Surface and Interfacial Approaches for the Characterization of Biomolecular Interactions and to Optimize Desi-MS Performance

This work involves the surface and interfacial characterization of biomolecular interactions by atomic force microscopy and optimization of desorption electrospray ionization mass spectrometry. In the first part (part I), suitable surface of functionalized porous silicon are created and characterized to enhance Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) capability. DESI-MS is a powerful emerging analytical tool that finds applications in fundamental research and as a diagnostic tool. Enhancement of the performance of this technique will make it superior and improve the scope of its applications. The use of super hydrophobic porous silicon proved to enhance the desorption/ionization mechanism of DESI. It improves the ionization efficiency almost two fold when compared to the traditionally used glass slide and porous polytetraflouroethylene surfaces under the same conditions. The functionalized porous surfaces showed incredible stability, which is suitable for long time and high throughput analysis. We proposed a mechanism whereby the porous silicon acts as a barrier for the spray solvent, and creates a pool of analyte during desorption, leading to greater stability. On the other hand, the super hydrophobic functionality improves the ionization power of the technique by increasing analyte concentration over the area sampled and preventing filing of the pores. The functionalized porous surfaces are also suitable for DESI-imaging of biomolecules and tissue cells. In the second part (part II) of this thesis, Atomic Force Microscopy (AFM) was used to confirm site-specific protein-DNA interaction of the vancomycin resistance associated regulatory protein (VraR) from S. aureus. The protein stoichiometry at the binding site was confirmed as being mostly dimer for VraR, and as oligomers for phosphorylated VraR. In another project, AFM proved to be a very useful technique for the visualization and characterization of RNA. It enable us to visualized for the first time, the three dimensional structural architecture of genomic RNA of tomato bushy stunt virus. AFM allowed us to confirm the proposed long range-RNA-RNA interaction existing within the genome, leading to a more compact structure. Volume analyses enable to confirm the existence of compact structures as visualized in the AFM images. These results are consistent with expected conformations utilized by the TBSV virus for different viral processes. Sub-genomic RNA expressed by the TBSV virus, also exhibited compact structures with different degree of protrusions. All observed RNA and sub-genomic RNA structures from our AFM images were consistent with the selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) predicted structures.