Fluorogenic Aptamer Optimizations on a Massively Parallel Sequencing Platform.

Area of Science:

• Biotechnology
• Molecular Biology
• Biophysics

Background:

• Fluorogenic aptamers (FAPs) are crucial for cellular sensing and pathogen diagnostics.
• Optimizing FAP performance is challenging, even with structural data from X-ray crystallography.

Purpose of the Study:

• To develop a novel approach for optimizing DNA-based FAPs (D-FAPs) using next-generation sequencing (NGS) chips.
• To enhance the performance of the D-FAP, Lettuce, by screening variants and optimizing its interaction with a new chromophore.

Main Methods:

• Repurposed Illumina NGS chips for high-throughput screening of 8,821 Lettuce variants with the TO1-biotin chromophore.
• Utilized molecular dynamic simulations to understand aptamer-chromophore interactions.
• Characterized FAP variants by measuring fluorescence emission, quantum yield, fluorescence lifetime, and apparent dissociation constant.

Main Results:

• Substitution of the cognate chromophore with TO1-biotin resulted in a 53 nm red-shifted emission and a 4-fold fluorescence enhancement.
• The C14T mutation in Lettuce showed improved binding affinity (lower Kd), increased quantum yield (0.62), and longer fluorescence lifetime (6.00 ns).
• The optimized C14T variant exhibited 45% greater ensemble fluorescence compared to the original Lettuce/TO1-biotin complex.

Conclusions:

• A screening-and-simulation pipeline effectively optimizes FAPs without prior structural knowledge.
• The optimized Lettuce D-FAP with TO1-biotin serves as an improved molecular probe for fluorescence sensing.
• The study provides insights into the critical role of π-π stacking in aptamer-chromophore complex structures.