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CROWDASSAY (2018-2020)

Funding: H2020-MSCA-IF-2017-799695

Researcher: Dr. Anirban Ghosh

Secondment: National Institute of Chemistry, Prof. Janez Plavec group, Ljubljana, Slovenia

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Project:

Understanding G-quadruplex (G4) conformations/folding pathway in cellular mimicking conditions is of prime importance. This will help to design ligands for stabilizing a particular topology among different conformations in order to affect G4 mediated biochemical pathways. Until now most of the in vitro biophysically assays were performed in aqueous solutions (with ~100 mM K+) thus did not account for the effect of the cellular crowding conditions (macromolecular crowding and co-solutes). The main aim of the project was to understand whether and how conformation/folding and ligand binding aspects of G quadruplexes differ in dilute aqueous solution and presence of co-solutes. 

In this work, on the methodological aspect, we tried to develop native MS of G quadruplex in presence of co-solutes (compatible with electrospray ionization). Then we demonstrated the conformational transition of different topologies of quadruplexes in the presence of two different co-solutes with a focus on telomeric G-quadruplexes (G4). Next, we screened different telomeric G4 sequences against two well-known G4 binders (PhenDC3 & 360A) and found one lead complex [23TAG (PDB: 2JSM) & PhenDC3 complex] for further in-depth structural characterization by solution NMR (Secondment in Slovenia). It is noteworthy that in the literature there are no high-resolution structures of ligand-bound 2-quartet G4. Therefore, in this study, we showed for the first time the atomistic details of ligand bound-2 quartet antiparallel human telomeric G-quadruplex in K+ solution.

Further, we tried to push the limits of in vitro screening of G4 specific ligands by adding the co-solutes in the solution to make them more relevant to the cellular condition. The differential binding modes/stoichiometry of ligands in presence of co-solutes pinpoints that current screening strategies in dilute aqueous solutions are inadequate, which needs to be revised by taking into account the co-solutes.                                                              

Another advantage of our approach was to integrate orthogonal solution spectroscopic techniques (CD, 1HNMR) with native MS (coupled with ion mobility spectrometry) to give complementary information on the different co-existing states and their structures. We incorporate them in a unified manner to provide a robust model of G-quadruplex folding.

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Main outcomes:

We have shown state-of-the-art native MS conditions for studying G-quadruplexes in K+ solutions in presence of co-solutes. In a broader application these conditions can be used for studying other types of nucleic acid (Duplex DNA & RNA, Riboswitches, etc.) and intrinsically disordered protein (IDP) folding in MS-compatible volatile buffer. So the native MS conditions can be a starting point for the study of protein folding and aggregation in presence of co-solutes.

On the other hand, during secondment it created strong collaboration between two European institutes (IECB in France and the NIC in Slovenia). In the future, this will build a long-term collaboration between those to solve complex scientific problems and thus an exchange of researchers.

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