Lipid membranes modulate the activity of RNA through sequence-dependent interactions

Czerniak, T.; Sáenz, J. P. (2022) Proceedings of the National Academy of Sciences, 119(4), e2119235119. DOI: 10.1073/pnas.2119235119

Summary

We showed that lipid membranes can act as RNA organization platforms, directly modulating ribozyme activity through sequence-dependent RNA-lipid interactions. The activity of the R3C ligase ribozyme is altered by the presence of lipid membranes, with binding dependent on RNA nucleotide content, base pairing, and length. Guanine content in short RNAs is crucial for lipid binding, and G-quadruplex formation further promotes the interaction. By engineering the R3C substrate sequence to enhance membrane binding, we generated a lipid-sensitive ribozyme with riboswitch-like behavior — demonstrating that RNA-lipid interactions can be harnessed for synthetic riboregulation.

Key Findings

• Lipid membranes modify R3C ribozyme activity — the membrane sequesters substrate RNA, reducing its availability for the catalytic reaction.
• RNA-lipid interactions are sequence-dependent: guanine content, base pairing state, and RNA length all determine binding affinity.
• Short, single-stranded RNAs rich in guanine bind lipid membranes most effectively. G-quadruplex structures further enhance binding.
• By modifying the R3C substrate to increase guanine content, we engineered a lipid-sensitive ribozyme whose activity responds to membrane presence — a synthetic riboswitch-like system.
• RNA-lipid binding occurs with zwitterionic phospholipid membranes (DOPC), not only charged lipids, indicating that the interaction is not purely electrostatic.

Methods

• R3C ribozyme assay: fluorescence-based ligation assay to measure ribozyme activity in the presence/absence of liposomes.
• RNA-lipid binding: fluorescence quenching and gel electrophoresis to quantify RNA partitioning to liposome surfaces.
• Sequence engineering: systematic modification of R3C substrate nucleotide content to tune membrane binding affinity.
• G-quadruplex analysis: circular dichroism and fluorescence to detect G-quadruplex formation in membrane-binding RNAs.

Significance

This paper opened an entirely new research direction for the lab — RNA-lipid interactions as a mechanism for riboregulation. It demonstrated that the two most ancient classes of biomolecules (RNA and lipids) can interact directly, with functional consequences for catalysis. The implications span synthetic biology (engineerable riboswitches), RNA World scenarios (membrane-based RNA regulation before proteins), and potentially modern cell biology (undiscovered RNA-membrane interactions).