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<p>RNAs are growing in their importance as regulators in multiple biological processes.
A deep understanding of how RNAs function within cells requires an understanding of
their dynamic behavior to enable the targeting of RNA in drug discovery as therapeutics.
The HIV-1 Rev response element (RRE) RNA which is a known drug target mediates the
export of incompletely-spliced viral RNA to express viral proteins required for HIV-1
replication and spread. Rev protein first recognizes the purine-rich region on RRE
stem IIB (RREIIB), and then other Rev monomers cooperatively assemble on RRE to form
ribonucleoprotein complex. Conformational flexibility at this stem IIB region has
been shown to be important for Rev binding. In addition, m6A modifications on HIV-1
RNA has shown their critical roles to HIV-1 replication, and two probably essential
m6A sites on purine-rich region on RREIIB was discovered. However, the nature of the
flexibility and m6A modification to RREIIB have remained elusive. The work in this
thesis is aiming to characterize the conformational dynamics of RRE stem IIB as potential
drug targets, and to discover small molecules binding to RRE using computational and
experimental drug screening. </p><p>First, nuclear magnetic resonance (NMR) techniques
are applied to identify the native and non-native conformations of RREIIB. Including
relaxation dispersion NMR and a new strategy for directly observing transient conformational
states in large RNAs, we find that stem IIB alone or when part of the larger stem
II three-way junction robustly exists in dynamic equilibrium with non-native excited
state (ES) conformations that have a combined population of around 20%. The ESs disrupt
the Rev binding site by changing local secondary structure and their stabilization
via point substitution mutations decreases the binding affinity to the Rev arginine-rich
motif (ARM) by 15 to 80 fold. The ensemble clarifies the conformational flexibility
observed in stem IIB, reveals long-range conformational coupling between stem IIB
and the three-way junction that may play important roles in the cooperative Rev binding
and the development of anti-HIV therapeutics. </p><p>Secondly, m6A has also been found
in viral RNAs where it is proposed to modulate host-pathogen interactions. Two m6A
sites have been reported in the RREIIB, one of which was shown to enhance binding
to the viral protein Rev and viral RNA export. However, because these m6A sites have
not been observed in other studies mapping m6A in HIV-1 RNA, their significance remains
to be firmly established. We show that m6A minimally impacts the stability, structure,
and dynamics of RRE stem IIB as well as its binding affinity to the Rev-ARM using
optical melting experiments, NMR spectroscopy, and in vitro binding assays. Our results
indicate that if present in stem IIB, m6A is unlikely to substantially alter the conformational
properties of the RNA. </p><p>Next, to confirm the RRE ESs ensemble visualized in
vitro can recapitulate in cells, we show that stabilizing ESs using point substitution
mutations leads to potent conformation-dependent inhibition of RNA cellular activity.
The point substitution mutations with invert the equilibrium so that the ES becomes
the dominant (>50% population) conformation and secondary rescue mutations to restore
the GS conformation and control for sequence effects. We then demonstrate that the
degree to which increasing the population of the ES at the expense of the GS leads
to a corresponding decrease in cellular activity. The results also support that stabilizing
non-native ESs potentially provides an alternative therapeutic strategy for targeting
RNA. </p><p>Finally, we construct atomic resolution ensembles for the RRE ground state
using RDC-SAS; perform computational docking against the ensemble of RRE GS ,and validate
selected hits using in vitro and cell-based assays. We will also generate the dynamic
ensembles of RRE ESs as alternative targets for ensemble-based virtual screening.
The final goal is to identify small molecules that stabilize RRE GS or inactive ESs
and thereby inhibit Rev-RRE interaction.</p>
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