Fluorogenic Aptamer Optimizations on a Massively Parallel Sequencing Platform

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Fluorogenic Aptamer Optimizations on a Massively Parallel Sequencing Platform


Kuo, Y.-A.; Chen, Y.-I.; Wang, Y.; Korkmaz, Z.; Yonas, S.; He, Y.; Nguyen, T. D.; Hong, S.; Nguyen, A.-T.; Kim, S.; Seifi, S.; Fan, P.-H.; Wu, Y.; Yang, Z.; Liu, H.-W.; Lu, Y.; Ren, P.; Yeh, H.-C.


Fluorogenic aptamers (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 (Kdapp: 0.63 vs. 0.82 uM), an increased quantum yield (QY: 0.62 vs. 0.59) and an elongated fluorescence lifetime (tau: 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 pi-pi 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.

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