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Structure of short Lettuce aptamer (A5T variant) bound with TO1-biotin.
Structural highlights
Publication Abstract from PubMedFluorogenic aptamers (FAPs) are increasingly important tools for cellular sensing and pathogen diagnostics. However, enhancing their performance remains a significant challenge. Here, we introduce a massively parallel approach to optimize the DNA-based FAP Lettuce using repurposed next-generation sequencing flow cells. By replacing Lettuce's cognate fluorogen, DFHBI-1T, with TO1-biotin, we achieve a 4-fold ensemble fluorescence enhancement, and a broader fluorescence lifetime modulation range from 4.7 to 6.0 ns. Through screening 8821 Lettuce variants complexed with TO1-biotin on a MiSeq chip, we identify the C14T mutant which exhibits an improved dissociation constant, increased quantum yield, extended fluorescence lifetime, and enhanced emission intensity. Co-crystal structures of the aptamer/fluorogen complexes reveal that pi-pi stacking interactions are critical for the stable coordination of TO1-biotin within Lettuce. When tested in a cellular environment, both the screening-identified C14T mutant and a structure-guided rationally designed variant, C14dU, exhibit 15% and 18% stronger fluorescence intensities, respectively, compared with canonical Lettuce. Our massive screening and molecular dynamics simulation pipeline enable efficient FAP optimization without prior structural knowledge, yielding not only improved probes for fluorescence sensing but also deeper insights into aptamer-fluorogen interactions. Fluorogenic Aptamer Optimization on a Massively Parallel Sequencing Platform.,Kuo YA, Chen YI, Siraj N, He Y, Yang Z, Wang Y, Batchelder-Schwab EJ, Korkmaz Z, Yonas S, Nguyen TD, Hong S, Nguyen AT, Kim S, Seifi S, Fan PH, Wu Y, Liu HW, Lu Y, Ren P, Mao C, Yeh HC ACS Sens. 2026 May 22;11(5):3587-3600. doi: 10.1021/acssensors.5c04046. Epub 2026 , May 11. PMID:42112875[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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