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Structural Hairpin Anchoring-Mediated TtAgo Activity Regulation for Programmable Biosensing.

Lixuan Guan¹, Haoran Shen¹, Kangling Tang^1,2

¹Key Laboratory for Biobased Materials and Energy of Ministry of Education, College of Materials and Chemical Engineering, South China Agricultural University, Guangzhou 510642, China.

Analytical Chemistry

|July 8, 2026

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The structural hairpin anchor regulates endonuclease cleavage activity (SHARE) effect optimizes Thermus thermophilus Argonaute (TtAgo) activity. This discovery enables precise TtAgo biosensor engineering for molecular diagnostics.

Area of Science:

Biochemistry
Molecular Biology
Biotechnology

Background:

Thermus thermophilus Argonaute (TtAgo) is a DNA-guided enzyme with potential in biosensing and diagnostics.
TtAgo's activity is sensitive to guide DNA (gDNA) structure, with optimal function within a specific linear gDNA length range.
Current strategies for regulating TtAgo activity via gDNA modification are limited.

Purpose of the Study:

To investigate the impact of gDNA structural modifications on TtAgo activity.
To introduce a novel regulatory mechanism for TtAgo based on gDNA conformation.
To engineer a TtAgo biosensor utilizing this regulatory mechanism for biomarker detection.

Main Methods:

Rational design of gDNA structures, specifically introducing hairpin modifications at the 3' terminus.

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Characterization of TtAgo cleavage activity with varying gDNA structures.

Engineering a TtAgo-based biosensor for apurinic/apyrimidinic endonuclease 1 detection.

Main Results:

TtAgo exhibits optimal activity with linear gDNA of 15-21 nt; longer gDNA or gDNA with extended hairpin structures can inhibit activity.
A hairpin structure at the gDNA 3' terminus acts as a physical anchor, regulating TtAgo binding and cleavage.
This regulatory mechanism, termed the structural hairpin anchor regulates endonuclease cleavage activity (SHARE) effect, was successfully applied in a TtAgo biosensor.
The engineered biosensor demonstrated sensitive and specific detection of apurinic/apyrimidinic endonuclease 1.

Conclusions:

The SHARE effect provides a novel method for precisely controlling TtAgo activity through gDNA conformational engineering.
This mechanism broadens the strategies for TtAgo-based biosensing and molecular diagnostics.
The developed TtAgo biosensor shows promise for sensitive and specific biomarker detection.