Dynamics of an RNase H-Responsive Tetrahedral DNA Nanostructure for Efficient Intracellular microRNA Inhibition

Tetrahedral DNA nanostructures (TDNs) are emerging as next-generation platforms for delivering therapeutic oligonucleotides. This study introduces a novel strategy embedding antisense oligonucleotide (ASO) sequences directly within the structural framework of TDNs, not requiring external extensions as hybridization handles and preserving flexibility for functionalization. The integration of a gapmer-based design enables structural reconfiguration upon cellular delivery, promoting ASO accessibility and efficient target engagement. To validate this approach, we engineered a TDN-gapmer targeting microRNA-21 (miR-21), a dysregulated biomarker linked to glioblastoma. Without requiring transfection agents, TDN-gapmer demonstrated autonomous delivery capacity into glioblastoma cells, leading to robust miR-21 inhibition. Mechanistic studies revealed that the integrated gapmer can potentially recruit ribonuclease (RNase) H, facilitating RNA cleavage and enhancing target suppression. Coarse-grained modeling provided a detailed view of the predicted structural transitions and thermodynamic parameters that demonstrate that cleavage initiates toehold formation, enabling strand displacement and potential catalytic reconfiguration of the nanostructures. The TDN-gapmer demonstrated stability in serum, withstanding degradation while maintaining its therapeutic potential. The ability to integrate active sequences into the structural framework increases availability for further potential multifunctionalization. This innovative TDN design establishes a versatile and transformative platform with promising implications for precision RNA-targeting therapeutics.