2′-fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165) -: Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain
2′-fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165) -: Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain
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DOI：
10.1074/jbc.273.32.20556
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发表时间：
1998-08-07
影响因子：
4.8
通讯作者：
Janjic, N
中科院分区：
文献类型：
--
作者：
Ruckman, J;Green, LS;Janjic, N
血管内皮生长因子（VEGF）在多种增殖性疾病中与新血管生长的病理诱导有关。使用SELEX方法（通过指数富集的配体的系统进化），我们已经分离了人VEGF的2 '-F-嘧啶RNA寡核苷酸配体（适体）（165）。将来自三个不同序列家族的代表性适体截短为能够高亲和力结合VEGF的最小序列（23-29个核苷酸），并通过在所有核糖嘌呤位置用2 ′-O-甲基取代2 ′-OH来进一步修饰，其中所述取代是耐受的。VEGF与截短的2 ′-O-甲基修饰的适体相互作用的平衡解离常数在49和130 pM之间。这些适体与鼠VEGF（164）同样良好地结合，不与VEGF（121）或胎盘生长因子的较小同种型（PlGF（129））结合，并且显示出对VEGF（165）/PlGF（129）异二聚体的降低但显著的亲和力。VEGF外显子7编码结构域中的半胱氨酸137（165）与三种适体中的单个尿苷残基形成光诱导交联。适体有效抑制VEGF与转染的猪主动脉内皮细胞表达的人VEGF受体KDR和Flt-1的结合。此外，其中一种适体能够显著降低皮内VEGF诱导的体内血管通透性。
Vascular endothelial growth factor (VEGF) has been implicated in the pathological induction of new blood vessel growth in a variety of proliferative disorders. Using the SELEX process (Systematic evolution of ligands by exponential enrichment), we have isolated 2'-F-pyrimidine RNA oligonucleotide ligands (aptamers) to human VEGF(165). Representative aptamers from three distinct sequence families were truncated to the minimal sequence capable of high affinity binding to VEGF (23-29 nucleotides) and were further modified by replacement of 2'-O-methyl for 2'-OH at all ribopurine positions where the substitution was tolerated. Equilibrium dissociation constants for the interaction of VEGF with the truncated, 2'-O-methyl-modified aptamers range between 49 and 130 pM, These aptamers bind equally well to murine VEGF(164), do not bind to VEGF(121) or the smaller isoform of placenta growth factor (PlGF(129)), and show reduced, but significant affinity for the VEGF(165)/PlGF(129) heterodimer. Cysteine 137 in the exon 7-encoded domain of VEGF(165) forms a photo-inducible cross-link to a single uridine residue in each of the three aptamers. The aptamers potently inhibit the binding of VEGF to the human VEGF receptors, KDR and Flt-1, expressed by transfected porcine aortic endothelial cells. Furthermore, one of the aptamers is able to significantly reduce intradermal VEGF-induced vascular permeability in vivo.