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

• Published in: Analytical chemistry (Washington) 2012-09, Vol.84 (18), p.7603-7606
• Main Authors: Liu, Yanming, Wang, Chuan, Li, Feng, Shen, Shengwen, Tyrrell, D. Lorne J, Le, X. Chris, Li, Xing-Fang
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Aptamers, Nucleotide - chemistry; Aptamers, Nucleotide - metabolism; Blood Proteins - chemistry; Blood Proteins - metabolism; Chemistry; Deoxyribonucleases - metabolism; Deoxyribonucleic acid; DNA; DNA, Single-Stranded - metabolism; Electrophoresis, Polyacrylamide Gel; Exact sciences and technology; Hepatitis; Hepatitis B Core Antigens - isolation & purification; Hepatitis B Core Antigens - metabolism; Membranes; Molecules; Polyvinyls - chemistry; Proteins; Real-Time Polymerase Chain Reaction
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac302047e

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.