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  • DNase-mediated single-cycle selection of aptamers for proteins blotted on a membrane.

DNase-mediated single-cycle selection of aptamers for proteins blotted on a membrane.

Analytical chemistry (2012-08-31)
Yanming Liu, Chuan Wang, Feng Li, Shengwen Shen, D Lorne J Tyrrell, X Chris Le, Xing-Fang Li
ABSTRACT

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 10(15) 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Poly(vinylidene fluoride), average Mw ~534,000 by GPC, powder
Sigma-Aldrich
Poly(vinylidene fluoride), average Mw ~275,000 by GPC, average Mn ~107,000, pellets
Sigma-Aldrich
Poly(vinylidene fluoride), average Mw ~530,000, pellets
Sigma-Aldrich
Poly(vinylidene fluoride), average Mw ~180,000 by GPC, average Mn ~71,000, beads or pellets