Year: 2026
Type: article
Abstract: F luorogenic ap tamers (FAPs) have become an increasingly important tool in cellular sensing and pathogen diagnostics. However, fine-tuning FAPs for enhanced performance remains challenging even with the structural details provided by X-ray crystallography. Here we present a novel approach to optimize a DNA-based FAP (D-FAP), Lettuce, on repurposed Illumina next-generation sequencing (NGS) chips. When substituting its cognate chromophore, DFHBI-1T, with TO1-biotin, Lettuce not only shows a red-shifted emission peak by 53 nm (from 505 to 558 nm), but also a 4-fold bulk fluorescence enhancement. After screening 8,821 Lettuce variants complexed with TO1-biotin, the C14T mutation is found to exhibit an improved apparent dissociated constant ( vs. 0.82 µM), an increased quantum yield (QY: 0.62 vs. 0.59) and an elongated fluorescence lifetime (τ: 6.00 vs. 5.77 ns), giving 45% more ensemble fluorescence than the canonical Lettuce/TO1-biotin complex. Molecular dynamic simulations further indicate that the π-π stacking interaction is key to determining the coordination structure of TO1-biotin in Lettuce. Our screening-and-simulation pipeline can effectively optimize FAPs without any prior structural knowledge of the canonical FAP/chromophore complexes, providing not only improved molecular probes for fluorescence sensing but also insights into aptamer-chromophore interactions.
Source: ACS Sensors
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: National Science Foundation, Welch Foundation, Cancer Prevention and Research Institute of Texas, Purdue University, University of Texas at Austin, National Institutes of Health, National Institute on Drug Abuse, National Institute of General Medical Sciences, Division of Computing and Communication Foundations, Division of Chemical, Bioengineering, Environmental, and Transport Systems, Division of Chemistry
Awards: G1475642905, G148752694, Novel tools for point-of-care therapeutic drug monitoring, G1833730492, Studying synaptic transmission under opioid exposure using hiPSC-derived midbrain model and hyperspectral fluorescence lifetime imaging, G2579277240, G262977785, G3049165437, Collaborative Research: Developing a Polarizable 3DRISM Implicit Solvent Model for AMOEBA Solutes to Enable Efficient and Accurate RNA Simulations, Chemical Measurements and Imaging Using Novel Functional Nucleic Acids, Collaborative Research: FTE: Medium: Three Dimensional Algorithmic Assembly and Information Storage, G5748168713, Targeted Therapeutic Drug Discovery & Development Program, DREAM Sentinels: Selection of aptamers that target viral variants with high specificity, G8123439633, New X-ray Diffractometer and Detector for Purdue Macromolecular Crystallography, G9000030800, G9000217152, G9000230098, G921166742
OpenAlex ID: W7160878779