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Updated: Jun 7, 2025
Primer-Free Aptamer Selection Using A Random DNA Library
Published on: July 26, 2010
Real-time visualization of "small Molecules-ssDNA" complexes for aptamer screening based on online competition
Ge Yang1, Wenzhe Tian2, Yangyang Hu2
1Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, 5 South Zhongguancun Street, Beijing, 100081, China; CAMS Key Laboratory of Antiviral Drug Research, Beijing Key Laboratory of Antimicrobial Agents, NHC Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China.
Abstract:
Capillary electrophoresis-based systematic evolution of ligands by exponential enrichment (CE-SELEX) is one of the most efficient techniques for aptamers screening. However, CE-SELEX is generally considered challenging for small molecule targets due to a slight charge-to-mass ratio (z/m) difference that fails to create a clear separation between small molecule-ssDNA complexes and ssDNA libraries, thus heavily restricting the screening target scopes. Herein, a novel online competition CE-SELEX (ocCE-SELEX) strategy was introduced for real-time visualization of "small molecules-ssDNA" complexes to screen aptamers for small molecule targets in free solution. To achieve this vision, the "pair-wise" interaction-based "Catcher-ssDNA-Target" ternary system was flexibly designed via online CE mode. First, we introduced a conceptual protein of an ssDNA catcher (Catcher) that is capable of capturing all sequences in ssDNA library completely through an online controllable procedure by adjusting the injection time of different zones, resulting in the disappearance of original peaks of the ssDNA library in electropherogram. Then, the "pair-wise" interaction would restore the peaks through an online competition process, in which the Catcher with a faster migration rate would traverse the equilibrium zone of small molecule/ssDNA mixture and capture free ssDNA, as well as compete with small molecules to capture weakly bound ssDNA sequences. Due to the different z/m changes, the ssDNA captured by Catcher continues to migrate rapidly and the retained ssDNA in small molecule-ssDNA complex approximately maintains its original migration rate. Consequently, the peaks of Catcher-captured ssDNA and small molecule-ssDNA complex achieved complete separation and real-time visualization, allowing the complex to be collected readily. Furthermore, the implementation of ocCE-SELEX strategy was verified with two small molecules (vitamin B12 and ofloxacin) and six polypeptides. The study expands the application of CE-SELEX in small molecules in terms of real-time visualization and accurate collection of small molecules-ssDNA complexes, opening a new path for aptamer screening for small molecule targets in their natural state.
