SELEX screen for zona pellucida-binding DNA aptamers.
SELEX screen for zona pellucida-binding DNA aptamers.
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SELEX 筛选透明带结合 DNA 适体。
DOI：
10.1093/biolre/ioy123
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发表时间：
2018
影响因子：
3.6
通讯作者：
Evans,JaniceP
中科院分区：
文献类型：
--
作者：
Miller,PaulS;Evans,JaniceP
Dear Editor, We report here on a potential method to develop a novel agent for oocyte-specific delivery of agents such as siRNAs. This work focused on identification of an aptamer that will interact with the oocyte’s zona pellucida (ZP), the extracellular coat of the egg and early embryo. The proteins of the ZP are oocyte-specific, and expressed on the oocyte surface as the oocyte grows in the ovarian follicle [1]. Aptamers are nucleic acid-based molecules that bind with high affinity to target molecules [2]. For example, Macugen is an RNA aptamer that binds to and has antagonistic action on an isoform of vascular endothelial growth factor and is an FDA-approved therapeutic for macular degeneration [2]. Aptamers also can be used for delivery of agents such as siRNAs into cells, and thus aptamers are being developed as therapeutics themselves or to target drugs, siRNAs, and other agents to specific cell types for treatment of a variety of diseases. In a proof-of-principle study, aptamer+ siRNA chimeric molecules are targeted to prostate cancer cells in mice through the aptamer binding to a specific prostate cancer surface marker; the delivery of this aptamer+ siRNA into prostate cancer cells leads to knockdown of the siRNA’s target [3]. Aptamers for targets of interest are identified by a procedure called SELEX, for systemic evolution of ligands by exponential enrichment [4]. We prepared an aptamer library that consists of single-stranded oligodeoxyribonucleotides (Figure 1). The central portion of the oligos contains 30 random nucleotides (N30; A, G, C or T), and thus the library comprises approximately 1.2× 1018 oligo-2-deoxyribonucleotide sequence isomers. The first and last 20 nucleotides are binding sites for PCR forward and reverse primers that contain KpnI and XbaI restriction sites, respectively (Figure 1). ZP proteins were isolated from frozen mouse ovaries according to standard protocols, involving centrifugation on Percoll gradients [5, 6]. The purified proteins were characterized by SDS-PAGE and by immunoblotting using monoclonal antibodies specific to mouse ZP1, ZP2, and ZP3 [7, 8].A nitrocellulose filter binding procedure was used to select aptamers that bind to ZP proteins (Figure 1). Nitrocellulose filters (0.45 μm MF filters, MilliporeSigma, Burlington, MA) were pretreated with KOH to reduce non-specific binding; however, we found that∼ 25% of the library still bound to these treated filters. Therefore, we first incubated the library with a KOH-pre-treated filter, and collected unbound aptamers; this fraction of the library was then used for the selection procedure shown in Figure 1. We carried out five rounds of selection in binding buffer (50 mM MOPS, pH 7.0; 2 mM MgCl2) that contained increasing of concentrations of NaCl (100–300 mM in 50 mM increments). A final round of selection was carried out in the presence or absence of ZP proteins in binding buffer containing 500 mM NaCl; quantitative PCR detected the same amount of aptamer recovered from both filters, suggesting
