Affinity-Guided Coevolution of Aptamers for Guanine, Xanthine, Hypoxanthine, and Adenine

• DOI: 10.1021/acschembio.3c00660
• Publication Date: 2024-01-09T17:37:12Z
• Authors: Yuzhe Ding; Lide Gu; Xiaoqin Wang; Ziyu Zhang; Hanxiao Zhang; Juewen Liu
• Keywords: Aptamer; Hypoxanthine; Xanthine; Riboswitch; Isothermal Titration Calorimetry

The simultaneous evolution of multiple aptamers can drastically increase the speed aptamer discovery. Most previous studies used same concentration for different targets, leading to dominance libraries by one or a few and low success rate. To foster best grow independently in sequence space, it is important (1) use target concentrations close their dissociation constants (2) stop at an early round before any starts dominate. In this study, we demonstrate affinity-guided selection concept using capture-SELEX method isolate four purines: guanine (5 μM), xanthine (50 hypoxanthine (10 adenine μM). 9 library was split, 10, targets were individually elute binding sequences. Using thioflavin T fluorescence spectroscopy isothermal titration calorimetry, confirmed highly selective xanthine, guanine, adenine. These have Kd values below 1 μM around 100-fold selectivity against most competing analytes, they compare favorably with existing RNA riboswitches. A separate performed alone, no achieved, even negative selection, explaining lack its our mixed selection. This multiplex SELEX study offers fundamental insights into provides high-quality three purines.