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

Liu, Yanming, Wang, Chuan, Li, Feng, Shen, Shengwen, Tyrrell, D. Lorne J., Le, X. Chris, Li, Xing-Fang
Analytical chemistry 2012 v.84 no.18 pp. 7603-7606
Hepatitis B virus, blood proteins, buffers, deoxyribonuclease I, hepatitis B antigens, humans, oligonucleotides, polyacrylamide gel electrophoresis, single-stranded DNA, thermoplastics, urea
We describe a single-cycle DNA aptamer selection strategy that is able to obtain high affinity aptamers (Kd 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 (Kd ∼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.