Atom-Modified gDNA Enhances Cleavage Activity of TtAgo Enabling Ultra-Sensitive Nucleic Acid Testing

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Thermus thermophilus Argonaute (TtAgo) exhibits limited double-stranded DNA (dsDNA) cleavage at lower temperatures, hindering its application in nucleic acid detection and gene editing.
• Enhancing TtAgo's dsDNA cleavage activity is crucial for developing practical and sensitive molecular diagnostic tools.

Purpose of the Study:

• To enhance the dsDNA cleavage activity of TtAgo using modified guide DNA (gDNA).
• To develop a novel, highly sensitive nucleic acid testing strategy based on the enhanced TtAgo activity.

Main Methods:

• Utilized 2 ac-fluorine (2 ac'F)-modified gDNA to promote TtAgo-guide-target ternary complex assembly.
• Developed a one-pot real-time fluorescence assay (FAST) integrating 2 ac'F-gDNA/TtAgo with Bst DNA polymerase, G-quadruplex, and Thioflavin T.

Main Results:

• Achieved approximately 40-fold enhancement in TtAgo activity with 2 ac'F-gDNA.
• Reduced the minimum reaction temperature from 65 ac °C to 60 ac °C.
• Demonstrated ultra-sensitive detection of miR-21 with a limit of detection as low as 5 copies using the FAST assay.

Conclusions:

• Atom modification of gDNA is an effective strategy to enhance TtAgo cleavage activity and expand its utility.
• The developed FAST assay offers a highly sensitive and specific TtAgo-based platform for nucleic acid testing.