Cleaving Folded RNA with DNAzyme Agents.

Area of Science:

• Molecular Biology
• Biochemistry
• Oligonucleotide Therapeutics

Background:

• DNAzymes (Dz) are catalytic DNA molecules with potential in oligonucleotide gene therapy (OGT) for cleaving specific mRNA targets.
• Traditional Dz design for OGT emphasizes low-affinity RNA-binding arms (Tm near 37°C) to prevent product inhibition and ensure specificity.
• However, RNA secondary structures can impede cleavage, necessitating high-affinity binding for efficient RNA targeting.

Purpose of the Study:

• To optimize 10-23 DNAzyme (Dz) agents for mRNA cleavage under multiple turnover conditions.
• To investigate the impact of RNA-binding arm affinity on Dz performance against RNA targets with varying folding energies.
• To re-evaluate the design principles for DNAzyme-based oligonucleotide gene therapy agents.

Main Methods:

• Optimization of 10-23 DNAzyme sequences targeting three distinct RNA substrates with differing folding energies.
• Assay conditions included 2 mM Mg2+ at 37°C under multiple turnover conditions.
• Analysis of DNAzyme-RNA binding affinity using melting temperature (Tm) measurements and assessment of product inhibition and selectivity.

Main Results:

• Unexpectedly, an optimized DNAzyme with high-affinity RNA-binding arms (Tm ≥60°C) demonstrated efficient cleavage without product inhibition or loss of selectivity.
• The observed high performance was attributed to the stable secondary structures formed by the cleaved RNA products, which prevented re-binding.
• This finding contradicts the conventional design strategy favoring low-affinity binding arms.

Conclusions:

• DNAzymes with high-affinity RNA-binding arms are viable candidates for oligonucleotide gene therapy (OGT).
• The folding propensity of RNA cleavage products significantly influences DNAzyme efficacy and should be considered in agent design.
• Future DNAzyme selection algorithms and optimization workflows must incorporate testing with folded RNA substrates and analyze product folding characteristics.