Nicked Invader probes: multistranded and sequence-unrestricted recognition of double-stranded DNA

Area of Science:

• Molecular Biology
• Biophysics
• Genetics

Background:

• Developing sequence-specific double-stranded DNA (dsDNA) recognition tools is crucial for molecular biology, diagnostics, and medicine.
• Previous Invader probes and toehold Invader probes offered limited recognition of long dsDNA targets due to probe stability.

Purpose of the Study:

• To investigate the biophysical properties and dsDNA-targeting capabilities of a novel nicked Invader probe architecture.
• To enhance dsDNA recognition affinity and enable targeting of previously inaccessible DNA sequences.

Main Methods:

• Design and synthesis of nicked Invader probes featuring intercalator-modified auxiliary strands.
• Assessment of probe biophysical properties, including dsDNA binding affinity.
• Application of nicked Invader probes in non-denaturing Fluorescence In Situ Hybridization (FISH) experiments.

Main Results:

• Nicked Invader probes exhibit increased dsDNA affinity compared to toehold or blunt-ended Invader probes.
• The novel probes successfully recognized chromosomal DNA targets that were refractory to conventional Invader probes.
• Demonstrated utility in non-denaturing FISH, highlighting effective dsDNA targeting.

Conclusions:

• Nicked Invader probes represent a significant advancement in dsDNA recognition technology.
• This enhanced probe design broadens the scope of applications for Invader probes in molecular diagnostics and research.
• The study validates nicked Invader probes as effective tools for targeting challenging dsDNA sequences.