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Structure of human telomere G-quadruplex in the presence of a model drug

G-rich DNA sequences are able to fold into structures called G-quadruplexes. These structures are the focus of a number of studies in both fundamental and applied research, from cancer biology and novel therapeutics through to nanotechnology. G4 may fold in a variety of different topologies, depending on a range of factors, such as oligonucleotide sequence, the particular cation used, the presence of crowding agents, the DNA concentration and the specific interaction with ligands. This last point is key for therapeutic applications, as different conformers can be visited during the binding of G4 with ligands. 
A team of researchers from Italy (University of Perugia, IOM-CNR, Elettra Sincrotrone Trieste – IUVS beamline) and Germany (JCNS & ICS, JCNS at Heinz Maier-Leibnitz Zentrum) has deployed a multi-technique approach, by combining circular dichroism spectroscopy (CD), ultraviolet resonance Raman spectroscopy (UVRR) and small angle scattering techniques (SAS), to elucidate how the structural features of the human telomeric G-quadruplex d[A(GGGTTA)3GGG] (Tel22) change upon thermal unfolding. The system was studied both in the free form and when it is bound to Actinomycin D (ActD), an anticancer ligand with remarkable conformational flexibility. UVRR and CD experiments revealed the molecular details of stacking of Tel22 and Tel22+ActD upon melting, as highlighted by the spectral changes observed for temperature dependent UV Raman spectra collected on free and complexed Tel22 (Fig. 1).
Singular value decomposition analysis applied to CD and UVRR data allowed to identify a temperature region populated with intermediate conformers along the path from the native to the unfolded state, for both the quadruplex alone and the complex. Small angles Neutron Scattering (SANS) technique was decisively used to provide evidence for ligand-induced quadruplex dimerization. On the other hand, small angles X-ray Scattering (SAXS) was exploited to characterize the dimer dissociation and the trend of the characteristic size of Tel22 and Tel22+ActD with the increasing temperature.

Figure 1.   Temperature dependent UVRR spectra collected for (a) Tel22 and (b) Tel22+ActD. The arrows highlight the three bands mainly assigned to normal in-plane modes of dG residues, as represented in the central picture.


The emerging comprehensive view is that the interaction between ActD and Tel22 is not only able to perturb the equilibrium between coexisting conformations in the temperature range where the quadruplex is still in its folded state, but it can also preserve a compact structure in conditions where Tel22 undergoes thermal unfolding (Fig. 2).
These findings prove that ActD is able to set conformational constraints to the complex even in the presence of large structural fluctuations. The ability of ActD to form non-quadruplex compact structures even in single stranded G-rich DNA sequences may be of importance for the transcriptional regulation within promoter regions of oncogenes. 
 

Figure 2.  Unfolding picture for Tel22+ActD. The Folded and Intermediate 1 states present a high level of order, with stacked G-tetrads (colored in red and blue). Actinomycin D (colored in orange) is end-stacking over the extremal tetrads. After the last transition (Tm3 as shown by SVD in CD data) the correlation between tetrads is lost, but the interaction of nucleobases with the drug allows the persistence of a compact, although disordered, structure.

 

This research was conducted by the following research team:

F. Bianchi 1, L. Comez 2, R. Biehl 3, F. D’Amico 4, A. Gessini 4, M. Longo 5, C. Masciovecchio 4, C. Petrillo 1, A. Radulescu 5, B. Rossi 4, F. Sacchetti 1, F. Sebastiani 6, N. Violini 7and A. Paciaroni 1

 

1Dipartimento di Fisica e Geologia, Universita di Perugia, 06123 Perugia, Italy,

IOM-CNR c/o Dipartimento di Fisica e Geologia, Universita di Perugia, 06123 Perugia, Italy 

JCNS & ICS, Forschungszentrum Juelich, GmbH, Leo-Brandt Strasse, 52425 Juelich, Germany

Elettra-Sincrotrone Trieste, Strada Statale 14 - km 163,5 in AREA Science Park, 34149 Basovizza, Trieste, Italy

JCNS Forschungszentrum Juelich GmbH at Heinz Maier-Leibnitz Zentrum (MLZ), Lichtenbergstrasse 1, 85748 Garching, Germany

Lehrstuhl fur Physikalische Chemie 2, Ruhr-Universitat Bochum, 44780 Bochum, Germany

JCNS, Forschungszentrum Juelich GmbH, Leo-Brandt Strasse, 52425 Juelich, Germany


Contact persons:

Alessandro Paciaroni, e-mail: alessandro.paciaroni@unipg.it
Lucia Comez, e-mail: comez@iom.cnr.it 
 

 The authors acknowledge the CERIC-ERIC Consortium for the access to experimental facilities and financial support.


Reference

F. Bianchi, L. Comez, R. Biehl, F. D'Amico, A. Gessini, M. Longo, C. Masciovecchio, C. Petrillo, A. Radulescu, B. Rossi, F. Sacchetti, F. Sebastiani, N. Violini, A. Paciaroni “Structure of human telomere G-quadruplex in the presence of a model drug along the thermal unfolding pathway”, Nucleic Acids Research 22, 11927 (2019), DOI: doi.org/10.1093/nar/gky1092

 
Last Updated on Tuesday, 16 April 2019 18:06