The organization of biological materials into versatile three-dimensional assemblies could be used to build multifunctional therapeutic scaffolds for use in nanomedicine. Here, we report a strategy to design three-dimensional nanoscale scaffolds that can be self-assembled from RNA with precise control over their shape, size and composition. These cubic nanoscaffolds are only ∼13 nm in diameter and are composed of short oligonucleotides, making them amenable to chemical synthesis, point modifications and further functionalization. Nanocube assembly is verified by gel assays, dynamic light scattering and cryogenic electron microscopy. Formation of functional RNA nanocubes is also demonstrated by incorporation of a light-up fluorescent RNA aptamer that is optimally active only upon full RNA assembly. Moreover, we show that the RNA nanoscaffolds can self-assemble in isothermal conditions (37 °C) during in vitro transcription, which opens a route towards the construction of sensors, programmable packaging and cargo delivery systems for biomedical applications.

Our recent advancements in RNA nanotechnology introduced novel nanoscaffolds (nanorings); however, the potential of their use for biomedical applications was never fully revealed. As presented here, besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous embedment of assorted RNA aptamers, fluorescent dyes, proteins, as well as recently developed RNA–DNA hybrids aimed to conditionally activate multiple split functionalities inside cells.

Using the massively parallel genetic algorithm for RNA folding, we show that the core region of the 3'-untranslated region of the dengue virus (DENV) RNA can form two dumbbell structures (5'- and 3'-DBs) of unequal frequencies of occurrence. These structures have the propensity to form two potential pseudoknots between identical five-nucleotide terminal loops 1 and 2 (TL1 and TL2) and their complementary pseudoknot motifs, PK2 and PK1. Mutagenesis using a DENV2 replicon RNA encoding the Renilla luciferase reporter indicated that all four motifs and the conserved sequence 2 (CS2) element within the 3'-DB are important for replication. However, for translation, mutation of TL1 alone does not have any effect; TL2 mutation has only a modest effect in translation, but translation is reduced by ∼60% in the TL1/TL2 double mutant, indicating that TL1 exhibits a cooperative synergy with TL2 in translation. Despite the variable contributions of individual TL and PK motifs in translation, WT levels are achieved when the complementarity between TL1/PK2 and TL2/PK1 is maintained even under conditions of inhibition of the translation initiation factor 4E function mediated by LY294002 via a noncanonical pathway. Taken together, our results indicate that the cis-acting RNA elements in the core region of DENV2 RNA that include two DB structures are required not only for RNA replication but also for optimal translation.

Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use.