Interaction between hammerhead ribozyme and RNA substrates measured by a surface plasmon resonance biosensor

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Hammerhead ribozymes are RNA molecules with catalytic activity.
• Understanding ribozyme-substrate interactions is crucial for RNA biology and therapeutics.
• Surface Plasmon Resonance (SPR) is a label-free technology for studying molecular interactions.

Purpose of the Study:

• To investigate the dynamic interactions between hammerhead ribozymes and immobilized RNA substrates using SPR.
• To differentiate the binding affinities and catalytic activities of two hammerhead ribozymes with varying complementarity to their substrates.
• To assess the utility of SPR for studying ribozyme-substrate interactions.

Main Methods:

• Immobilization of in vitro transcribed RNA substrates (non-cleavable and cleavable) onto streptavidin-coated dextran matrices.
• Binding assays using two hammerhead ribozymes with different target complementarity (16-nucleotide vs. 9-nucleotide).
• Monitoring binding kinetics and cleavage activity in real-time using SPR technology.

Main Results:

• SPR successfully measured dynamic interactions between hammerhead ribozymes and RNA substrates.
• The two ribozymes exhibited distinct binding affinities, differentiating them based on complementarity.
• Cleavage activity was observed for the ribozyme with full target complementarity on the cleavable substrate, while the lower-affinity ribozyme showed no cleavage.

Conclusions:

• SPR is a valuable tool for quantifying ribozyme-substrate binding affinities and kinetics.
• The study demonstrates the feasibility of using SPR to investigate RNA-based catalysis.
• SPR can differentiate ribozymes based on their interaction strength and functional activity, aiding in the study of RNA enzymes.