Direct in vitro selection of a 2′-O-methyl aptamer to VEGF
Direct in vitro selection of a 2′-O-methyl aptamer to VEGF
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DOI：
10.1016/j.chembiol.2004.10.017
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发表时间：
2005-01-01
影响因子：
--
通讯作者：
Keefe, AD
中科院分区：
文献类型：
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作者：
Burmeister, PE;Lewis, SD;Keefe, AD
适体（蛋白质结合寡核苷酸）具有作为一类新的靶向治疗剂的潜力。对于需要长期全身给药的应用，适体必须实现高亲和力的靶标结合，同时保持高的体内稳定性、耐受性和易于化学合成。为此，我们描述了一种用于产生完全由2 '-O-甲基核苷酸（mRmY）组成的适体的方法。我们提出了可以直接产生2 '-O-甲基转录物的条件，并使用这些条件从3 × 10（15）个独特的2'-O-甲基转录物的文库中选择完全的2 '-O-甲基适体。该适体ARC 245长23个核苷酸，以2 nM的Kd与血管内皮生长因子（VEGF）结合，并在细胞测定中抑制VEGF活性。值得注意的是，ARC 245是如此稳定，以至于在37 ℃下在血浆中96小时后或在125 ℃下高压灭菌后不能检测到降解。我们相信ARC 245作为抗血管生成治疗剂具有相当大的潜力。
Aptamers (protein binding oligonucleotides) have potential as a new class of targeted therapeutics. For applications requiring chronic systemic administration, aptamers must achieve high-affinity target binding while simultaneously retaining high in vivo stability, tolerability, and ease of chemical synthesis. To this end, we describe a method for generating aptamers composed entirely of 2'-O-methyl nucleotides (mRmY). We present conditions under which 2'-O-methyl transcripts can be generated directly and use these conditions to select a fully 2'-O-methyl aptamer from a library of 3 x 10(15) unique 2'-O-methyl transcripts. This aptamer, ARC245, is 23 nucleotides in length, binds to vascular endothelial growth factor (VEGF) with a Kd of 2 nM, and inhibits VEGF activity in cellular assays. Notably, ARC245 is so stable that degradation cannot be detected after 96 hr in plasma at 37degreesC or after autoclaving at 125degreesC. We believe ARC245 has considerable potential as an antiangio-genesis therapeutic.