DNase-Mediated Single-Cycle Selection of Aptamers for Proteins Blotted on a Membrane

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• 作者：Yanming Liu ; Chuan Wang ; Feng Li ; Shengwen Shen ; D. Lorne J. Tyrrell ; X. Chris Le ; Xing-Fang Li
• 刊名：Analytical Chemistry
• 出版年：2012
• 出版时间：September 18, 2012
• 年：2012
• 卷：84
• 期：18
• 页码：7603-7606
• 全文大小：246K
• 年卷期：v.84,no.18(September 18, 2012)
• ISSN：1520-6882

文摘

We describe a single-cycle DNA aptamer selection strategy that is able to obtain high affinity aptamers (K_d of sub-nM) directly from a protein blotted on membrane. The key to the success of this strategy is the unique use of DNase I digestion to remove unwanted ssDNA from the membrane, leaving only the strongest bound aptamers. A crude Hepatitis B virus core protein (HBcAg) was separated using polyacrylamide gel electrophoresis (PAGE) and electro-blotted onto a polyvinylidene fluoride (PVDF) membrane. The membrane strip containing HBcAg and a second membrane strip containing human serum proteins were coincubated with a ssDNA library consisting of 10 copies each of 10¹⁵ random sequences. Unbound and weakly bound sequences were efficiently removed from the membrane containing HBcAg using DNase I digestion and gradient wash with urea buffers. The remaining ssDNA bound to the target consisted of approximately 500 molecules, from which two aptamers with high affinity (K_d 100 and 200 pM) were identified. This technique can be potentially used for selection of aptamers directly from multiple proteins that are separated by gel electrophoresis from a biological mixture.