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Updated: May 12, 2026
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Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis
Published on: July 8, 2025
Fluorogenic Aptamer Optimization on a Massively Parallel Sequencing Platform
Yu-An Kuo1, Yuan-I Chen1, Naseem Siraj2
1Department of Biomedical Engineering, University of Texas at Austin, Austin, Texas 78712, USA.
ACS Sensors
|May 11, 2026
Summary
We developed a high-throughput method to optimize fluorogenic aptamers (FAPs) for cellular sensing. This approach significantly enhances FAP performance, leading to improved diagnostic tools.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Fluorogenic aptamers (FAPs) are crucial for cellular sensing and pathogen diagnostics.
- Enhancing FAP performance is essential for advancing these applications.
- Current optimization methods face significant challenges.
Purpose of the Study:
- To develop a massively parallel screening approach for optimizing DNA-based FAPs.
- To improve the fluorescence properties and cellular sensing capabilities of FAPs.
- To gain deeper insights into aptamer-fluorogen interactions.
Main Methods:
- Utilized repurposed next-generation sequencing flow cells for high-throughput screening.
- Screened 8821 variants of the DNA-based FAP Lettuce with a novel fluorogen, TO1-biotin.
- Employed co-crystal structure analysis and molecular dynamics simulations.
Main Results:
- Achieved a 4-fold ensemble fluorescence enhancement and broader fluorescence lifetime modulation.
- Identified the C14T mutant with improved dissociation constant, quantum yield, and emission intensity.
- Demonstrated enhanced fluorescence intensity in cellular environments for optimized FAPs.
Conclusions:
- Massively parallel screening enables efficient FAP optimization, even without prior structural knowledge.
- Optimized FAPs show superior performance for fluorescence sensing applications.
- The study provides valuable insights into aptamer-fluorogen complex stability and interactions.
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