G-quadruplexes (G4s) formed within RNA are emerging as promising targets for therapeutic intervention in cancer, neurodegenerative disorders and infectious diseases. Sequences containing a succession of short GG blocks, or uneven G-tract lengths unable to form three-tetrad G4s (GG motifs), are overwhelmingly more frequent than canonical motifs involving multiple GGG blocks. We recently showed that DNA is not able to form stable two-tetrad intramolecular parallel G4s. Whether RNA GG motifs can form intramolecular G4s under physiological conditions and play regulatory roles remains a burning question. In this study, we performed a systematic analysis and experimental evaluation of a number of biologically important RNA regions involving RNA GG motifs. We show that most of these motifs do not form stable intramolecular G4s but need to dimerize to form stable G4 structures. The strong tendency of RNA GG motif G4s to associate may participate in RNA-based aggregation under conditions of cellular stress.

Center for Molecular Medicine Cologne CMMC University of Cologne Robert Koch Str 21 50931 Cologne Germany

Central European Institute of Technology Masaryk University Kamenice 735 5 625 00 Brno Czech Republic

Department of Biochemistry Masaryk University Kamenice 735 5 625 00 Brno Czech Republic

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

Laboratoire d'Optique et Biosciences Ecole Polytechnique CNRS INSERM Institut Polytechnique de Paris 91120 Palaiseau France

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 735 5 625 00 Brno Czech Republic

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Gilbert D.E., Feigon J.. Multistranded DNA structures. Curr. Opin. Struct. Biol. 1999; 9:305–314. PubMed

Neidle S., Balasubramanian S.. Quadruplex Nucleic Acids. Royal Society of Chemistry. 2006; London, Cambridge.

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

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

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

Salgado G.F., Cazenave C., Kerkour A., Mergny J.-L.. G-quadruplex DNA and ligand interaction in living cells using NMR spectroscopy. Chem. Sci. 2015; 6:3314–3320. PubMed PMC

Di Antonio M., Ponjavic A., Radzevičius A., Ranasinghe R.T., Catalano M., Zhang X., Shen J., Needham L.-M., Lee S.F., Klenerman D.et al. .. Single-molecule visualization of DNA G-quadruplex formation in live cells. Nat. Chem. 2020; 12:832–837. PubMed PMC

Hänsel-Hertsch R., Di Antonio M., Balasubramanian S.. DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential. Nat. Rev. Mol. Cell Biol. 2017; 18:279–284. PubMed

Maizels N. G4-associated human diseases. EMBO Rep. 2015; 16:910–922. 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

Abiri A., Lavigne M., Rezaei M., Nikzad S., Zare P., Mergny J.-L., Rahimi H.-R.. Unlocking G-quadruplexes as antiviral targets. Pharmacol. Rev. 2021; 73:897–923. PubMed

Mergny J.L., De Cian A., Ghelab A., Sacca B., Lacroix L.. Kinetics of tetramolecular quadruplexes. Nucleic Acids Res. 2005; 33:81–94. PubMed PMC

Schultze P., Macaya R.F., Feigon J.. Three-dimensional solution structure of the thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG). J. Mol. Biol. 1994; 235:1532–1547. PubMed

Lim K.W., Amrane S., Bouaziz S., Xu W., Mu Y., Patel D.J., Luu K.N., Phan A.T.. Structure of the human telomere in K+ solution: a stable basket-type G-quadruplex with only two G-tetrad layers. J. Am. Chem. Soc. 2009; 131:4301–4309. PubMed PMC

Kotar A., Rigo R., Sissi G., Plavec J.. Two-quartet kit* G-quadruplex is formed via double-stranded pre-folded structure. Nucleic Acids Res. 2019; 47:2641–2653. PubMed PMC

Chen J., Cheng M., Salgado G.F., Stadlbauer P., Zhang X., Amrane S., Guedin A., He F., Sponer J., Ju H.et al. .. The beginning and the end: flanking nucleotides induce a parallel G-quadruplex topology. Nucleic Acids Res. 2021; 49:9548–9559. PubMed PMC

Cheng M., Cheng Y., Hao J., Jia G., Zhou J., Mergny J.-L., Li C.. Loop permutation affects the topology and stability of G-quadruplexes. Nucleic Acids Res. 2018; 46:9264–9275. PubMed PMC

Guédin A., Gros J., Alberti P., Mergny J.-L.. How long is too long? Effects of loop size on G-quadruplex stability. Nucleic Acids Res. 2010; 38:7858–7868. PubMed PMC

Parkinson G.N., Lee M.P., Neidle S.. Crystal structure of parallel quadruplexes from human telomeric DNA. Nature. 2002; 417:876–880. PubMed

Sket P., Plavec J.. Tetramolecular DNA quadruplexes in solution: insights into structural diversity and cation movement. J.Am. Chem. Soc. 2010; 132:12724–12732. PubMed

Webba da Silva M. Geometric formalism for DNA quadruplex folding. Chem. Eur. J. 2007; 13:9738–9745. PubMed

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

Lech C.J., Heddi B., Phan A.T.. Guanine base stacking in G-quadruplex nucleic acids. Nucleic Acids Res. 2013; 41:2034–2046. PubMed PMC

Chan K.L., Peng B., Umar M.I., Chun C.Y., Sahakyan A.B., Le M.T.N., Kwok C.K.. Structural analysis reveals the formation and role of RNA G-quadruplex structures in human mature microRNAs. Chem. Commun. 2018; 54:10878–10881. PubMed

Qin M.Y., Chen Z.X., Luo Q.C., Wen Y., Zhang N.X., Jiang H.L., Yang H.Y.. Two-quartet G-quadruplexes formed by DNA sequences containing four contiguous GG runs. J. Phys. Chem. B. 2015; 119:3706–3713. PubMed

Chung W.J., Heddi B., Schmitt E., Lim K.W., Mechulam Y., Phan A.T.. Structure of a left-handed DNA G-quadruplex. Proc. Natl. Acad. Sci. USA. 2015; 112:2729–2733. PubMed PMC

Do N.Q., Chung W.J., Truong T., Hong A., Heddi B., Phan A.T.. G-quadruplex structure of an anti-proliferative DNA sequence. Nucleic Acids Res. 2017; 45:7487–7493. PubMed PMC

Maity A., Winnerdy F.R., Chang W.D., Chen G., Phan A.T.. Intra-locked G-quadruplex structures formed by irregular DNA G-rich motifs. Nucleic Acids Res. 2020; 48:3315–3327. PubMed PMC

Kejnovska I., Stadlbauer P., Trantirek L., Renciuk D., Gajarsky M., Krafcik D., Palacky J., Bednarova K., Sponer J., Mergny J.L.et al. .. G-quadruplex formation by DNA sequences deficient in guanines: two tetrad parallel quadruplexes do not fold intramolecularly. Chem. 2021; 27:12115–12125. PubMed

Islam B., Stadlbauer P., Vorlickova M., Mergny J.L., Otyepka M., Sponer J.. Stability of two-quartet G-quadruplexes and their dimers in atomistic simulations. J. Chem. Theory Comput. 2020; 16:3447–3463. PubMed

Binas O., Bessi I., Schwalbe H.. Structure validation of G-rich RNAs in noncoding regions of the human genome. ChemBioChem. 2020; 21:1656–1663. PubMed PMC

Gomez D., Guédin A., Mergny J.L., Salles B., Riou J.F., Teulade-Fichou M.P., Calsou P.. A G-quadruplex structure within the 5'-UTR of TRF2 mRNA represses translation in human cells. Nucleic Acids Res. 2010; 38:7187–7198. PubMed PMC

Zhao C., Qin G., Niu J., Wang Z., Wang C., Ren J., Qu X.. Targeting RNA G-quadruplex in SARS-CoV-2: a promising therapeutic target for COVID-19?. Angew. Chem. Int. Ed Engl. 2021; 60:432–438. PubMed

Hon J., Martínek T., Zendulka J., Lexa M.. pqsfinder: an exhaustive and imperfection-tolerant search tool for potential quadruplex-forming sequences in R. Bioinformatics. 2017; 33:3373–3379. PubMed

O’Leary N.A., Wright M.W., Brister J.R., Ciufo S., Haddad D., McVeigh R., Rajput B., Robbertse B., Smith-White B., Ako-Adjei D.et al. .. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 2016; 44:D733–D745. PubMed PMC

The RNAcentral Consortium RNAcentral: a hub of information for non-coding RNA sequences. Nucleic Acids Res. 2018; 47:D1250–D1251. PubMed PMC

Ma L., Cao J., Liu L., Du Q., Li Z., Zou D., Bajic V.B., Zhang Z.. LncBook: a curated knowledgebase of human long non-coding RNAs. Nucleic Acids Res. 2019; 47:2699. PubMed PMC

Raney B.J., Barber G.P., Benet-Pagès A., Casper J., Clawson H., Cline M.S., Diekhans M., Fischer C., Navarro Gonzalez J., Hickey G.et al. .. The UCSC Genome Browser database: 2024 update. Nucleic Acids Res. 2024; 52:D1082–D1088. PubMed PMC

Kent W.J., Sugnet C.W., Furey T.S., Roskin K.M., Pringle T.H., Zahler A.M., Haussler D. The human genome browser at UCSC. Genome Res. 2002; 12:996–1006. PubMed PMC

Quinlan A.R., Hall I.M.. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26:841–842. PubMed PMC

Team, T.B.D. BSgenome.Hsapiens.UCSC.Hg38: full genomic sequences for homo sapiens (UCSC genome hg38. 2023;

Nurk S., Koren S., Rhie A., Rautiainen M., Bzikadze A.V., Mikheenko A., Vollger M.R., Altemose N., Uralsky L., Gershman A.et al. .. The complete sequence of a human genome. Science. 2022; 376:44–53. PubMed PMC

Kejnovska I., Renciuk D., Palacky J., Vorlickova M.. Yang D., Lin C.. G-Quadruplex Nucleic Acids: Methods and Protocols. 2019; 2035:Totowa, NJ, USA: Humana Press; 25–44.

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 assayfor studying G4 structures in vitro. Biopolymers. 2021; 112:e23415. 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

Sklenář V., Bax A.. Spin-echo water suppression for the generation of pure-phase two-dimensional NMR spectra. J. Magn. Reson. 1987; 74:469–479.

Vorlíčková M., Kejnovská I., Bednářová K., Renčiuk D., Kypr J.. Circular dichroism spectroscopy of DNA: from duplexes to quadruplexes. Chirality. 2012; 24:691–698. PubMed

Vorlíčková M., Kejnovská I., Sagi J., Renčiuk D., Bednářová K., Motlová J., Kypr J.. Circular dichroism and guanine quadruplexes. Methods. 2012; 57:64–75. PubMed

Holoubek J., Bednářová K., Haviernik J., Huvarová I., Dvořáková Z., Černý J., Outlá M., Salát J., Konkol’ová E., Boura E.et al. .. Guanine quadruplexes in the RNA genome of the tick-borne encephalitis virus: their role as a new antiviral target and in virus biology. Nucleic Acids Res. 2022; 50:4574–4600. PubMed PMC

Malgowska M., Gudanis D., Kierzek R., Wyszko E., Gabelica V., Gdaniec Z.. Distinctive structural motifs of RNA G-quadruplexes composed of AGG, CGG and UGG trinucleotide repeats. Nucleic Acids Res. 2014; 42:10196–10207. PubMed PMC

Yett A., Lin L.Y., Beseiso D., Miao J., Yatsunyk L.A.. N-methyl mesoporphyrin IX as a highly selective light-up probe for G-quadruplex DNA. J. Porphyr. Phthalocya. 2019; 23:1195–1215. PubMed PMC

Renciuk D., Rynes J., Kejnovska I., Foldynova-Trantirkova S., Andang M., Trantirek L., Vorlickova M.. G-quadruplex formation in the Oct4 promoter positively regulates Oct4 expression. Biochim. Biophys. Acta-Gene Regulat. Mech. 2017; 1860:175–183. PubMed

Bednářová K., Vorlíčková M., Renčiuk D. Diversity of parallel guanine quadruplexes induced by guanine substitutions. Int. J. Mol. Sci. 2020; 21:6123. PubMed PMC

Cervenak M., Molnár O.R., Horváth P., Smeller L.. Stabilization of G-quadruplex structures of the SARS-CoV-2 genome by TMPyP4, BRACO19, and PhenDC3. Int. J. Mol. Sci. 2024; 25:2482. PubMed PMC

Kypr J., Fialová M., Chládková J., Tůmová M., Vorlícková M.. Conserved guanine-guanine stacking in tetraplex and duplex DNA. Eur. Biophys. J. 2001; 30:555–558. PubMed

Kypr J., Kejnovska I., Renciuk D., Vorlickova M.. Circular dichroism and conformational polymorphism of DNA. Nucleic Acids Res. 2009; 37:1713–1725. PubMed PMC

Georgakopoulos-Soares I., Parada G.E., Wong H.Y., Medhi R., Furlan G., Munita R., Miska E.A., Kwok C.K., Hemberg M.. Alternative splicing modulation by G-quadruplexes. Nat. Commun. 2022; 13:2404. PubMed PMC

Lee C.-Y., Joshi M., Wang A., Myong S.. 5′UTR G-quadruplex structure enhances translation in size dependent manner. Nat. Commun. 2024; 15:3963. PubMed PMC

Yang S.Y., Lejault P., Chevrier S., Boidot R., Robertson A.G., Wong J.M.Y., Monchaud D. Transcriptome-wide identification of transient RNA G-quadruplexes in human cells. Nat. Commun. 2018; 9:4730. PubMed PMC

Yang S.Y., Monchaud D., Wong J.M.Y.. Global mapping of RNA G-quadruplexes (G4-RNAs) using G4RP-seq. Nat. Protoc. 2022; 17:870–889. PubMed

Kwok C.K., Marsico G., Sahakyan A.B., Chambers V.S., Balasubramanian S.. rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome. Nat. Methods. 2016; 13:841–844. PubMed

Xiao C.-D., Ishizuka T., Xu Y.. Antiparallel RNA G-quadruplex formed by human telomere RNA containing 8-bromoguanosine. Sci. Rep. 2017; 7:6695. PubMed PMC

Arora A., Suess B.. An RNA G-quadruplex in the 3' UTR of the proto-oncogene PIM1 represses translation. RNA Biol. 2011; 8:802–805. PubMed

Beaudoin J.-D., Perreault J.-P.. 5′-UTR G-quadruplex structures acting as translational repressors. Nucleic Acids Res. 2010; 38:7022–7036. PubMed PMC

Agarwala P., Pandey S., Maiti S.. Role of G-quadruplex located at 5′ end of mRNAs. Biochim. Biophys. Gen. Sub. 2014; 1840:3503–3510. PubMed

Hacht A.v., Seifert O., Menger M., Schütze T., Arora A., Konthur Z., Neubauer P., Wagner A., Weise C., Kurreck J.. Identification and characterization of RNA guanine-quadruplex binding proteins. Nucleic Acids Res. 2014; 42:6630–6644. PubMed PMC

Zhang Y., Liu S., Jiang H., Deng H., Dong C., Shen W., Chen H., Gao C., Xiao S., Liu Z.-F.et al. .. G2-quadruplex in the 3’UTR of IE180 regulates Pseudorabies virus replication by enhancing gene expression. RNA Biol. 2020; 17:816–827. PubMed PMC

Van Treeck B., Protter D.S.W., Matheny T., Khong A., Link C.D., Parker R.. RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome. Proc. Natl. Acad. Sci. USA. 2018; 115:2734–2739. PubMed PMC

Fay M.M., Anderson P.J., Ivanov P.. ALS/FTD-associated C9ORF72 repeat RNA promotes phase transitions in vitro and in cells. Cell Rep. 2017; 21:3573–3584. PubMed PMC

Danino Y.M., Molitor L., Rosenbaum-Cohen T., Kaiser S., Cohen Y., Porat Z., Marmor-Kollet H., Katina C., Savidor A., Rotkopf R.et al. .. BLM helicase protein negatively regulates stress granule formation through unwinding RNA G-quadruplex structures. Nucleic Acids Res. 2023; 51:9369–9384. PubMed PMC

Sauer M., Juranek S.A., Marks J., De Magis A., Kazemier H.G., Hilbig D., Benhalevy D., Wang X., Hafner M., Paeschke K.. DHX36 prevents the accumulation of translationally inactive mRNAs with G4-structures in untranslated regions. Nat. Commun. 2019; 10:2421. PubMed PMC