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Light-regulated catalysis by an RNA-cleaving deoxyribozyme.

Yong Liu1, Dipankar Sen

  • 1Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada.

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Researchers developed light-controlled DNA enzymes (DNAzymes) using azobenzene. Visible light enhanced activity in some designs, while UV light boosted others, enabling precise control over gene expression.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • The 8-17 deoxyribozyme (DNAzyme) is a small, RNA-cleaving enzyme.
  • Azobenzene (Az) moieties can be incorporated into DNA, exhibiting distinct structural effects based on their cis-trans isomerization state.
  • Trans-azobenzene stabilizes DNA helices, while cis-azobenzene destabilizes them.

Purpose of the Study:

  • To engineer light-switchable DNAzymes by incorporating azobenzene moieties.
  • To investigate how azobenzene placement affects light-induced control of DNAzyme catalytic activity.
  • To explore the potential of light-responsive DNAzymes for gene expression regulation.

Main Methods:

  • Covalent tethering of azobenzene (Az) moieties into the 8-17 DNAzyme structure at specific locations.
  • Design of two classes of Az-modified DNAzyme constructs: one with Az in the substrate-binding arm (SBA) and another in the catalytic core.
  • Measurement of single-turnover kinetics for RNA cleavage under different light conditions (visible vs. ultraviolet).

Main Results:

  • Az-modified DNAzymes exhibited light-dependent catalytic activity.
  • SBA constructs (E11, E13) showed 5-6 fold higher activity under visible light (trans-Az), suggesting stabilization of the enzyme-substrate complex.
  • Catalytic core construct (E17) surprisingly showed 5-6 fold higher activity under UV light (cis-Az).

Conclusions:

  • Az-modified DNAzymes can be switched on/off using light, with activity dependent on azobenzene location.
  • The differential response of SBA and catalytic core constructs highlights the nuanced role of Az isomerization in DNAzyme function.
  • Light-responsive DNAzymes offer a novel platform for spatiotemporal control of gene expression in biological systems.