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.