Title

Paranemic and Receptor-Loop RNA Motifs: Versatile Interactions for Biosensing Platforms and Nanotechnology Scaffolds

Date of Award

2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Chemistry

First Advisor

Neocles Leontis, PhD

Second Advisor

Neal Carothers (Committee Member)

Third Advisor

R. Marshall Wilson, PhD (Committee Member)

Fourth Advisor

H. Peter Lu, PhD (Committee Member)

Abstract

RNA has favorable properties for use as a medium for constructing multivalent, modular nanoparticles to deliver therapeutic agents targeting virus-infected or cancer cells or for serving as scaffolds to organize the matter at nanoscale. RNA nanoparticles possessing diverse functionalities can be engineered to self-associate via interaction motifs to increase their in vivo stability and propensity for cellular uptake or form various materials of fibrous architecture.

High affinity and specificity RNA-RNA binding interfaces can be constructed by combining pairs of GNRA loop/loop-receptor interaction motifs. By fusing these RNA interactions and 4-way junction motifs, we have developed tecto-RNA complexes possessing favorable properties for drug delivery applications such as enhanced nuclease protection and hetero-multimerization amenability desirable for multi-functionality aims. We demonstrated that these RNA molecules can be programmed for uncompensated assembly to form closed, ring-shaped complexes of defined and predictable stoichiometries that assemble cooperatively. We provided a step-by-step description how the stoichiometry can be controlled at the RNA monomer level from ring-closed dimeric, trimeric and tetrameric complexes to polymeric structures where ring formation is no longer possible. Structure-probing studies of optimally designed dimer and trimer complexes provided strong experimental evidence that RNA systems self-associate as intended by design. Detailed thermodynamic analysis of tecto-RNA self-assembly allowed us to disclose the binding affinities, to quantify the cooperativity of assembly and to elucidate the energy of four-way junction conformational adjustments for interaction.

Alternative interaction motifs such as paranemic crossover (PX) motifs provide specific, programmable and reversible binding interactions between pre-folded nucleic acid molecules. We explored their potential for RNA biosensing and RNA nanotechnology applications. Sequence-specific, label-free RNA biosensors targeting pre-folded internal loop motifs were constructed by coupling paranemic binding motifs to a Malachite Green aptamer. We showed that this binding is sequence-specific as single-basepair mismatches in the paranemic binding motif disrupt the sensor-target interaction. We also explored the use of the paranemic motif as a cohesion tool for engineering linear RNA fibrils and for recognition of asymmetric, artificially-designed and natural internal loops.