The interaction of small regulatory RNA with model globular proteins: binding and effects on aggregation
The interaction of proteins with a variety of biomolecular targets is central to the entire cellular machinery and, in a way, the mechanistic essence of life. Proteins can cooperatively bind with other polypeptides or oligonucleotides or assemble into aggregates prompted by specific environmental conditions. In the case of protein-oligonucleotide (RNA) binding, much research has been focused on specific RNA-binding proteins to the exclusion of more fundamental questions: 1) can model globular proteins non-covalently bind to double-stranded microRNA (miRNA) regardless of sequence identity or protein size and function and 2) can these model proteins be leveraged as transporters to deliver these short oligonucleotides for genetic therapy? In this dissertation, three works are presented, with an emphasis on biomolecular interactions, as investigated by fluorescence spectroscopy. The first study demonstrates the aggregation extent of bovine serum albumin over time, incubated in highly acidic (pH 2) or basic (pH 9) solvents at room temperature. The role of protein structural and charge density changes are discussed, with an emphasis on intrinsic fluorescence spectroscopic analysis. The second study investigates the appropriateness of the segmental mobility and rigid rotor fitting models applied to time-resolved fluorescence decay analysis. After a MD simulation of the fluorophore Atto 390 attached via the N-terminus to hen egg white lysozyme, intramolecular residual effects on the ligand's mobility are presented, with subsequent emphasis on the comparison of the experimental anisotropy decay with the computed theoretical decay. The last study examines the binding affinity of human serum albumin and hen egg white lysozyme to mimic double-stranded microRNA, miR106a, in physiological in vitro conditions before investigating the roles of salt and concentration in binding. Finally, a new way to induce miRNA binding to HSA via the F-N transition is introduced, making targeted miRNA delivery possible using a native blood transport protein.