Parasitic helminths infecting humans are highly prevalent infecting ∼2 billion people worldwide, causing inflammatory responses, malnutrition and anemia that are the primary cause of morbidity. In addition, helminth infections of cattle have a significant economic impact on livestock production, milk yield and fertility. The etiological agents of helminth infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms). G-quadruplexes (G4) are unusual nucleic acid structures formed by G-rich sequences that can be recognized by specific G4 ligands. Here we used the G4Hunter Web Tool to identify and compare potential G4 sequences (PQS) in the nuclear and mitochondrial genomes of various helminths to identify G4 ligand targets. PQS are nonrandomly distributed in these genomes and often located in the proximity of genes. Unexpectedly, a Nematode, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This species can tolerate high-stability G4 structures, which are not counter selected at all, in stark contrast to most other species. We experimentally confirmed G4 formation for sequences found in four different parasitic helminths. Small molecules able to selectively recognize G4 were found to bind to Schistosoma mansoni G4 motifs. Two of these ligands demonstrated potent activity both against larval and adult stages of this parasite.

ARNA Laboratory Université de Bordeaux INSERM U1212 CNRS UMR 5320 UFR des Sciences Pharmaceutiques Bordeaux France

CNRS UMR9187 INSERM U1196 Institut Curie PSL Research University F 91405 Orsay France

CNRS UMR9187 INSERM U1196 Université Paris Saclay F 91405 Orsay France

Department of Experimental Biology Faculty of Science Masaryk University Kamenice 5 62500 Brno Czech Republic

Department of Medical Parasitology and Infection Biology Swiss Tropical and Public Health Institute Basel Switzerland

Faculty of Chemistry Brno University of Technology Purkyňova 118 612 00 Brno Czech Republic

Institute of Biophysics Czech Academy of Sciences Královopolská 135 612 65 Brno Czech Republic

Laboratoire d'Optique et Biosciences Ecole Polytechnique CNRS INSERM Institut Polytechnique de Paris 91128 Palaiseau France

Université Sorbonne Paris Nord UFR SMBH Bobigny France

University of Basel Basel Switzerland

See more in PubMed

Hotez P.J., Bundy D.A.P., Beegle K., Brooker S., Drake L., de Silva N., Montresor A., Engels D., Jukes M., Chitsulo L.et al. .. Jamison D.T., Breman J.G., Measham A.R., Alleyne G., Claeson M., Evans D.B., Jha P., Mills A., Musgrove P.. Helminth Infections: soil-transmitted helminth infections and schistosomiasis. Disease Control Priorities in Developing Countries. 2006; 2nd ednWashington (DC).

Bethony J., Brooker S., Albonico M., Geiger S.M., Loukas A., Diemert D., Hotez P.J.. Soil-transmitted helminth infections: ascariasis, trichuriasis and hookworm. The Lancet. 2006; 367:1521–1532. PubMed

Verjee MA. Schistosomiasis: still a cause of significant morbidity and mortality. Res. Rep. Trop. Med. 2019; 10:153–163. PubMed PMC

Anderson T.J.C., Duraisingh M.T.. Transformative tools for parasitic flatworms. Science. 2020; 369:1562–1564. PubMed

Christian P., Khatry S.K., West K.P.. Antenatal anthelmintic treatment, birthweight, and infant survival in rural Nepal. Lancet. 2004; 364:981–983. PubMed

Charlier J., De Waele V., Ducheyne E., van der Voort M., Vande Velde F., Claerebout E.. Decision making on helminths in cattle: diagnostics, economics and human behaviour. Irish Vet. J. 2016; 69:14. PubMed PMC

de Silva N.R., Brooker S., Hotez P.J., Montresor A., Engels D., Savioli L.. Soil-transmitted helminth infections: updating the global picture. Trends Parasitol. 2003; 19:547–551. PubMed

Nutman T.B. Human infection with Strongyloides stercoralis and other related Strongyloides species. Parasitology. 2017; 144:263–273. PubMed PMC

Colley D.G., Bustinduy A.L., Secor W.E., King C.H.. Human schistosomiasis. Lancet. 2014; 383:2253–2264. PubMed PMC

Thétiot-Laurent S.A.-L., Boissier J., Robert A., Meunier B.. Schistosomiasis chemotherapy. Angew. Chem. Int. Ed. 2013; 52:7936–7956. PubMed

Furtado L.F.V., de Paiva Bello A.C.P., Rabelo É.M.L.. Benzimidazole resistance in helminths: from problem to diagnosis. Acta Tropica. 2016; 162:95–102. PubMed

Bergquist R., Zhou X.-N., Rollinson D., Reinhard-Rupp J., Klohe K.. Elimination of schistosomiasis: the tools required. Infect. Dis. Poverty. 2017; 6:158. PubMed PMC

Berriman M., Haas B.J., LoVerde P.T., Wilson R.A., Dillon G.P., Cerqueira G.C., Mashiyama S.T., Al-Lazikani B., Andrade L.F., Ashton P.D.et al. .. The genome of the blood fluke Schistosoma mansoni. Nature. 2009; 460:352–358. PubMed PMC

Zhou Y., Zheng H., Chen Y., Zhang L., Wang K., Guo J., Huang Z., Zhang B., Huang W., Jin K.et al. .. The Schistosoma japonicum genome reveals features of host–parasite interplay. Nature. 2009; 460:345–351. PubMed PMC

Young N.D., Jex A.R., Li B., Liu S., Yang L., Xiong Z., Li Y., Cantacessi C., Hall R.S., Xu X.et al. .. Whole-genome sequence of Schistosoma haematobium. Nat. Genet. 2012; 44:221–225. PubMed

Kolesnikova S., Curtis E.A.. Structure and function of multimeric G-quadruplexes. Molecules. 2019; 24:3074. PubMed PMC

Burge S., Parkinson G.N., Hazel P., Todd A.K., Neidle S.. Quadruplex DNA: sequence, topology and structure. Nucleic Acids Res. 2006; 34:5402–5415. PubMed PMC

Lane A.N., Chaires J.B., Gray R.D., Trent J.O.. Stability and kinetics of G-quadruplex structures. Nucleic Acids Res. 2008; 36:5482–5515. PubMed PMC

Yoshida W., Saikyo H., Nakabayashi K., Yoshioka H., Bay D.H., Iida K., Kawai T., Hata K., Ikebukuro K., Nagasawa K.et al. .. Identification of G-quadruplex clusters by high-throughput sequencing of whole-genome amplified products with a G-quadruplex ligand. Sci. Rep. 2018; 8:3116. PubMed PMC

Carvalho J., Mergny J.L., Salgado G.F., Queiroz J.A., Cruz C.. G-quadruplex, friend or foe: the role of the G-quartet in anticancer strategies. Trends Mol. Med. 2020; 26:848–861. PubMed

Balasubramanian S., Hurley L.H., Neidle S.. Targeting G-quadruplexes in gene promoters: a novel anticancer strategy. Nat. Rev. Drug Discov. 2011; 10:261–275. PubMed PMC

Siddiqui-Jain A., Grand C.L., Bearss D.J., Hurley L.H.. Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc. Natl. Acad. Sci. U.S.A. 2002; 99:11593–11598. PubMed PMC

Sun Z.-Y., Wang X.-N., Cheng S.-Q., Su X.-X., Ou T.-M.. Developing novel G-quadruplex ligands: from interaction with nucleic acids to interfering with nucleic acid–protein interaction. Molecules. 2019; 24:396. PubMed PMC

Zhao J., Bacolla A., Wang G., Vasquez K.M.. Non-B DNA structure-induced genetic instability and evolution. Cell. Mol. Life Sci. 2010; 67:43–62. PubMed PMC

Wong H.M., Huppert J.L.. Stable G-quadruplexes are found outside nucleosome-bound regions. Mol. BioSys. 2009; 5:1713–1719. PubMed

Marsico G., Chambers V.S., Sahakyan A.B., McCauley P., Boutell J.M., di Antonio M., Balasubramanian S.. Whole genome experimental maps of DNA G-quadruplexes in multiple species. Nucleic Acids Res. 2019; 47:3862–3874. PubMed PMC

Hoffmann R.F., Moshkin Y.M., Mouton S., Grzeschik N.A., Kalicharan R.D., Kuipers J., Wolters A.H.G., Nishida K., Romashchenko A.V., Postberg J.et al. .. Guanine quadruplex structures localize to heterochromatin. Nucleic Acids Res. 2016; 44:152–163. PubMed PMC

Yu Q.-Q., Gao J.-J., Lang X.-X., Li H.-Y., Wang M.-Q.. Microenvironment-sensitive fluorescent ligand binds ascaris telomere antiparallel G-quadruplex DNA with blue-shift and enhanced emission. ChemBioChem. 2021; 22:1042–1048. PubMed

Craven H.M., Bonsignore R., Lenis V., Santi N., Berrar D., Swain M., Whiteland H., Casini A., Hoffmann K.F.. Identifying and validating the presence of guanine-quadruplexes (G4) within the blood fluke parasite Schistosoma mansoni. PLoS Negl. Trop. Dis. 2021; 15:e0008770. PubMed PMC

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

Mergny J.L., Phan A.T., Lacroix L.. Following G-quartet formation by UV-spectroscopy. FEBS Lett. 1998; 435:74–78. PubMed

Luo Y., Granzhan A., Verga D., Mergny J.-L.. FRET-MC: a fluorescence melting competition assay for studying G4 structures in vitro. Biopolymers. 2021; 112:e23415. PubMed

Mergny J.-L., Li J., Lacroix L., Amrane S., Chaires J.B.. Thermal difference spectra: a specific signature for nucleic acid structures. Nucleic Acids Res. 2005; 33:e138. PubMed PMC

Renaud de la Faverie A., Guédin A., Bedrat A., Yatsunyk L.A., Mergny J.-L.. Thioflavin T as a fluorescence light-up probe for G4 formation. Nucleic Acids Res. 2014; 42:e65. PubMed PMC

Guillon J., Denevault-Sabourin C., Chevret E., Brachet-Botineau M., Milano V., Guédin-Beaurepaire A., Moreau S., Ronga L., Savrimoutou S., Rubio S.et al. .. Design, synthesis, and antiproliferative effect of 2,9-bis[4-(pyridinylalkylaminomethyl)phenyl]-1,10-phenanthroline derivatives on human leukemic cells by targeting G-quadruplex. Archiv. Pharmazie. 2021; 354:e2000450. PubMed

Guillon J., Cohen A., Das R.N., Boudot C., Gueddouda N.M., Moreau S., Ronga L., Savrimoutou S., Basmaciyan L., Tisnerat C.et al. .. Design, synthesis, and antiprotozoal evaluation of new 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline derivatives. Chem. Biol. Drug Des. 2018; 91:974–995. PubMed

Guillon J., Cohen A., Boudot C., Valle A., Milano V., Das R.N., Guédin A., Moreau S., Ronga L., Savrimoutou S.et al. .. Design, synthesis, and antiprotozoal evaluation of new 2,4-bis[(substituted-aminomethyl)phenyl]quinoline, 1,3-bis[(substituted-aminomethyl)phenyl]isoquinoline and 2,4-bis[(substituted-aminomethyl)phenyl]quinazoline derivatives. J. Enz. Inhib. Med. Chem. 2020; 35:432–459. PubMed PMC

Lombardo F.C., Pasche V., Panic G., Endriss Y., Keiser J.. Life cycle maintenance and drug-sensitivity assays for early drug discovery in Schistosoma mansoni. Nat. Protoc. 2019; 14:461–481. PubMed

Crooks G.E., Hon G., Chandonia J.-M., Brenner S.E.. WebLogo: a sequence logo generator. Genome Res. 2004; 14:1188–1190. PubMed PMC

Coghlan A., Tyagi R., Cotton J.A., Holroyd N., Rosa B.A., Tsai I.J., Laetsch D.R., Beech R.N., Day T.A., Hallsworth-Pepin K.et al. .. Comparative genomics of the major parasitic worms. Nat. Gen. 2019; 51:163–174. PubMed PMC

Jourdan P.M., Lamberton P.H.L., Fenwick A., Addiss D.G.. Soil-transmitted helminth infections. Lancet. 2018; 391:252–265. PubMed

Bartas M., Čutová M., Brázda V., Kaura P., Šťastný J., Kolomazník J., Coufal J., Goswami P., Červen J., Pečinka P.. The presence and localization of G-quadruplex forming sequences in the domain of bacteria. Molecules. 2019; 24:1711. PubMed PMC

Brázda V., Luo Y., Bartas M., Kaura P., Porubiaková O., Šťastný J., Pečinka P., Verga D., Da Cunha V., Takahashi T.S.et al. .. G-Quadruplexes in the archaea domain. Biomolecules. 2020; 10:1349. PubMed PMC

Wang J., Gao S., Mostovoy Y., Kang Y., Zagoskin M., Sun Y., Zhang B., White L.K., Easton A., Nutman T.B.et al. .. Comparative genome analysis of programmed DNA elimination in nematodes. Genome Res. 2017; 27:2001–2014. PubMed PMC

Bedrat A., Lacroix L., Mergny J.-L.. Re-evaluation of G-quadruplex propensity with G4Hunter. Nucleic Acids Res. 2016; 44:1746–1759. PubMed PMC

Sengar A., Heddi B., Phan A.T.. Formation of G-quadruplexes in poly-G sequences: structure of a propeller-type parallel-stranded G-quadruplex formed by a G15 stretch. Biochemistry. 2014; 53:7718–7723. PubMed

Largy E., Granzhan A., Hamon F., Verga D., Teulade-Fichou M.P.. Visualizing the quadruplex: from fluorescent ligands to light-up probes. Top. Curr. Chem. 2013; 330:111–177. PubMed

Sabharwal N.C., Savikhin V., Turek-Herman J.R., Nicoludis J.M., Szalai V.A., Yatsunyk L.A.. N -methylmesoporphyrin IX fluorescence as a reporter of strand orientation in guanine quadruplexes. FEBS J. 2014; 281:1726–1737. PubMed PMC

McKinney J.A., Wang G., Mukherjee A., Christensen L., Subramanian S.H.S., Zhao J., Vasquez K.M.. Distinct DNA repair pathways cause genomic instability at alternative DNA structures. Nat Commun. 2020; 11:236. PubMed PMC

Spiegel J., Adhikari S., Balasubramanian S.. The structure and function of DNA G-quadruplexes. Trends Chem. 2020; 2:123–136. PubMed PMC

Brázda V., Laister R.C., Jagelská E.B., Arrowsmith C.. Cruciform structures are a common DNA feature important for regulating biological processes. BMC Mol Biol. 2011; 12:33. PubMed PMC

Petruska J., Arnheim N., Goodman MF.. Stability of intrastrand hairpin structures formed by the CAG/CTG class of DNA triplet repeats associated with neurological diseases. Nucleic Acids Res. 1996; 24:1992–1998. PubMed PMC

Helma R., Bažantová P., Petr M., Adámik M., Renčiuk D., Tichý V., Pastuchová A., Soldánová Z., Pečinka P., Bowater R.P.et al. .. p53 binds preferentially to non-B DNA structures formed by the pyrimidine-rich strands of GAA·TTC trinucleotide repeats associated with Friedreich's ataxia. Molecules. 2019; 24:2078. PubMed PMC

Cimino-Reale G., Zaffaroni N., Folini M.. Emerging role of G-quadruplex DNA as target in anticancer therapy. Curr. Pharm. Des. 2016; 22:6612–6624. PubMed

Hänsel-Hertsch R., Simeone A., Shea A., Hui W.W.I., Zyner K.G., Marsico G., Rueda O.M., Bruna A., Martin A., Zhang X.et al. .. Landscape of G-quadruplex DNA structural regions in breast cancer. Nat. Genet. 2020; 52:878–883. PubMed

Poggi L., Richard G-F.. Alternative DNA structures in vivo: molecular evidence and remaining questions. Microbiol. Mol. Biol. Rev. 2021; 85:e00110-20. PubMed PMC

Yang C., Hu R., Li Q., Li S., Xiang J., Guo X., Wang S., Zen Y., Yang G.. Visualization of parallel G-quadruplexes in cells with a series of new developed Bis(4-aminobenzylidene)acetone derivatives. ACS Omega. 2018; 3:10487–10492. PubMed PMC

Bochman M.L., Paeschke K., Zakian V.A.. DNA secondary structures: stability and function of G-quadruplex structures. Nat. Rev. Genet. 2012; 13:770–780. PubMed PMC

Takahashi S., Sugimoto N.. Effect of pressure on thermal stability of G-quadruplex DNA and double-stranded DNA structures. Molecules. 2013; 18:13297–13319. PubMed PMC

Yadav P., Kim N., Kumari M., Verma S., Sharma T.K., Yadav V., Kumar A.. G-Quadruplex structures in bacteria - biological relevance and potential as antimicrobial target. J. Bacteriol. 2021; 203:e0057720. PubMed PMC

Brázda V., Fojta M., Bowater R.P.. Structures and stability of simple DNA repeats from bacteria. Biochem J. 2020; 477:325–339. PubMed PMC

Ruggiero E., Richter S.N.. Viral G-quadruplexes: new frontiers in virus pathogenesis and antiviral therapy. Annu. Rep. Med. Chem. 2020; 54:101–131. PubMed PMC

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

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

Bartas M., Brázda V., Bohálová N., Cantara A., Volná A., Stachurová T., Malachová K., Jagelská E.B., Porubiaková O., Červeň J.et al. .. In-depth bioinformatic analyses of human SARS-CoV-2, SARS-CoV, MERS-CoV, and other nidovirales suggest important roles of noncanonical nucleic acid structures in their lifecycles. Front. Microbiol. 2020; 11:1583. PubMed PMC

Ruggiero E., Richter S.N.. Viral G-quadruplexes: new frontiers in virus pathogenesis and antiviral therapy. Annu. Rep. Med. Chem. 2020; 54:101–131. PubMed PMC

Li R., Hsieh C.-L., Young A., Zhang Z., Ren X., Zhao Z.. Illumina synthetic long read sequencing allows recovery of missing sequences even in the «Finished» C. elegans genome. Sci Rep. 2015; 5:10814. PubMed PMC

Kruisselbrink E., Guryev V., Brouwer K., Pontier D.B., Cuppen E., Tijsterman M.. Mutagenic capacity of endogenous G4 DNA underlies genome instability in FANCJ-defective C. elegans. Curr Biol. 2008; 18:900–905. PubMed

Vannutelli A., Belhamiti S., Garant J.-M., Ouangraoua A., Perreault J.-P.. Where are G-quadruplexes located in the human transcriptome. NAR Genom. Bioinform. 2020; 2:lqaa035. PubMed PMC

Maltby C.J., Schofield J.P.R., Houghton S.D., O’Kelly I., Vargas-Caballero M., Deinhardt K., Coldwell M.J.. A 5’ UTR GGN repeat controls localisation and translation of a potassium leak channel mRNA through G-quadruplex formation. Nucleic Acids Res. 2020; 48:9822–9839. PubMed PMC

Katsuda Y., Sato S.-I., Asano L., Morimura Y., Furuta T., Sugiyama H., Hagihara M., Uesugi M.. A small molecule that represses translation of G-quadruplex-containing mRNA. J. Am. Chem. Soc. 2016; 138:9037–9040. PubMed

Hirai H. Chromosomal differentiation of schistosomes: what is the message. Front. Genet. 2014; 5:301. PubMed PMC

Müller F., Wicky C., Spicher A., Tobler H.. New telomere formation after developmentally regulated chromosomal breakage during the process of chromatin diminution in Ascaris lumbricoides. Cell. 1991; 67:815–822. PubMed

Phan A.T. Human telomeric G-quadruplex: structures of DNA and RNA sequences. FEBS J. 2010; 277:1107–1117. PubMed

Hänsel R., Foldynová-Trantírková S., Löhr F., Buck J., Bongartz E., Bamberg E., Schwalbe H., Dötsch V., Trantirek L.. Evaluation of parameters critical for observing nucleic acids inside living Xenopus laevis oocytes by in-cell NMR spectroscopy. J. Am. Chem. Soc. 2009; 131:15761–15768. PubMed

Biffi G., Tannahill D., McCafferty J., Balasubramanian S.. Quantitative visualization of DNA G-quadruplex structures in human cells. Nat. Chem. 2013; 5:182–186. PubMed PMC

Školáková P., Foldynová-Trantírková S., Bednářová K., Fiala R., Vorlíčková M., Trantírek L.. Unique C. elegans telomeric overhang structures reveal the evolutionarily conserved properties of telomeric DNA. Nucleic Acids Res. 2015; 43:4733–4745. PubMed PMC

Puig Lombardi E., Holmes A., Verga D., Teulade-Fichou M.-P., Nicolas A., Londoño-Vallejo A.. Thermodynamically stable and genetically unstable G-quadruplexes are depleted in genomes across species. Nucleic Acids Res. 2019; 47:6098–6113. PubMed PMC

Ribeiro de Almeida C., Dhir S., Dhir A., Moghaddam A.E., Sattentau Q., Meinhart A., Proudfoot N.J.. RNA helicase DDX1 converts RNA G-quadruplex structures into R-loops to promote IgH class switch recombination. Mol. Cell. 2018; 70:650–662. PubMed PMC

Ingram K., Yaremenko I.A., Krylov I.B., Hofer L., Terent’ev A.O., Keiser J.. Identification of antischistosomal leads by evaluating bridged 1,2,4,5-tetraoxanes, alphaperoxides, and tricyclic monoperoxides. J. Med. Chem. 2012; 55:8700–8711. PubMed

Asymmetric distribution of G-quadruplex forming sequences in genomes of retroviruses

Abundance of G-Quadruplex Forming Sequences in the Hepatitis Delta Virus Genomes

A sodium/potassium switch for G4-prone G/C-rich sequences

Genomic Analysis of Non-B Nucleic Acids Structures in SARS-CoV-2: Potential Key Roles for These Structures in Mutability, Translation, and Replication?

The Newly Sequenced Genome of Pisum sativum Is Replete with Potential G-Quadruplex-Forming Sequences-Implications for Evolution and Biological Regulation