A modified X10-23 DNAzyme that can better access large, structured RNA targets

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Therapeutics

Background:

• The 10-23 DNA enzyme is highly efficient at cleaving RNA but struggles with large, structured RNA (lsRNA) targets.
• Modifications like Xeno nucleic acids (XNAs) and antisense DNA oligonucleotides (ASOs) have been explored to improve accessibility but present limitations.
• Current strategies face challenges in balancing accessibility, cleavage efficiency, and product release for lsRNA.

Purpose of the Study:

• To develop an improved 10-23 DNA enzyme variant with enhanced accessibility and activity against lsRNA targets.
• To investigate the efficacy of incorporating specific XNA patterns into the substrate recognition arms of the 10-23 enzyme.
• To evaluate the performance of the novel enzyme variant against SARS-CoV-2 lsRNA targets.

Main Methods:

• Designed and synthesized a novel 10-23 enzyme variant (XdZ-2) featuring a specific 2'F-RNA-LNA-FANA arm pattern.
• Tested the cleavage activity of XdZ-2 against various lsRNA targets from SARS-CoV-2.
• Compared the performance of XdZ-2 with the unmodified X10-23 enzyme and ASO strategies.

Main Results:

• XdZ-2 demonstrated significantly enhanced cleavage rates, up to 82-fold faster than X10-23 for a specific SARS-CoV-2 lsRNA target.
• The XdZ-2 enzyme showed improved accessibility to lsRNA targets compared to the X10-23 variant.
• While ASO strategies showed higher rates in some cases, XdZ-2 offered advantages in low Mg2+ conditions and product release.

Conclusions:

• The XdZ-2 enzyme represents a promising advancement in RNA-cleaving technology for targeting lsRNA.
• XdZ-2 offers a potential alternative to existing methods, particularly in challenging biological environments.
• This modified enzyme holds potential for applications in RNA therapeutics and diagnostics, especially against viral targets like SARS-CoV-2.