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Physical properties of R-Loops and viral capsids : A single molecule approach based on AFM high resolution imaging and nano-indentation

mar 06 avr 2021



Soutenance de thèse de Mme Yéraldinne CARRASCO SALAS sous la direction de thèse de Mme Cendrine MOSKALENKO

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Description générale

By combining high-resolution AFM imaging and nano-mechanical measurements at the single molecule level, we studied the physical properties of two different biological objects:
R-Loops are DNA/RNA hybrid structures arising from the DNA transcription process. The RNA hybridizes with its DNA template by Watson-Crick interactions while the non-template DNA strand remains single stranded. The R-loops structures have been associated with genomic instability phenotypes in vivo. We characterized by AFM imaging the conformation of these structures generated in vitro. Through the analysis of quantitative physical parameters, we showed that these R-loops generated for different genes differ in their architecture. We could conclude that the non-template DNA strand organization is a fundamental characteristic to lead to the formation of a compact hybrid object, which could then generate DNA breaks dependent on replication.
We studied the physical properties of two viral capsids, the AAV vector and the Hepatitis B capsid in connection with their disassembly. By combining the imaging and mechanical response measurement in a liquid medium using AFM, we obtained quantitative information on their mechanical properties. An indentation profile over the capsid allows highlighting spatial heterogeneities, which origin is partly geometric and results from an intrinsic heterogeneity linked to the capsid organization. We also compared the capsid mechanical properties to the ability to release their genome in vitro by temperature-induced. Finally, we studied the Inverted Terminal Repeats (ITRs) structure of AAV. These ITRs adopt a functional structure that we characterized by AFM imaging.

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