Argonaute with stepwise endonuclease activity promotes specific and multiplex nucleic acid detection

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Argonaute proteins (Agos) are crucial for host defense, acting as endonucleases guided by nucleic acids.
• Understanding the guide-dependent catalytic specificity of Agos is key to their application in molecular diagnostics.
• Thermophilic Agos offer unique enzymatic properties for biotechnological advancements.

Purpose of the Study:

• To elucidate the molecular mechanism of endonuclease activity in Pyrococcus furiosus Argonaute (PfAgo).
• To develop a novel platform for sensitive and specific gene detection utilizing PfAgo's unique properties.
• To demonstrate the application of this platform in multiplex gene detection and pathogen identification.

Main Methods:

• Investigated the endonuclease activity of PfAgo using single guide DNA (gDNA) for double-stranded DNA cleavage.
• Characterized the stepwise cleavage mechanism where products can act as new guides.
• Developed and validated the Renewed-gDNA Assisted DNA cleavage by Argonaute (RADAR) platform for gene detection.

Main Results:

• PfAgo exhibits stepwise endonuclease activity, cleaving DNA with a single gDNA.
• Cleavage products with 5'-phosphorylated ends can re-initiate cleavage, enabling a cascade effect.
• The RADAR platform achieved femtomolar sensitivity and di-nucleotide resolution for gene detection.
• RADAR successfully discriminated between four human papillomavirus serotypes in patient samples.

Conclusions:

• The stepwise endonuclease activity of PfAgo is a unique characteristic that can be harnessed for biotechnological applications.
• The RADAR platform offers a rapid, sensitive, and specific method for unambiguous multiplex gene detection.
• This approach provides a versatile tool for molecular diagnostics, including pathogen identification from clinical samples.