Direct Cytosolic Delivery of Amphiphilic Framework Nucleic Acids for RNA Interference

The efficient cytosolic delivery of nucleic acid molecular machines remains a major challenge due to the dual barriers of the cell membrane and endosomal sequestration. Here, we report a class of amphiphilic framework nucleic acids (ampFNAs) that enable direct cytosolic delivery involving energy-independent traversal of lipid membranes. Among several designed geometries, a rigid rod-like six-helix bundle functionalized with a single cholesterol moiety exhibits superior cellular binding and internalization. We find that this ampFNA can enter cells through a cholesterol-dependent, lipid raft-mediated pathway, capable of bypassing endosomal entrapment. Compared to the commercial transfection reagent Lipofectamine 3000 (Lipo3000), the ampFNA platform exhibits reduced lysosomal entrapment. When delivering small interfering RNAs (siRNAs), the ampFNA-mediated enhanced green fluorescent protein (EGFP) gene silencing was achieved with efficiency comparable to Lipo3000. By targeting the proto-oncogene Bcl-2, the ampFNA induced an apoptotic rate of 31.3% in the tumor cell population. Our work establishes ampFNAs as a programmable, efficient, and biocompatible platform for the development of next‑generation smart nucleic acid delivery machines for precision medicine.