Small Molecule-Controlled Gene Expression: Design of Drug-like High-Affinity Modulators of a Custom-Made Riboswitch.

Area of Science:

• Biochemistry
• Synthetic Biology
• Molecular Biology

Background:

• Riboswitches are RNA molecules that regulate gene expression in response to small molecules.
• Current research on ligand analogs for riboswitches primarily focuses on antibiotic development.
• Riboswitches offer a promising platform for gene therapy due to their precise control and lack of immunogenicity.

Purpose of the Study:

• To design a synthetic small molecule ligand for the thiamine pyrophosphate (TPP) riboswitch aptamer.
• To engineer an RNA aptamer with reduced affinity for its natural ligand (TPP) but maintained binding to the novel ligand.
• To develop ribozyme-based gene expression ON/OFF switches for potential gene therapy applications.

Main Methods:

• Structure-based design of a novel small molecule ligand (BI-5232) targeting the TPP riboswitch aptamer.
• Molecular Dynamics simulations to understand ligand-RNA interactions.
• Engineering the aptamer sequence to achieve selective binding.
• Construction and testing of ribozyme-based ON/OFF gene expression switches in human cell lines.

Main Results:

• A novel small molecule ligand, BI-5232, was designed with high binding affinity (K_D = 1.0 nM) to the TPP aptamer, structurally distinct from TPP.
• The pyrophosphate moiety of TPP was replaced by an uncharged heterocycle, revealing a novel binding interaction.
• Engineered aptamer showed significantly reduced TPP affinity while retaining BI-5232 binding.
• Functional ribozyme-based ON/OFF gene expression switches were successfully constructed and demonstrated in human cells.

Conclusions:

• A novel, drug-like small molecule ligand and engineered RNA aptamer pair was developed.
• This engineered riboswitch system enables precise, conditional control of gene expression.
• These synthetic RNA switches represent a valuable tool for gene therapy and synthetic biology.