title: DNase-mediated single-cycle selection of aptamers for proteins blotted on a membrane.
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.
authors.affiliation: Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Canada.
authors.forename: Yanming
authors.initials: Y
authors.lastname: Liu
authors.forename: Chuan
authors.initials: C
authors.lastname: Wang
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.forename: Shengwen
authors.initials: S
authors.lastname: Shen
authors.forename: D Lorne J
authors.initials: DL
authors.lastname: Tyrrell
authors.forename: X Chris
authors.initials: XC
authors.lastname: Le
authors.forename: Xing-Fang
authors.initials: XF
authors.lastname: Li
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006512:Hepatitis B Core Antigens / Q000302:isolation & purification / Q000378:metabolism*; D011145:Polyvinyls / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction; D005465:Fluorocarbon Polymers
chemical_list: D052157:Aptamers, Nucleotide; D001798:Blood Proteins; D004277:DNA, Single-Stranded; D003851:Deoxyribonucleases; D006512:Hepatitis B Core Antigens; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers