Site-selective faithful monitoring at the single molecule level of local changes in nucleic acids – SMFLUONA

Numerous cell mechanisms and pathways rely on dynamic interactions of RNAs or DNAs with proteins that induce local and transient changes in their secondary and tertiary structures. Though structural methods, such as X-ray diffraction, NMR or electron microscopy provide invaluable information on the structure of the protein/nucleic acid complexes, they are less suited for monitoring the dynamics of these interactions, especially in diluted solutions. Due to their exquisite sensitivity, fluorescence-based techniques are highly potent for this purpose. However, in the case of nucleic acids, these techniques suffer from the fact that natural nucleobases are almost not fluorescent, so that labeling with external and generally bulky probes is requested. A breakthrough has been recently achieved with the introduction of thienoguanosine (thG), a truly faithful emissive and responsive surrogate for G, which actually reproduces the structural context and dynamics of the parent native nucleoside. In addition, this fluorescent G analogue remains well fluorescent when incorporated in ODNs, and exhibits environment sensitive fluorescence properties. thG is expected to transform nucleic acid biophysics by allowing for the first time to selectively and faithfully monitor the conformations and dynamics of a given G residue in a nucleic acid sequence. To further extend the applications of this outstanding nucleoside analogue and provide for the first time relevant information on the local and transient modifications of nucleic acids at the single molecule level, the objective of the SMFLUONA project is to implement and apply thG-based single molecule experiments. To this end, one of the main challenges is to increase thG brightness and photostability. This will be achieved by using the surface plasmon resonances of Al and Mg nanoparticles (NPs) that match with thG absorption (300-400 nm), but require a precise control of the distance between the dye and the metallic surface. This control will be achieved by synthesizing Al and Mg NPs of controlled size and surface, and then grafting them at a 1:1 stoichiometry with thG-labeled ODNs of appropriate length or by accurately positioning them together with thG-labeled ODNs on DNA origami platforms. To validate and apply this thG-based single molecule approach, we will monitor the base flipping steps of the DNA methylation replication by the tandem formed by the DNA methyltransferase 1 (DNMT1) and its guide, the UHRF1 protein (Ubiquitin-like, containing PHD and RING finger domains). DNA methylation of cytosines in CpG sites at very precise positions on the genome provides key epigenetic marks that allow a cell to express a well-defined number of genes which determine the cell identity. When the cells multiply, they must transmit this information by faithfully copying the methylation marks. Therefore, thG-based single molecule experiments should provide for the first time a complete picture of the SRA- and DNMT1-induced base flipping processes, as well as their dependence on the CpG context, DNA length and key protein residues. This thG-based single molecule fluorescence approach will help solving a large range of biological questions involving local and transient structural nucleic acid changes. Moreover, the deciphering of the molecular mechanism of the base flipping steps in DNA methylation replication by the UHRF1/DNMT1 tandem should provide new clues on its possible blockage and thus, lead to major therapeutic applications in pathologies, such as cancers and neurodegenerative diseases, where the methylation profile is modified. This interdisciplinary project will be performed by two highly complementary partners with expertise in fluorescence, advanced fluorescence techniques and UHRF1/DNMT1 (Partner 1, Y. Mély, U. Strasbourg) and metal-induced fluorescence enhancement and synthesis of Al NPs (Partner 2, J. Plain, UTT Troyes).