult P, Kümmerer BM, Hafner M, Paeschke K. 2024. Viral hijacking of hnRNPH1 unveils a G-quadruplex-driven mechanism of stress control. Cell Host Microbe 32:1579–1593. doi: 10.1016/j.chom.2024.07.006 PubMed DOI PMC

Brázda V, Bartas M, Bowater RP. 2021. Evolution of diverse strategies for promoter regulation. Trends Genet 37:730–744. doi: 10.1016/j.tig.2021.04.003 PubMed DOI

Mahmoudabadi G, Phillips R. 2018. A comprehensive and quantitative exploration of thousands of viral genomes. eLife 7:e31955. doi: 10.7554/eLife.31955 PubMed DOI PMC

White J. 2020. PubMed 2.0. Med Ref Serv Q 39:382–387. doi: 10.1080/02763869.2020.1826228 PubMed DOI

Zheng A-L, Thermou A, Guixens Gallardo P, Malbert-Colas L, Daskalogianni C, Vaudiau N, Brohagen P, Granzhan A, Blondel M, Teulade-Fichou M-P, Martins RP, Fahraeus R. 2022. The different activities of RNA G-quadruplex structures are controlled by flanking sequences. Life Sci Alliance 5:e202101232. doi: 10.26508/lsa.202101232 PubMed DOI PMC

Amrane S, Jaubert C, Bedrat A, Rundstadler T, Recordon-Pinson P, Aknin C, Guédin A, De Rache A, Bartolucci L, Diene I, Lemoine F, Gascuel O, Pratviel G, Mergny J-L, Andreola M-L. 2022. Deciphering RNA G-quadruplex function during the early steps of HIV-1 infection. Nucleic Acids Res 50:12328–12343. doi: 10.1093/nar/gkac1030 PubMed DOI PMC

Tong Q, Liu G, Sang X, Zhu X, Fu X, Dou C, Jian Y, Zhang J, Zou S, Zhang G, Du X, Liu D, Qi S, Cheng W, Tian Y, Fu X. 2023. Targeting RNA G-quadruplex with repurposed drugs blocks SARS-CoV-2 entry. PLoS Pathog 19:e1011131. doi: 10.1371/journal.ppat.1011131 PubMed DOI PMC

Nicoletto G, Richter SN, Frasson I. 2023. Presence, location and conservation of putative G-quadruplex forming sequences in arboviruses infecting humans. Int J Mol Sci 24:9523. doi: 10.3390/ijms24119523 PubMed DOI PMC

Xu J, Huang H, Zhou X. 2021. G-quadruplexes in neurobiology and virology: functional roles and potential therapeutic approaches. JACS Au 1:2146–2161. doi: 10.1021/jacsau.1c00451 PubMed DOI PMC

Butovskaya E, Soldà P, Scalabrin M, Nadai M, Richter SN. 2019. HIV-1 nucleocapsid protein unfolds stable RNA G-quadruplexes in the viral genome and is inhibited by G-quadruplex ligands. ACS Infect Dis 5:2127–2135. doi: 10.1021/acsinfecdis.9b00272 PubMed DOI PMC

Ji D, Juhas M, Tsang CM, Kwok CK, Li Y, Zhang Y. 2021. Discovery of G-quadruplex-forming sequences in SARS-CoV-2. Brief Bioinform 22:1150–1160. doi: 10.1093/bib/bbaa114 PubMed DOI PMC

Park D, Chung W-C, Gong S, Ravichandran S, Lee GM, Han M, Kim KK, Ahn J-H. 2024. G-quadruplex as an essential structural element in cytomegalovirus replication origin. Nat Commun 15:7353. doi: 10.1038/s41467-024-51797-6 PubMed DOI PMC

Bose D, Panda S, Banerjee N, Chatterjee S. 2025. Dynamic G-quadruplexes in the Rous sarcoma virus genome: scaffolds for protein interaction and potential anti-viral target. Chembiochem 26:e202400941. doi: 10.1002/cbic.202400941 PubMed DOI

Meier-Stephenson V, Badmalia MD, Mrozowich T, Lau KCK, Schultz SK, Gemmill DL, Osiowy C, van Marle G, Coffin CS, Patel TR. 2021. Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA. J Biol Chem 296:100589. doi: 10.1016/j.jbc.2021.100589 PubMed DOI PMC

Brázda V, Dobrovolná M, Bohálová N, Mergny J-L. 2023. G-quadruplexes in the evolution of hepatitis B virus. Nucleic Acids Res 51:7198–7204. doi: 10.1093/nar/gkad556 PubMed DOI PMC

Brázda V, Valková N, Dobrovolná M, Mergny J-L. 2024. Abundance of G-quadruplex forming sequences in the hepatitis delta virus genomes. ACS Omega 9:4096–4101. doi: 10.1021/acsomega.3c09288 PubMed DOI PMC

Kledus F, Dobrovolná M, Mergny J-L, Brázda V. 2025. Asymmetric distribution of G-quadruplex forming sequences in genomes of retroviruses. Sci Rep 15:76. doi: 10.1038/s41598-024-82613-2 PubMed DOI PMC

Biswas B, Kandpal M, Jauhari UK, Vivekanandan P. 2016. Genome-wide analysis of G-quadruplexes in herpesvirus genomes. BMC Genomics 17:949. doi: 10.1186/s12864-016-3282-1 PubMed DOI PMC

Bohálová N, Cantara A, Bartas M, Kaura P, Šťastný J, Pečinka P, Fojta M, Mergny J-L, Brázda V. 2021. Analyses of viral genomes for G-quadruplex forming sequences reveal their correlation with the type of infection. Biochimie 186:13–27. doi: 10.1016/j.biochi.2021.03.017 PubMed DOI

Bohálová N, Cantara A, Bartas M, Kaura P, Šťastný J, Pečinka P, Fojta M, Brázda V. 2021. Tracing dsDNA virus-host coevolution through correlation of their G-quadruplex-forming sequences. Int J Mol Sci 22:3433. doi: 10.3390/ijms22073433 PubMed DOI PMC

Ruggiero E, Richter SN. 2023. G-quadruplexes in human viruses: a promising route to innovative antiviral therapies, p 2465–2492. In Handbook of chemical biology of nucleic acids. Springer, Singapore.

Ryazantsev DY, Myshkin MY, Alferova VA, Tsvetkov VB, Shustova EY, Kamzeeva PN, Kovalets PV, Zaitseva ER, Baleeva NS, Zatsepin TS, Shenkarev ZO, Baranov MS, Kozlovskaya LI, Aralov AV. 2021. Probing GFP chromophore analogs as anti-HIV agents targeting LTR-III G-quadruplex. Biomolecules 11:1409. doi: 10.3390/biom11101409 PubMed DOI PMC

Gao C, Wei S, Xu Y, Mohamed HI, Liu W, Wang Z, Wu W, Wang M, He Y. 2025. BRACO-19 targeted the G-quadruplex in the 3’UTR of the Cucumber mosaic virus 1a, 2b, and CP genes to inhibit viral proliferation. Pest Manag Sci 81:4027–4034. doi: 10.1002/ps.8769 PubMed DOI

Kuś K, Rakus K, Boutier M, Tsigkri T, Gabriel L, Vanderplasschen A, Athanasiadis A. 2015. The structure of the Cyprinid herpesvirus 3 ORF112-Zα•Z-DNA complex reveals a mechanism of nucleic acids recognition conserved with E3L, a poxvirus inhibitor of interferon response. J Biol Chem 290:30713–30725. doi: 10.1074/jbc.M115.679407 PubMed DOI PMC

Peterson JM, Becker ST, O’Leary CA, Juneja P, Yang Y, Moss WN. 2024. Structure of the SARS-CoV-2 frameshift stimulatory element with an upstream multibranch loop. Biochemistry 63:1287–1296. doi: 10.1021/acs.biochem.3c00716 PubMed DOI PMC

Terrell JR, Le TT, Paul A, Brinton MA, Wilson WD, Poon GMK, Germann MW, Siemer JL. 2024. Structure of an RNA G-quadruplex from the West Nile virus genome. Nat Commun 15:5428. doi: 10.1038/s41467-024-49761-5 PubMed DOI PMC

Zaccaria F, Fonseca Guerra C. 2018. RNA versus DNA G-quadruplex: the origin of increased stability. Chemistry 24:16315–16322. doi: 10.1002/chem.201803530 PubMed DOI PMC

Robinson J, Raguseo F, Nuccio SP, Liano D, Di Antonio M. 2021. DNA G-quadruplex structures: more than simple roadblocks to transcription? Nucleic Acids Res 49:8419–8431. doi: 10.1093/nar/gkab609 PubMed DOI PMC

Sato K, Martin-Pintado N, Post H, Altelaar M, Knipscheer P. 2021. Multistep mechanism of G-quadruplex resolution during DNA replication. Sci Adv 7:eabf8653. doi: 10.1126/sciadv.abf8653 PubMed DOI PMC

van Wietmarschen N, Merzouk S, Halsema N, Spierings DCJ, Guryev V, Lansdorp PM. 2018. BLM helicase suppresses recombination at G-quadruplex motifs in transcribed genes. Nat Commun 9:271. doi: 10.1038/s41467-017-02760-1 PubMed DOI PMC

Perrone R, Nadai M, Frasson I, Poe JA, Butovskaya E, Smithgall TE, Palumbo M, Palù G, Richter SN. 2013. A dynamic G-quadruplex region regulates the HIV-1 long terminal repeat promoter. J Med Chem 56:6521–6530. doi: 10.1021/jm400914r PubMed DOI PMC

Saranathan N, Vivekanandan P. 2019. G-quadruplexes: more than just a kink in microbial genomes. Trends Microbiol 27:148–163. doi: 10.1016/j.tim.2018.08.011 PubMed DOI PMC

Jovin TM. 2023. The origin of left-handed poly[d(G-C)], p 1–32. In Kim KK, Subramani VK (ed), Z-DNA: methods and protocols. Springer US, New York, NY. PubMed

Boyd DF, Jordan SV, Balachandran S. 2025. ZBP1-driven cell death in severe influenza. Trends Microbiol 33:521–532. doi: 10.1016/j.tim.2024.12.008 PubMed DOI PMC

Evdokimova M, Feng S, Caobi A, Moreira FR, Jones D, Alysandratos K-D, Tully ES, Kotton DN, Boyd DF, Banach BS, Kirchdoerfer RN, Saeed M, Baker SC. 2025. Coronavirus endoribonuclease antagonizes ZBP1-mediated necroptosis and delays multiple cell death pathways. Proc Natl Acad Sci USA 122:e2419620122. doi: 10.1073/pnas.2419620122 PubMed DOI PMC

Herbert A. 2024. The ancient Z-DNA and Z-RNA specific Zα fold has evolved modern roles in immunity and transcription through the natural selection of flipons. R Soc Open Sci 11:240080. doi: 10.1098/rsos.240080 PubMed DOI PMC

Romero MF, Krall JB, Nichols PJ, Vantreeck J, Henen MA, Dejardin E, Schulz F, Vicens Q, Vögeli B, Diallo MA. 2024. Novel Z-DNA binding domains in giant viruses. J Biol Chem 300:107504. doi: 10.1016/j.jbc.2024.107504 PubMed DOI PMC

Xie F, Wu D, Huang J, Liu X, Shen Y, Huang J, Su Z, Li J. 2024. ZBP1 condensate formation synergizes Z-NAs recognition and signal transduction. Cell Death Dis 15:487. doi: 10.1038/s41419-024-06889-y PubMed DOI PMC

Brazda V, Fojta M, Bowater RP. 2020. Structures and stability of simple DNA repeats from bacteria. Biochem J 477:325–339. doi: 10.1042/BCJ20190703 PubMed DOI PMC

Brázda V, Laister RC, Jagelská EB, Arrowsmith C. 2011. Cruciform structures are a common DNA feature important for regulating biological processes. BMC Mol Biol 12:33. doi: 10.1186/1471-2199-12-33 PubMed DOI PMC

Gupta S, Pal D. 2021. Clusters of hairpins induce intrinsic transcription termination in bacteria. Sci Rep 11:16194. doi: 10.1038/s41598-021-95435-3 PubMed DOI PMC

Bowater RP, Bohálová N, Brázda V. 2022. Interaction of proteins with inverted repeats and cruciform structures in nucleic acids. Int J Mol Sci 23:6171. doi: 10.3390/ijms23116171 PubMed DOI PMC

Goswami P, Bartas M, Lexa M, Bohálová N, Volná A, Červeň J, Červeňová V, Pečinka P, Špunda V, Fojta M, Brázda V. 2021. SARS-CoV-2 hot-spot mutations are significantly enriched within inverted repeats and CpG island loci. Brief Bioinformatics 22:1338–1345. doi: 10.1093/bib/bbaa385 PubMed DOI PMC

Dobrovolná M, Brázda V, Warner EF, Bidula S. 2023. Inverted repeats in the monkeypox virus genome are hot spots for mutation. J Med Virol 95:e28322. doi: 10.1002/jmv.28322 PubMed DOI PMC

Shen W, Wang Z, Ning K, Cheng F, Engelhardt JF, Yan Z, Qiu J. 2021. Hairpin transfer-independent parvovirus DNA replication produces infectious virus. J Virol 95:e01108-21. doi: 10.1128/JVI.01108-21 PubMed DOI PMC

Lkharrazi A, Tobler K, Marti S, Bratus-Neuenschwander A, Vogt B, Fraefel C. 2024. AAV2 can replicate its DNA by a rolling hairpin or rolling circle mechanism, depending on the helper virus. J Virol 98:e01282-24. doi: 10.1128/jvi.01282-24 PubMed DOI PMC

Brázda V, Kolomazník J, Lýsek J, Bartas M, Fojta M, Šťastný J, Mergny J-L. 2019. G4Hunter web application: a web server for G-quadruplex prediction. Bioinformatics 35:3493–3495. doi: 10.1093/bioinformatics/btz087 PubMed DOI PMC

Hon J, Martínek T, Zendulka J, Lexa M. 2017. Pqsfinder: an exhaustive and imperfection-tolerant search tool for potential quadruplex-forming sequences in R. Bioinformatics 33:3373–3379. doi: 10.1093/bioinformatics/btx413 PubMed DOI

Garant J-M, Perreault J-P, Scott MS. 2018. G4RNA screener web server: user focused interface for RNA G-quadruplex prediction. Biochimie 151:115–118. doi: 10.1016/j.biochi.2018.06.002 PubMed DOI

Beknazarov N, Jin S, Poptsova M. 2020. Deep learning approach for predicting functional Z-DNA regions using omics data. Sci Rep 10:19134. doi: 10.1038/s41598-020-76203-1 PubMed DOI PMC

Puig Lombardi E, Londoño-Vallejo A. 2020. A guide to computational methods for G-quadruplex prediction. Nucleic Acids Res 48:1–15. doi: 10.1093/nar/gkz1097 PubMed DOI PMC

Luo D, Zheng Y, Huang Z, Wen Z, Guo L, Deng Y, Li Q, Bai Y, Haider S, Wei D. 2025. Exploiting functional regions in the viral RNA genome as druggable entities. eLife 13:RP103923. doi: 10.7554/eLife.103923 PubMed DOI PMC

Yang SY, Monchaud D, Wong JMY. 2022. Global mapping of RNA G-quadruplexes (G4-RNAs) using G4RP-seq. Nat Protoc 17:870–889. doi: 10.1038/s41596-021-00671-6 PubMed DOI

Artusi S, Ruggiero E, Nadai M, Tosoni B, Perrone R, Ferino A, Zanin I, Xodo L, Flamand L, Richter SN. 2021. Antiviral activity of the G-quadruplex ligand TMPyP4 against herpes simplex virus-1. Viruses 13:196. doi: 10.3390/v13020196 PubMed DOI PMC

Majee P, Pattnaik A, Sahoo BR, Shankar U, Pattnaik AK, Kumar A, Nayak D. 2021. Inhibition of Zika virus replication by G-quadruplex-binding ligands. Mol Ther Nucleic Acids 23:691–701. doi: 10.1016/j.omtn.2020.12.030 PubMed DOI PMC

Xu H, Di Antonio M, McKinney S, Mathew V, Ho B, O’Neil NJ, Santos ND, Silvester J, Wei V, Garcia J, et al. 2017. CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours. Nat Commun 8:14432. doi: 10.1038/ncomms14432 PubMed DOI PMC

Tosoni B, Naghshineh E, Zanin I, Gallina I, Di Pietro L, Cleris L, Nadai M, Lecchi M, Verderio P, Pratesi P, Pasquali S, Zaffaroni N, Neidle S, Folini M, Richter SN. 2025. The G-quadruplex experimental drug QN-302 impairs liposarcoma cell growth by inhibiting MDM2 expression and restoring p53 levels. Nucleic Acids Res 53:gkaf085. doi: 10.1093/nar/gkaf085 PubMed DOI PMC

Sun Y, Zhao C, Liu Y, Wang Y, Zhang C, Yang J, Qin G, Song H, Postings M, Scott P, Ren J, Qu X. 2025. Screening of metallohelices for enantioselective targeting SARS-CoV-2 RNA G-quadruplex. Nucleic Acids Res 53:gkaf199. doi: 10.1093/nar/gkaf199 PubMed DOI PMC

Ruggiero E, Richter SN. 2018. G-quadruplexes and G-quadruplex ligands: targets and tools in antiviral therapy. Nucleic Acids Res 46:3270–3283. doi: 10.1093/nar/gky187 PubMed DOI PMC

Monchaud D. 2024. Translating G-quadruplex ligands from bench to bedside: a Stephen Neidle’s legacy. Med Chem Res 33:2020–2029. doi: 10.1007/s00044-024-03310-3 DOI

Oh J, Kim H, Lee J, Kim S, Shin S, Kim Y-E, Park S, Lee S. 2025. Korean red ginseng enhances ZBP1-mediated cell death to suppress viral protein expression in host defense against influenza A virus. J Microbiol 63:e. doi: 10.71150/jm.2409007 PubMed DOI