Lupold Shawn E, Hicke Brian J, Lin Yun, Coffey Donald S
Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-2101, USA.
Cancer Res. 2002 Jul 15;62(14):4029-33.
We have identified two synthetic oligonucleotides (aptamers) that bind to prostate cancer cells,with low nanomolar affinity, via the extracellular portion of the prostate-specificmembrane antigen (PSMA). These two specific aptamers were selected from an initial 40mer library of approximately 6 x 10(14) random-sequence RNA molecules for their ability to bind to a recombinant protein representing the extracellular 706 amino acids of PSMA, termed xPSM. Six rounds of in vitro selection were performed, enriching for xPSM binding as monitored by aptamer inhibition of xPSM N-acetyl-alpha-linked acid dipeptidase (NAALADase) enzymatic activity. By round six, 95% of the aptamer pool consisted of just two sequences. These two aptamers, termed xPSM-A9 and xPSM-A10, were cloned and found to be unique, sharing no consensus sequences. The affinity of each aptamer for PSMA was quantitated by its ability to inhibit NAALADase activity. Aptamer xPSM-A9 inhibits PSMA noncompetitively with an average K(i) of 2.1 nM, whereas aptamer xPSM-A10 inhibits competitively with an average K(i) of 11.9 nM. Distinct modes of inhibition suggest that each aptamer identifies a unique extracellular epitope of xPSM. One aptamer was truncated from 23.4 kDa to 18.5 kDa and specifically binds LNCaP human prostate cancer cells expressing PSMA but not PSMA-devoid PC-3 human prostate cancer cells. These are the first reported RNA aptamers selected to bind a tumor-associated membrane antigen and the first application of RNA aptamers to a prostate specific cell marker. These aptamers may be used clinically as NAALADase inhibitors or be modified to carry imaging agents and therapeutic agents directed to prostate cancer cells.
我们已经鉴定出两种合成寡核苷酸（适体），它们通过前列腺特异性膜抗原（PSMA）的细胞外部分，以低纳摩尔亲和力与前列腺癌细胞结合。这两种特异性适体是从一个初始的40聚体文库中筛选出来的，该文库包含约6×10¹⁴个随机序列RNA分子，筛选依据是它们与一种代表PSMA细胞外706个氨基酸的重组蛋白（称为xPSM）结合的能力。进行了六轮体外筛选，通过适体对xPSM N - 乙酰 - α - 连接酸性二肽酶（NAALADase）酶活性的抑制作用来监测，富集能与xPSM结合的适体。到第六轮时，95%的适体文库仅由两个序列组成。这两种适体，称为xPSM - A9和xPSM - A10，被克隆并发现是独特的，没有共同的共有序列。每种适体对PSMA的亲和力通过其抑制NAALADase活性的能力来定量。适体xPSM - A9以非竞争性方式抑制PSMA，平均抑制常数（K(i)）为2.1 nM，而适体xPSM - A10以竞争性方式抑制，平均K(i)为11.9 nM。不同的抑制模式表明每种适体识别xPSM独特的细胞外表位。一种适体从23.4 kDa截短至18.5 kDa，特异性结合表达PSMA的LNCaP人前列腺癌细胞，而不结合不表达PSMA的PC - 3人前列腺癌细胞。这些是首次报道的被选择用于结合肿瘤相关膜抗原的RNA适体，也是RNA适体在前列腺特异性细胞标志物上的首次应用。这些适体可在临床上用作NAALADase抑制剂，或被修饰以携带针对前列腺癌细胞的成像剂和治疗剂。
