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Screening of Clenbuterol Hydrochloride Aptamers Based on Capillary Electrophoresis

Author:
Ge YANG  Chao ZHU  Xiao-Hui LIU  Yong WANG  Feng QU  


Journal:
Chinese Journal of Analytical Chemistry


Issue Date:
2018


Abstract(summary):

Abstract Capillary electrophoresis based systematic evolution of ligands via exponential enrichment (CE-SELEX) was reported as a homogeneous efficient method for high-affinity selection of aptamer, with several merits involving screening in free solution without nonspecific binding, capable of high-efficient separation, low-sample consumption, and saving money. There are few studies regarding the aptamer selection against small molecule using CE-SELEX, resulting from the aspects of less binding sites and the negligible variety of its complex with nucleic acid in the electrophoretic mobility. In this study, we performed the aptamer selection towards a small molecule target of clenbuterol hydrochloride (Clen) by CE-SELEX. In brief, Clen were first incubated with an 80 nt ssDNA library, and CZE-UV approach was used to separate complex and random ssDNA. The complex was then collected into a vial followed by PCR amplification. Through three round selections, the third library was selected to clone and ten sequences were finally obtained. The dissociation constant ( K d ) of three potential candidates (Apt 4, Apt 7 and Apt 12) were determined by CE-LIF, and showed high affinities of 9.315 × 10 −7 M, 1.040 × 10 −6 M and 1.143 × 10 −5 M, respectively. The result of m-Fold software analysis showed that the above three sequences could form stem-loop structure, and the Apt 4 gave the lowest free energy and the most stable structure. Using salbutamol as a control, three selected aptamers were verified with high specificity. Graphical abstract Clen were first incubated with an 80 nt ssDNA library, and CZE-UV approach was used to separate complex and random ssDNA. The complex was then collected into a vial followed by PCR amplification. Through three round selections, the third library was selected to clone and ten sequences were finally obtained. Aptamer affinity and specificity were analyzed and compared by AuNPs colorimetry, GO-capillary electrophoresis. Using salbutamol as a control, three selected aptamers were verified with high affinity and specificity.


Page:
1595-1595


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