We introduce a new concept that utilizes cognate nucleic acid nanoparticles which are fully complementary and functionally-interdependent to each other. In the described approach, the physical interaction between sets of designed nanoparticles initiates a rapid isothermal shape change which triggers the activation of multiple functionalities and biological pathways including transcription, energy transfer, functional aptamers and RNA interference. The individual nanoparticles are not active and have controllable kinetics of re-association and fine-tunable chemical and thermodynamic stabilities. Computational algorithms were developed to accurately predict melting temperatures of nanoparticles of various compositions and trace the process of their re-association in silico. Additionally, tunable immunostimulatory properties of described nanoparticles suggest that the particles that do not induce pro-inflammatory cytokines and high levels of interferons can be used as scaffolds to carry therapeutic oligonucleotides, while particles with strong interferon and mild pro-inflammatory cytokine induction may qualify as vaccine adjuvants. The presented concept provides a simple, cost-effective and straightforward model for the development of combinatorial regulation of biological processes in nucleic acid nanotechnology.

In the past few years, the study of therapeutic RNA nanotechnology has expanded tremendously to encompass a large group of interdisciplinary sciences. It is now evident that rationally designed programmable RNA nanostructures offer unique advantages in addressing contemporary therapeutic challenges such as distinguishing target cell types and ameliorating disease. However, to maximize the therapeutic benefit of these nanostructures, it is essential that we understand the immunostimulatory aptitude of such tools and identify potential complications. We present a set of 16 nanoparticle platforms that are highly configurable. These novel nucleic acid-based polygonal platforms are programmed for controllable self-assembly from RNA and/or DNA strands via canonical Watson-Crick interactions. We demonstrate that the immunostimulatory properties of these particular designs can be tuned to elicit the desired immune response or lack thereof. To advance our current understanding of the nanoparticle properties that contribute to the observed immunomodulatory activity and establish corresponding designing principles, we conducted QSAR (quantitative structure-activity relationships) modeling. The results demonstrate that molecular weight, together with melting temperature and half-life, strongly predict the observed immunomodulatory activity. This framework provides the fundamental guidelines necessary for the development of a new library of nanoparticles with predictable immunomodulatory activity.

RNA nanotechnology employs synthetically modified ribonucleic acid (RNA) to engineer highly stable nanostructures in one, two, and three dimensions for medical applications. Despite the tremendous advantages in RNA nanotechnology, unmodified RNA itself is fragile and prone to enzymatic degradation. In contrast to use traditionally modified RNA strands e.g. 2'-fluorine, 2'-amine, 2'-methyl, we studied the effect of RNA/DNA hybrid approach utilizing a computer-assisted RNA tetra-uracil (tetra-U) motif as a toolkit to address questions related to assembly efficiency, versatility, stability, and the production costs of hybrid RNA/DNA nanoparticles. The tetra-U RNA motif was implemented to construct four functional triangles using RNA, DNA and RNA/DNA mixtures, resulting in fine-tunable enzymatic and thermodynamic stabilities, immunostimulatory activity and RNAi capability. Moreover, the tetra-U toolkit has great potential in the fabrication of rectangular, pentagonal, and hexagonal NPs, representing the power of simplicity of RNA/DNA approach for RNA nanotechnology and nanomedicine community.

Precise shape control of architectures at the nanometer scale is an intriguing but extremely challenging facet. RNA has recently emerged as a unique material and thermostable building block for use in nanoparticle construction. Here, we describe a simple method from design to synthesis of RNA triangle, square, and pentagon by stretching RNA 3WJ native angle from 60° to 90° and 108°, using the three-way junction (3WJ) of the pRNA from bacteriophage phi29 dsDNA packaging motor. These methods for the construction of elegant polygons can be applied to other RNA building blocks including the utilization and application of RNA 4-way, 5-way, and other multi-way junctions.

Temperature gradient gel electrophoresis (TGGE) is a powerful tool used to analyze the thermal stabilities of nucleic acids. While TGGE is a decades-old technique, it has recently gained favor in the field of RNA nanotechnology, notably in assessing the thermal stabilities of RNA nanoparticles (NPs). With TGGE, an electrical current and a linear temperature gradient are applied simultaneously to NP-loaded polyacrylamide gel, separating the negatively charged NPs based on their thermal behavior (a more stable RNA complex will remain intact through higher temperature ranges). The linear temperature gradient can be set either perpendicular or parallel to the electrical current, as either will make the NPs undergo a transition from native to denatured conformations. Often, the melting transition is influenced by sequence variations, secondary/tertiary structures, concentrations, and external factors such as the presence of a denaturing agent (e.g., urea), the presence of monovalent or divalent metal ions, and the pH of the solvent. In this chapter, we describe the experimental setup and the analysis of the thermal stability of RNA NPs in native conditions using a modified version of a commercially available TGGE system.