Autonomously folded α-helical lockers promote RNAi

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Delivery

Background:

• RNA interference (RNAi) is a powerful research tool with significant therapeutic promise.
• The clinical application of RNAi is limited by challenges in siRNA delivery and understanding gene suppression mechanisms.
• The precise relationship between siRNA delivery and gene silencing efficacy requires further elucidation.

Purpose of the Study:

• To investigate the role of interfacial tertiary contacts between alpha-helices in regulating siRNA cytoplasmic delivery and RNAi.
• To introduce a novel rationale of helical amphipathic lockers to differentiate functional and non-functional helices in gene silencing.
• To develop structurally balanced amphipathic scaffolds for enhanced RNAi applications.

Main Methods:

• Design and synthesis of helical amphipathic lockers with specific interfacial properties.
• Cellular delivery studies using energy-dependent endocytosis to assess siRNA uptake.
• Gene silencing assays to quantify the efficacy of different helical designs in promoting RNAi.

Main Results:

• Both helical designs facilitated siRNA delivery into cells via energy-dependent endocytosis.
• Only autonomously folded helices with pre-locked hydrophobic interfaces demonstrated statistically significant gene silencing.
• The "amphipathic locking" of helices before siRNA binding was identified as crucial for enabling RNAi.

Conclusions:

• Interfacial tertiary contacts between alpha-helices can regulate siRNA cytoplasmic delivery and RNAi efficacy.
• Helical amphipathic lockers provide a rational design principle for developing effective RNAi therapeutics.
• This approach offers a pathway to advance the clinical exploitation of functional RNAi by optimizing siRNA delivery and gene suppression.