Multivalent DNAzyme agents for cleaving folded RNA

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Multivalent binding naturally enhances molecular interaction stability (avidity) without compromising specificity.
• DNAzymes (Dz) are catalytic DNA molecules with potential therapeutic applications, such as gene knockdown.
• Optimizing DNAzyme efficiency and specificity is crucial for developing effective gene-targeting agents.

Purpose of the Study:

• To enhance the efficiency and specificity of RNA cleavage using DNAzyme technology.
• To design and evaluate bivalent DNAzyme devices (BDD) that leverage multivalent binding principles.
• To explore the impact of cooperative catalytic action on RNA cleavage efficacy and selectivity.

Main Methods:

• Design and synthesis of bivalent DNAzyme devices (BDD) with two catalytic Dz agents.
• In vitro assessment of RNA cleavage efficiency and kinetics for BDD compared to conventional DNAzymes.
• Evaluation of BDD selectivity using single-base mismatched RNA targets.
• Investigation of trivalent DNAzyme constructs for comparison.

Main Results:

• One BDD design achieved up to a 17-fold increase in cleavage efficiency for a folded RNA fragment compared to conventional DNAzymes.
• The enhanced efficiency resulted from improved RNA binding and increased probability of cleavage by dual catalytic cores.
• BDD demonstrated excellent selectivity in distinguishing single-base mismatched RNA while maintaining high cleavage rates.
• A trivalent DNAzyme construct exhibited even greater cleavage efficiency than the BDD.

Conclusions:

• Cooperative action of multiple RNA-cleaving units in DNAzymes significantly boosts efficiency and maintains high specificity.
• Bivalent and trivalent DNAzyme designs represent a promising strategy for developing advanced Dz-based gene knockdown agents.
• The ability to moderate Dz agent association in BDD allows for fine-tuned selectivity in RNA targeting.