CROWDASSAY · Folding Pathways of DNA G-quadruplexes in Crowding Conditions, and Implications for Mass Spectrometry-based Ligand Screening Assays
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2018-06-01 → 2020-05-31
- EU contribution
- €173,076
- Participants
- 1
- Scheme
- MSCA-IF-EF-ST
Lines connect the coordinator with its partners.
Results in brief
Folding Pathways of DNA G-quadruplexes in Crowding Conditions, and Implications for Mass Spectrometry-based Ligand Screening Assays
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. In conclusion, this work contributed to reveal some fundamental principles of nucleic acid folding.
Data: CORDIS, © European Union
Project objective
Nucleic acids come in many forms aside from the Watson-Crick duplex. For example, guanine-rich DNA strands can form G-quadruplex structures, which have become attractive targets for small molecule ligands. It is now proven that G-quadruplexes can form in cells. Today most biophysical and structural studies on G-quadruplex folding are carried out in dilute aqueous solutions, but recent works suggest that the folding of some G-quadruplexes may differ in a crowded environment such as the cell environment. In the same way, most in vitro ligand binding assays are today carried out in dilute aqueous solutions. The aim of the project is to assess whether and how the folding pathways of G-quadruplexes differs in dilute and crowded conditions, and whether the community should revise the experimental design of ligand screening assays. We will focus on mass spectrometry-based assays, which have the unique advantage to give a direct read-out of ligand binding stoichiometry, quantity, and the variety of structural ensembles hiding behind the free and bound nucleic acid. The research program involves (1) developing mass spectrometry in more “native” conditions in the sense that we will add co-solutes to mimic cellular crowding, (2) comparing the G-quadruplex folding pathways in dilute and crowded conditions, by combining for the first time mass spectrometry, nuclear magnetic resonance, and single-molecule FRET (Förster resonance energy transfer), and (3) evaluating the impact of crowding on both traditional melting assays and mass spectrometry-based ligand screening assays. Our project will contribute to unveil fundamental principles of nucleic acid folding. It will also foster collaboration between three European institutes specialized in complementary biophysical and structural approaches to study G-quadruplexes. Finally, our project will also contribute to society by improving analytical approaches that are highly relevant to pharmacology.
Original text from CORDIS.
Participants
- INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE · ParisCoordinatorFrance
Links
Data: CORDIS, © European Union