Abstract
A multi-technique approach, combining circular dichroism spectroscopy, ultraviolet resonance Raman spectroscopy and small angle scattering techniques, has been deployed to elucidate how the structural features of the human telomeric G-quadruplex d[A(GGGTTA)3GGG] (Tel22) change upon thermal unfolding. The system is studied both in the free form and when it is bound to Actinomycin D (ActD), an anticancer ligand with remarkable conformational flexibility. We find that at room temperature binding of Tel22 with ActD involves end-stacking upon the terminal G-tetrad. Structural evidence for drug-driven dimerization of a significant fraction of the G-quadruplexes is provided. When the temperature is raised, both free and bound Tel22 undergo melting through a multi-state process. We show that in the intermediate states of Tel22 the conformational equilibrium is shifted toward the (3+1) hybrid-type, while a parallel structure is promoted in the complex. The unfolded state of the free Tel22 is consistent with a self-avoiding random-coil conformation, whereas the high-temperature state of the complex is observed to assume a quite compact form. Such an unprecedented high-temperature arrangement is caused by the persistent interaction between Tel22 and ActD, which stabilizes compact conformations even in the presence of large thermal structural fluctuations.
Highlights
Guanine-rich DNA and RNA sequences are prone to fold into stable helical four-stranded structures called Gquadruplexes [1]
By using circular dichroism (CD), we probe the changes of topology for Tel22 and Tel22+Actinomycin D (ActD) as a function of temperature on approaching the melting, as different geometry of quartet stacking gives rise to distinct spectroscopic signatures [9]
We presented and discussed the results of an experimental study on the structural, molecular and thermodynamic properties of the G-quadruplex formed by the human telomeric sequence AG3(TTAG3)3 in the presence of the ligand ActD, upon thermal unfolding
Summary
Guanine-rich DNA and RNA sequences are prone to fold into stable helical four-stranded structures called Gquadruplexes [1] These folds are the focus of a number of studies in both fundamental and applied research, from cancer biology and novel therapeutics [2] through to nanotechnology [3]. Stable putative G-quadruplex forming sequences have been identified in vivo mainly within cancer genes in human chromatin [6]. These findings greatly boosted the attention toward G-quadruplexes as attractive therapeutic targets for drugs like small molecule ligands that can stabilize their structure [1]. G-quadruplexes display quite similar structural properties in terms of rise and twist of the right-handed helical motif, while any of the four G-tracts can have a parallel or anti-
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