Abstract:
The structure/function relation of nucleic acids represents a fundamental challenge in modern molecular biology. While DNA and RNA were long studied mainly for their role in the storage and transmission of genetic information, it is now clearly established that their structural, dynamic and conformational properties condition wide diversity of their biological function. Their structural plasticity, particularly that of RNAs, allow the adoption of various secondary and tertiary structures, directly involved in genetic regulation, assembly of ribonucleoprotein, catalysis or even cellular communication. In this context, the identification and characterization of the dynamic structure of nucleic acids in solution appear as a major methodological challenge. This issue is particularly critical for messenger RNAs, whose interest has increased considerably with the development of mRNA vaccines. The stability, three-dimensional organization and structural homogeneity of mRNAs directly condition their biological efficiency, immunogenicity and robustness during production, formulation and storage steps. However, mRNA structural study, which are large and poorly structured, remains complex.
In this context, this thesis explores and develops the use of circular dichroism (CD) spectroscopy as an analysis tool for the secondary structure of complex nucleic acids. The main objective of this thesis work was to establish a complete methodological framework allowing the structural analysis of nucleic acids by CD, from the construction of reference resources enabling inter-experimental comparison to their application to biological system. The first contribution of this thesis lies in the establishment of the Nucleic Acid Circular Dichroism DataBase (NACDDB), the first database dedicated to CD spectra of nucleic acids. The NACDDB provides a unified format associating each spectrum with detailed information about the sequence, the chemical nature of the nucleic acid and the experimental conditions. Published with more than a hundred spectrum, covering DNA, RNA and DNA/RNA hybrids structures, the NACDDB is a rigorous reference resource allowing inter-experimental comparison.
Based on these CD references, an in-depth work was carried out in order to define spectral characteristics signatures of the main secondary structures adopted by nucleic acids. An original approach was therefore developed, based on the combined exploitation of CD spectra and known structural information from the literature and theoretical sequence design. From an extended and carefully annotated dataset, eighteen distinct spectral references were defined. These spectral references served as the basis for the development of an original method for the deconvolution of nucleic acid CD spectra. The method developed in this thesis allows a quantitative estimation of the secondary structure composition of oligonucleotides containing multiple distinct folding.
Finally, the methods developed during this thesis were applied to the study of the structure of viral mRNAs used in the development of mRNA vaccines. This analysis shows a satisfactory agreement between the proportions of double-stranded and disordered regions. Thus, this thesis establishes a coherent methodological framework for the exploitation of circular dichroism in the study of nucleic acids structure.
Keywords: Synchrotron, Database, Circular dichroism, Spectroscopy, RNA.