In vitro selection of allosteric ribozymes: theory and experimental validation.

Area of Science:

• Molecular Biology
• Biochemistry
• Synthetic Biology

Background:

• In vitro selection is a powerful technique for isolating functional nucleic acid sequences.
• Allosteric ribozymes offer precise molecular control through ligand-induced activity modulation.
• Quinolone antibiotics are widely used and understanding their interactions with biological molecules is important.

Purpose of the Study:

• To develop a de novo in vitro selection strategy for allosteric self-cleaving ribozymes.
• To isolate ribozymes that specifically respond to pefloxacin and other quinolone derivatives.
• To establish a theoretical model for predicting selection outcomes.

Main Methods:

• Employing an in vitro selection strategy over 16 cycles to isolate specific ribozyme sequences.
• Testing the sensitivity of selected ribozymes to various concentrations of quinolone derivatives.
• Analyzing the structure-activity relationship by modifying quinolone compounds.
• Developing and validating a theoretical model for in vitro selection prediction.

Main Results:

• Isolation of highly sensitive allosteric ribozymes responding to sub-micromolar concentrations of quinolones after 16 selection cycles.
• Identification of the morpholine moiety of quinolones as critical for self-cleavage inhibition.
• Demonstration that modifications to the aromatic system of quinolones are better tolerated than changes to the morpholine ring.
• A theoretical model accurately predicted experimental results for ribozyme selection.

Conclusions:

• The developed in vitro selection strategy is effective for de novo selection of allosteric ribozymes.
• The selected ribozymes show high sensitivity to quinolone antibiotics, with specific structural requirements for activity.
• The theoretical model can optimize conditions for selecting allosteric ribozymes with desired properties for specific applications.