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Multivalent DNAzyme agents for cleaving folded RNA.

Mikhail V Dubovichenko1, Michael Batsa1, Gleb A Bobkov1

  • 1Laboratory of Frontier Nucleic Acid Technologies in Gene Therapy of Cancer, SCAMT Institute, ITMO University, Saint-Petersburg, 191002, Russia.

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Researchers developed bivalent DNAzyme devices (BDD) for enhanced RNA cleavage. These bivalent DNAzymes (Dz) significantly increased cleavage efficiency and specificity, showing promise for gene knockdown applications.

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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.