Chemical modifications of RNA influence the micellar assembly stability and intracellular distribution of polycation/RNA complexes

RNA molecules play a crucial role in regulating genetic information within cells and have increasingly emerged as a promising class of therapeutic agents for a wide range of diseases. The delivery of RNA to intracellular targets remains a pivotal challenge in advancing RNA-based therapeutics. Current research on RNA delivery has primarily concentrated on the design of delivery carriers and chemical modification of RNA. However, the influence of RNA structural characteristics, particularly chemical modifications, on the assembly properties and intracellular fate of cationic carrier/RNA complexes has been relatively overlooked. Hence, we systematically investigated how chemical modification of RNA influenced the micellar assembly and delivery properties of polycation/RNA complexes, using a cationic polymer (TPE-PEG^2K-8R, TP8R) as a model carrier. Nine chemical modifications of RNA significantly altered the assembly, cellular uptake efficiency, and subcellular distribution patterns of polycation/RNA complexes by modulating interfacial interactions. Crucially, using anti-miR-21 as a therapeutic model, we have demonstrated that hydrophobic modifications (e.g., Cy5) induced mitochondrial uptake of the cargo, thus rendering it therapeutically inactive, while hydrophilic and metabolically stable modifications (e.g., 2'-OMe, PS) were essential for cytosolic delivery. This study is expected to establish RNA interfacial chemistry as a critical design parameter for optimizing nucleic acid delivery systems. STATEMENT OF SIGNIFICANCE: While RNA chemical modifications are widely used to enhance stability, their impact on carrier/RNA assembly remains unexplored. This study reveals RNA chemical modifications as pivotal regulators of polycation/RNA complex assembly and intracellular fate. Key findings reveal that: (1) The conformational flexibility of nucleic acids may lead to structural distortions, which could potentially affect the assembly of multimolecular complexes. (2) Nine common chemical modifications of RNA dramatically influenced the assembly structures, binding energies, cellular uptake, and intracellular distribution of complexes. (3) Determining the precise assembly and delivery efficacy of each RNA modification is critical for advancing RNA therapies. This study bridges the gap between RNA chemistry and biomaterials engineering, providing new insights that could guide the rational design of more effective and tunable RNA delivery platforms.