Controlling DNA-RNA strand displacement kinetics with base distribution

Area of Science:

• Molecular Biology
• Biophysics
• Biochemistry

Background:

• DNA-RNA hybrid strand displacement is fundamental to biological processes and synthetic systems.
• Controlling these reactions is crucial for applications such as CRISPR-Cas9 gene editing.

Purpose of the Study:

• To investigate the impact of base distribution on DNA-RNA strand displacement kinetics.
• To compare sequence dependence in DNA-RNA hybrids versus all-DNA systems.
• To validate computational models for predicting these reactions.

Main Methods:

• Multiscale modeling combined with experimental strand displacement assays.
• Characterization of reaction kinetics for RNA invasion of dsDNA and DNA invasion of hybrid duplexes.
• Utilizing the oxNA coarse-grained model and developing a simple kinetic model.

Main Results:

• Base distribution within the displacement domain strongly influences DNA-RNA reaction kinetics, unique to these hybrids.
• Sequence-dependent reaction rates spanning over four orders of magnitude were achieved by redistributing bases.
• All-DNA strand displacement showed predictable but weaker sequence dependence compared to DNA-RNA hybrids.
• The oxNA model accurately reproduced experimental trends; a predictive kinetic model was developed.

Conclusions:

• DNA-RNA strand displacement offers greater thermodynamic and kinetic control than all-DNA systems due to base distribution effects.
• Base distribution may be a critical factor in natural R-loop formation and CRISPR guide RNA function.