Single-molecule studies of surface-immobilised and freely diffusing RNA structures
Abstract
During the process of transcription, a messenger RNA (mRNA) copy of the genetic
information encoded in the DNA is built. As mRNA is constructed, secondary and
tertiary motifs may form, which combine into intricate structures through a
process called RNA folding, enabling RNA to perform biological functions beyond
transporting genetic information, including gene regulation and catalytic self-
cleaving processes. Facilitating RNA folding are divalent ions, located site-
specifically within the structure, and monovalent ions which bind non-specifically
to the phosphate backbone, shielding the negative charge and allowing the motifs
to move into close proximity and interact. Three-way RNA junctions are among the
smallest biologically-active RNA structures and are known to mediate both gene
regulatory and catalytic processes. In the first part of this thesis, I use single-
molecule total-internal reflection fluorescence microscopy with Förster resonance
energy transfer (TIRFM-FRET) to characterise the folding and function of two of
these structures: the adenine riboswitch and the hammerhead ribozyme. Using
single-molecule Förster resonance energy transfer (sm-FRET), I extract
information on both the prevalent conformations of these molecules at specific
chemical conditions and kinetic information on structural rearrangements which
occurr on both the molecular and global levels. Building on this knowledge built up
of the folding pathway of the adenine riboswitch induced by monovalent ions, I
moved to develop a method in which the competing interplay between monovalent
ions and urea, an unfolding reagent, is exploited to isolate and overpopulate a
transient intermediate state identified on the folding pathway. Although chemical denaturants are commonly used to investigate the structures of proteins, their
application to RNA folding is still in its infancy. For the first time, I demonstrate
that this approach allows the manipulation of the folding dynamics of RNA, forcing
the structure into a state which is ordinarily poorly-populated. I speculate that this
could enable a detailed characterisation of these states by NMR and other high
resolution ensemble techniques. Finally, I move on to expanding the range of
single-molecule techniques available in St Andrews. Despite the power of single-
molecule TIRFM-FRET, it requires surface immobilisation, which can compromise
biological function through further modifications to the natural form of the sample
under investigation. To overcome this problem, I implement single-molecule
fluorescence correlation spectroscopy (sm-FCS), which probes freely diffusing
samples in solution. After testing this sm-FCS system various test structures, I
upgrade it for dual-colour fluorescence cross-correlation spectroscopy (sm-FCCS),
and finally to multi-parameter fluorescence detection (sm-MFD), where the
fluorescence lifetime of the sample is also returned. The capabilities of these three
techniques are tested by examining protein-DNA interactions, RNA structure and
vesicle morphology.
Type
Thesis, PhD Doctor of Philosophy
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