Human telomeric G-quadruplexes are emerging targets in anticancer drug discovery since they are able to efficiently inhibit telomerase, an enzyme which is greatly involved in telomere instability and immortalization process in malignant cells. G-quadruplex (G4) DNA is highly polymorphic and can adopt different topologies upon addition of electrolytes, additives, and ligands. The study of G-quadruplex forms under various conditions, however, might be quite challenging. In this work, surface-enhanced Raman scattering (SERS) spectroscopy has been applied to study G-quadruplexes formed by human telomeric sequences, d[A3G3(TTAGGG)3A2] (Tel26) and d[(TTAGGG)4T2] (wtTel26), under dilute and crowding conditions. The SERS spectra distinctive of hybrid-1 and hybrid-2 G-quadruplexes of Tel26 and wtTel26, respectively, were observed for the sequences folded in the presence of K+ ions (110 mM) in a buffered solution, representing the diluted medium. Polyethylene glycol (5, 10, 15, 20, and 40% v/v PEG) was used to create a molecular-crowded environment, resulting in the formation of the parallel G-quadruplexes of both studied human telomeric sequences. Despite extensive overlap by the crowding agent bands, the SERS spectral features indicative of parallel G4 form of Tel26 were recognized. The obtained results implied that SERS of G-quadruplexes reflected not only the primary structure of the studied human telomeric sequence, including its nucleobase composition and sequence, but also its secondary structure in the sense of Hoogsteen hydrogen bonds responsible for the guanine tetrad formation, and finally its tertiary structure, defining a three-dimensional DNA shape, positioned close to the enhancing metallic surface.
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We thank Ente Cassa Risparmio Firenze for a grant to FP (ECR2014.0309) and the University of Florence for funding FP’s stay in Zagreb (Contributo di Ateneo per la Promozione delle Attività Internazionali Anno 2015 and Piano di Internazionalizzazione di Ateneo 2013-2015).
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Papi, F., Kenđel, A., Ratkaj, M. et al. Effect of structure levels on surface-enhanced Raman scattering of human telomeric G-quadruplexes in diluted and crowded media. Anal Bioanal Chem 411, 5197–5207 (2019). https://doi.org/10.1007/s00216-019-01894-z
- Structure level
- Human telomere