{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T14:34:23Z","timestamp":1776436463265,"version":"3.51.2"},"reference-count":231,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2021,8,5]],"date-time":"2021-08-05T00:00:00Z","timestamp":1628121600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Progress in the design of G-quadruplex (G4) binding ligands relies on the availability of approaches that assess the binding mode and nature of the interactions between G4 forming sequences and their putative ligands. The experimental approaches used to characterize G4\/ligand interactions can be categorized into structure-based methods (circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography), affinity and apparent affinity-based methods (surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and mass spectrometry (MS)), and high-throughput methods (fluorescence resonance energy transfer (FRET)-melting, G4-fluorescent intercalator displacement assay (G4-FID), affinity chromatography and microarrays. Each method has unique advantages and drawbacks, which makes it essential to select the ideal strategies for the biological question being addressed. The structural- and affinity and apparent affinity-based methods are in several cases complex and\/or time-consuming and can be combined with fast and cheap high-throughput approaches to improve the design and development of new potential G4 ligands. In recent years, the joint use of these techniques permitted the discovery of a huge number of G4 ligands investigated for diagnostic and therapeutic purposes. Overall, this review article highlights in detail the most commonly used approaches to characterize the G4\/ligand interactions, as well as the applications and types of information that can be obtained from the use of each technique.<\/jats:p>","DOI":"10.3390\/ph14080769","type":"journal-article","created":{"date-parts":[[2021,8,5]],"date-time":"2021-08-05T09:35:32Z","timestamp":1628156132000},"page":"769","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":119,"title":["G-Quadruplexes and Their Ligands: Biophysical Methods to Unravel G-Quadruplex\/Ligand Interactions"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4620-0174","authenticated-orcid":false,"given":"Tiago","family":"Santos","sequence":"first","affiliation":[{"name":"CICS-UBI\u2014Centro de Investiga\u00e7\u00e3o em Ci\u00eancias da Sa\u00fade, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilh\u00e3, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0296-5979","authenticated-orcid":false,"given":"Gilmar F.","family":"Salgado","sequence":"additional","affiliation":[{"name":"ARNA Laboratory, Universite\u0301 de Bordeaux, Inserm U1212, CNRS UMR 5320, IECB, 33607 Pessac, France"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0720-2751","authenticated-orcid":false,"given":"Eurico J.","family":"Cabrita","sequence":"additional","affiliation":[{"name":"UCIBIO, REQUIMTE, Departamento de Qu\u00edmica, Faculdade de Ci\u00eancias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal"},{"name":"Associate Laboratory i4HB\u2014Institute for Health and Bioeconomy, NOVA School of Science and Technology, NOVA University Lisbon, 2819-516 Caparica, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6630-1242","authenticated-orcid":false,"given":"Carla","family":"Cruz","sequence":"additional","affiliation":[{"name":"CICS-UBI\u2014Centro de Investiga\u00e7\u00e3o em Ci\u00eancias da Sa\u00fade, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilh\u00e3, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/j.trechm.2019.07.002","article-title":"The Structure and Function of DNA G-Quadruplexes","volume":"2","author":"Spiegel","year":"2020","journal-title":"Trends Chem."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2013","DOI":"10.1073\/pnas.48.12.2013","article-title":"Helix formation by guanylic acid","volume":"48","author":"Gellert","year":"1962","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1042\/bj1410537","article-title":"Structures for polyinosinic acid and polyguanylic acid","volume":"141","author":"Arnott","year":"1974","journal-title":"Biochem. 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