Aptamers are short DNA or RNA sequences that can fold into unique three-dimensional structures, enabling them to bind specifically to target molecules with high affinity, similar to antibodies. A distinctive feature of many aptamers is their ability to adopt a G-quadruplex (G4) fold, a four-stranded structure formed by guanine-rich sequences. While G4 formation has been proposed or demonstrated for some aptamers, we aimed to investigate how frequently quadruplex-prone motifs emerge from the SELEX process. To achieve this, we examined quadruplex candidate sequences from the UTexas Aptamer Database, which contains over 1400 aptamer sequences extracted from 400 publications spanning several decades. We analyzed the G4 and i-motif propensity of these sequences. While no likely i-motif forming candidates were found, nearly 1/4 of DNA aptamers and 1/6 of RNA aptamers were predicted to form G4 structures. Interestingly, many motifs capable of forming G4 structures were not previously reported or suspected. Out of 311 sequences containing a potential stable G4 motif, only 53 of them (17%) reported the word "quadruplex" in the corresponding article. We experimentally tested G4 formation for 30 aptamer sequences and were able to confirm G4 formation for all the sequences with a G4Hunter score of 1.31 or more. These observations suggest the need to reevaluate G4 propensity among aptamer sequences.

Faculty of Chemistry Brno University of Technology Purkyňova 118 61200 Brno Czech Republic

Institute of Biophysics Czech Academy of Sciences Královopolská 135 61200 Brno Czech Republic

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

See more in PubMed

Toulmé  J-J, Peyrin  E, Ducongé  F  Nucleic acid aptamers. Methods. 2016; 97:1–2.10.1016/j.ymeth.2016.02.015. PubMed DOI

Domsicova  M, Korcekova  J, Poturnayova  A  et al. .  New insights into aptamers: an alternative to antibodies in the detection of molecular biomarkers. Int J Mol Sci. 2024; 25:6833.10.3390/ijms25136833. PubMed DOI PMC

Stoltenburg  R, Reinemann  C, Strehlitz  B  SELEX–a (r) evolutionary method to generate high-affinity nucleic acid ligands. Biomol Eng. 2007; 24:381–403.10.1016/j.bioeng.2007.06.001. PubMed DOI

Egli  M, Manoharan  M  Chemistry, structure and function of approved oligonucleotide therapeutics. Nucleic Acids Res. 2023; 51:2529–73.10.1093/nar/gkad067. PubMed DOI PMC

Askari  A, Kota  S, Ferrell  H  et al. .  UTexas Aptamer Database: the collection and long-term preservation of aptamer sequence information. Nucleic Acids Res. 2024; 52:D351–9.10.1093/nar/gkad959. PubMed DOI PMC

Yu  H, Zhu  J, Shen  G  et al. .  Improving aptamer performance: key factors and strategies. Microchim Acta. 2023; 190:255.10.1007/s00604-023-05836-6. PubMed DOI

Chandola  C, Kalme  S, Casteleijn  MG  et al. .  Application of aptamers in diagnostics, drug-delivery and imaging. J Biosci. 2016; 41:535–61.10.1007/s12038-016-9632-y. PubMed DOI

Shaban  SM, Kim  D-H  Recent advances in aptamer sensors. Sensors. 2021; 21:979.10.3390/s21030979. PubMed DOI PMC

Joshi  R, Janagama  H, Dwivedi  HP  et al. .  Selection, characterization, and application of DNA aptamers for the capture and detection of Salmonella enterica serovars. Mol Cell Probes. 2009; 23:20–8.10.1016/j.mcp.2008.10.006. PubMed DOI

Suh  SH, Jaykus  L-A  Nucleic acid aptamers for capture and detection of Listeria spp. J Biotechnol. 2013; 167:454–61.10.1016/j.jbiotec.2013.07.027. PubMed DOI

Nimjee  SM, White  RR, Becker  RC  et al. .  Aptamers as therapeutics. Annu Rev Pharmacol Toxicol. 2017; 57:61–79.10.1146/annurev-pharmtox-010716-104558. PubMed DOI PMC

Gragoudas  ES, Adamis  AP, Cunningham  ET  et al. .  Pegaptanib for neovascular age-related macular degeneration. N Engl J Med. 2004; 351:2805–16.10.1056/NEJMoa042760. PubMed DOI

Mullard  A  FDA approves second RNA aptamer. Nat Rev Drug Discov. 2023; 22:774.10.1038/d41573-023-00148-z. PubMed DOI

Mergny  J-L, Sen  D  DNA quadruple helices in nanotechnology. Chem Rev. 2019; 119:6290–325.10.1021/acs.chemrev.8b00629. PubMed DOI

Nicholson  DA, Nesbitt  DJ  Kinetic and thermodynamic control of G-quadruplex polymorphism by Na+ and K+ cations. J Phys Chem B. 2023; 127:6842–55.10.1021/acs.jpcb.3c01001. PubMed DOI

Fujii  T, Podbevšek  P, Plavec  J  et al. .  Effects of metal ions and cosolutes on G-quadruplex topology. J Inorg Biochem. 2017; 166:190–8.10.1016/j.jinorgbio.2016.09.001. PubMed DOI

Macaya  RF, Schultze  P, Smith  FW  et al. .  Thrombin-binding DNA aptamer forms a unimolecular quadruplex structure in solution. Proc Natl Acad Sci USA. 1993; 90:3745–9.10.1073/pnas.90.8.3745. PubMed DOI PMC

Bates  PJ, Reyes-Reyes  EM, Malik  MT  et al. .  G-quadruplex oligonucleotide AS1411 as a cancer-targeting agent: uses and mechanisms. Biochim Biophys Gen Sub. 2017; 1861:1414–28.10.1016/j.bbagen.2016.12.015. PubMed DOI

Brázda  V, Kolomazník  J, Lýsek  J  et al. .  G4Hunter web application: a web server for G-quadruplex prediction. Bioinformatics. 2019; 35:3493–5.10.1093/bioinformatics/btz087. PubMed DOI PMC

Bedrat  A, Lacroix  L, Mergny  J-L  Re-evaluation of G-quadruplex propensity with G4Hunter. Nucleic Acids Res. 2016; 44:1746–59.10.1093/nar/gkw006. PubMed DOI PMC

Sahakyan  AB, Chambers  VS, Marsico  G  et al. .  Machine learning model for sequence-driven DNA G-quadruplex formation. Sci Rep. 2017; 7:14535.10.1038/s41598-017-14017-4. PubMed DOI PMC

Gruber  AR, Lorenz  R, Bernhart  SH  et al. .  The Vienna RNA Websuite. Nucleic Acids Res. 2008; 36:W70–4.10.1093/nar/gkn188. PubMed DOI PMC

Luo  Y, Granzhan  A, Verga  D  et al. .  FRET-MC: a fluorescence melting competition assay for studying G4 structures in vitro. Biopolymers. 2021; 112:e23415.10.1002/bip.23415. PubMed DOI

Luo  Y, Granzhan  A, Marquevielle  J  et al. .  Guidelines for G-quadruplexes: I. In vitro characterization. Biochimie. 2023; 214:5–23.10.1016/j.biochi.2022.12.019. PubMed DOI

Mergny  J-L, Li  J, Lacroix  L  et al. .  Thermal difference spectra: a specific signature for nucleic acid structures. Nucleic Acids Res. 2005; 33:e138.10.1093/nar/gni134. PubMed DOI PMC

Esnault  C, Magat  T, Zine  El Aabidine A  et al. .  G4access identifies G-quadruplexes and their associations with open chromatin and imprinting control regions. Nat Genet. 2023; 55:1359–69.10.1038/s41588-023-01437-4. PubMed DOI

Platella  C, Riccardi  C, Montesarchio  D  et al. .  G-quadruplex-based aptamers against protein targets in therapy and diagnostics. Biochim Biophys Gen Sub. 2017; 1861:1429–47.10.1016/j.bbagen.2016.11.027. PubMed DOI PMC

Liu  H, Zheng  K, He  Y  et al. .  RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation. Chem Sci. 2016; 7:4573–81.10.1039/C5SC03946K. PubMed DOI PMC

Yett  A, Lin  LY, Beseiso  D  et al. .  N-methyl mesoporphyrin IX as a highly selective light-up probe for G-quadruplex DNA. J Porphyrins Phthalocyanines. 2019; 23:1195–215.10.1142/S1088424619300179. PubMed DOI PMC

del Villar-Guerra  R, Trent  JO, Chaires  JB  G-quadruplex secondary structure obtained from circular dichroism spectroscopy. Angew Chem Int Ed. 2018; 57:7171–5.10.1002/anie.201709184. PubMed DOI PMC

Zhao  C, Ren  J, Qu  X  G-quadruplexes form ultrastable parallel structures in deep eutectic solvent. Langmuir. 2013; 29:1183–91.10.1021/la3043186. PubMed DOI

Kypr  J, Kejnovská  I, Renčiuk  D  et al. .  Circular dichroism and conformational polymorphism of DNA. Nucleic Acids Res. 2009; 37:1713–25.10.1093/nar/gkp026. PubMed DOI PMC

Chen  J, Cheng  M, Salgado  GF  et al. .  The beginning and the end: flanking nucleotides induce a parallel G-quadruplex topology. Nucleic Acids Res. 2021; 49:9548–59.10.1093/nar/gkab681. PubMed DOI PMC

Guerra-Pérez  N, Ramos  E, García-Hernández  M  et al. .  Molecular and functional characterization of ssDNA aptamers that specifically bind Leishmania infantum PABP. PLoS One. 2015; 10:e0140048.10.1371/journal.pone.0140048. PubMed DOI PMC

Low  SY, Hill  JE, Peccia  J  A DNA aptamer recognizes the asp f 1 allergen of Aspergillus fumigatus. Biochem Biophys Res Commun. 2009; 386:544–8.10.1016/j.bbrc.2009.06.089. PubMed DOI PMC

Eissa  S, Zourob  M  In vitro selection of DNA aptamers targeting β-lactoglobulin and their integration in graphene-based biosensor for the detection of milk allergen. Biosens Bioelectron. 2017; 91:169–74.10.1016/j.bios.2016.12.020. PubMed DOI

Elshafey  R, Siaj  M, Zourob  M  In vitro selection, characterization, and biosensing application of high-affinity cylindrospermopsin-targeting aptamers. Anal Chem. 2014; 86:9196–203.10.1021/ac502157g. PubMed DOI

Cheng  M, Qiu  D, Tamon  L  et al. .  Thermal and pH stabilities of i-DNA: confronting in vitroexperiments with models and In-cell NMR data. Angew Chem Int Ed. 2021; 60:10286–94.10.1002/anie.202016801. PubMed DOI

Tsvetkov  V, Mir  B, Alieva  R  et al. .  Unveiling the unusual i-motif-derived architecture of a DNA aptamer exhibiting high affinity for influenza A virus. Nucleic Acids Res. 2025; 53:gkae1282.10.1093/nar/gkae1282. PubMed DOI PMC

Saccà  B, Lacroix  L, Mergny  J-L  The effect of chemical modifications on the thermal stability of different G-quadruplex-forming oligonucleotides. Nucleic Acids Res. 2005; 33:1182–92.10.1093/nar/gki257. PubMed DOI PMC

Puig  Lombardi E, Londoño-Vallejo  A  A guide to computational methods for G-quadruplex prediction. Nucleic Acids Res. 2020; 48:1–15.10.1093/nar/gkz1097. PubMed DOI PMC

Thiel  WH, Bair  T, Wyatt  Thiel K  et al. .  Nucleotide bias observed with a short SELEX RNA aptamer library. Nucleic Acid Ther. 2011; 21:253–63.10.1089/nat.2011.0288. PubMed DOI PMC

Kohlberger  M, Gadermaier  G  SELEX: critical factors and optimization strategies for successful aptamer selection. Biotech App Biochem. 2022; 69:1771–92.10.1002/bab.2244. PubMed DOI PMC

Lucas  JK, Gruenke  PR, Burke  DH  Minimizing amplification bias during reverse transcription for in vitro selections. RNA. 2023; 29:1301–15.10.1261/rna.079650.123. PubMed DOI PMC

Brazda  V, Kolomaznik  J, Mergny  J-L  et al. .  G4Killer web application: a tool to design G-quadruplex mutations. Bioinformatics. 2020; 36:3246–7.10.1093/bioinformatics/btaa057. PubMed DOI PMC

Ngo  KH, Liew  CW, Lattmann  S  et al. .  Crystal structures of an HIV-1 integrase aptamer: formation of a water-mediated A•G•G•G•G pentad in an interlocked G-quadruplex. Biochem Biophys Res Commun. 2022; 613:153–8.10.1016/j.bbrc.2022.04.020. PubMed DOI

Niazi  JH, Lee  SJ, Gu  MB  Single-stranded DNA aptamers specific for antibiotics tetracyclines. Bioorg Med Chem. 2008; 16:7245–53.10.1016/j.bmc.2008.06.033. PubMed DOI

de la Renaud  Faverie A, Hamon  F, Di  Primo C  et al. .  Nucleic acids targeted to drugs: SELEX against a quadruplex ligand. Biochimie. 2011; 93:1357–67.10.1016/j.biochi.2011.05.022. PubMed DOI

Allali-Hassani  A, Pereira  MP, Navani  NK  et al. .  Isolation of DNA aptamers for CDP-ribitol synthase, and characterization of their inhibitory and structural properties. ChemBioChem. 2007; 8:2052–7.10.1002/cbic.200700257. PubMed DOI

Li  P, Zhou  L, Yu  Y  et al. .  Characterization of DNA aptamers generated against the soft-shelled turtle iridovirus with antiviral effects. BMC Vet Res. 2015; 11:245.10.1186/s12917-015-0559-6. PubMed DOI PMC

Ji  D, Yuan  J-H, Chen  S-B  et al. .  Selective targeting of parallel G-quadruplex structure using L-RNA aptamer. Nucleic Acids Res. 2023; 51:11439–52.10.1093/nar/gkad900. PubMed DOI PMC

Bourdon  S, Herviou  P, Dumas  L  et al. .  QUADRatlas: the RNA G-quadruplex and RG4-binding proteins database. Nucleic Acids Res. 2023; 51:D240–7.10.1093/nar/gkac782. PubMed DOI PMC

Brázda  V, Hároníková  L, Liao  JCC  et al. .  DNA and RNA quadruplex-binding proteins. Int J Mol Sci. 2014; 15:17493–517.10.3390/ijms151017493. PubMed DOI PMC

Luo  Y, Živković  ML, Wang  J  et al. .  A sodium/potassium switch for G4-prone G/C-rich sequences. Nucleic Acids Res. 2024; 52:448–61.10.1093/nar/gkad1073. PubMed DOI PMC

Ansari  N, Ghazvini  K, Ramezani  M  et al. .  Selection of DNA aptamers against mycobacterium tuberculosis Ag85A, and its application in a graphene oxide-based fluorometric assay. Microchim Acta. 2018; 185:21.10.1007/s00604-017-2550-3. PubMed DOI

Shahdordizadeh  M, Taghdisi  SM, Sankian  M  et al. .  Design, isolation and evaluation of the binding efficiency of a DNA aptamer against interleukin 2 receptor alpha, in vitro. Int Immunopharmacol. 2017; 53:96–104.10.1016/j.intimp.2017.10.011. PubMed DOI

Graham  JC, Zarbl  H  Use of cell-SELEX to generate DNA aptamers as molecular probes of HPV-associated cervical cancer cells. PLoS One. 2012; 7:e36103.10.1371/journal.pone.0036103. PubMed DOI PMC

Zhang  Y, Wu  F, Wang  M  et al. .  Single-stranded DNA aptamer targeting and neutralization of anti-D alloantibody: a potential therapeutic strategy for haemolytic diseases caused by Rhesus alloantibody. Blood Transfus. 2018; 16:184–92. PubMed PMC

Fukusaki  E, Kato  T, Maeda  H  et al. .  DNA aptamers that bind to chitin. Bioorg Med Chem Lett. 2000; 10:423–5.10.1016/S0960-894X(00)00013-5. PubMed DOI

Ninomiya  K, Kaneda  K, Kawashima  S  et al. .  Cell-SELEX based selection and characterization of DNA aptamer recognizing human hepatocarcinoma. Bioorg Med Chem Lett. 2013; 23:1797–802.10.1016/j.bmcl.2013.01.040. PubMed DOI

Waybrant  B, Pearce  TR, Wang  P  et al. .  Development and characterization of an aptamer binding ligand of fractalkine using domain targeted SELEX. Chem Commun. 2012; 48:10043–5.10.1039/c2cc34217k. PubMed DOI

Wang  T, Rahimizadeh  K, Veedu  RN  Development of a novel DNA oligonucleotide targeting low-density lipoprotein receptor. Mol Ther Nucleic Acids. 2020; 19:190–8.10.1016/j.omtn.2019.11.004. PubMed DOI PMC