Supramolecular RNAi with Multifunctional siRNA Nanostructures

Abstract

Supramolecular RNA nanostructures have recently emerged as powerful genetic tools in biomedical applications. The modular design of discrete RNA building block monomers for the self-assembly of genetically encoded short interfering RNA (siRNA) nanostructures has led to the precise silencing of oncogene targets in cancer gene therapy applications. In this study, we designed and synthesized branched V-and Y-shaped RNA templates to scaffold the assembly of well-defined 2D and 3Dshaped siRNA nanostructures targeting the oncogenic mRNA transcripts of the Glucose Regulated Proteins (GRP 75, 78, 94, 170) in cancer. Structure and stability analyses confirmed the formation of siRNA nano-squares, cubes and tubes of distinct supramolecular and biophysical properties. Bioconjugation strategies enabled the incorporation of fluorescein and coumarin fluorescent reporters, providing FRET-based biosensing of the dynamic assembly process of the siRNA nanostructures. In the human adenocarcinoma (A549) cell line, the fluorescently labelled siRNA nanostructures demonstrated cell uptake and intracellular localization for GRP silencing events that led to significant anti-proliferative effects, as an indicator of anticancer activity. This research will highlight the innovation of multifunctional siRNA nanostructures for applications as cancer gene-silencing therapies.