Psoralen photo-cross-linking by triplex-forming oligonucleotides at multiple sites in the human rhodopsin gene.
Psoralen photo-cross-linking by triplex-forming oligonucleotides at multiple sites in the human rhodopsin gene.
复制标题
补骨脂素通过人视紫红质基因中多个位点的三链体形成寡核苷酸进行光交联。
DOI：
10.1021/bi9902743
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发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Wilson,JH
中科院分区：
文献类型：
--
作者：
Perkins,BD;Wensel,TG;Vasquez,KM;Wilson,JH
通过三链体形成寡核苷酸（TFO）靶向DNA损伤代表了一种修饰基因表达和结构的方法，也是一种可能的基因治疗方法。我们已经确定，这种方法可以提供具有很大的特异性的G蛋白连接的受体视紫红质，其突变可以导致遗传疾病常染色体显性视网膜色素变性的人类基因的网站的损害。我们已经在基因内的多个靶位点引入了DNA单加合物和链间交联，使用在5 '端具有可光活化的peptide基团的TFO。光加合物的形成程度（即，在三链体-双链体连接处具有5 '-ApT序列的靶位点处以及在分别位于四个和七个核苷酸外的具有5'-ApT和5 '-TpA序列的靶位点处测量了单加合物和交联）。为了改善在更远位点的peptide反应性，将peptide部分用长度为2至9个核苷酸的核苷酸“接头”连接至TFO。高亲和力结合与高达10个核苷酸的接头保持，但由于更快的解离动力学，随着接头长度的增加，亲和力倾向于稍微降低。DNA酶I足迹法表明，很少，如果有的话，接头和双链体之间的相互作用。Pd-TFO结合物在位于三链体连接处的5 '-ApT序列处以高效率（56 - 65%）形成DNA交联。在4个核苷酸外的5 '-ApT位点，效率随接头长度而变化; 4个核苷酸的接头效率最高。具有两个核苷酸以外的5 ′-TpA和5 ′-ApT位点的双链体，在其他方面相同的序列中，分别以56%和38%的效率交联。这些结果表明，TFO-连接体-蛋白结合物允许同时有效靶向人视紫红质基因中的多个位点。
Targeting DNA damage by triplex-forming oligonucleotides (TFOs) represents a way of modifying gene expression and structure and a possible approach to gene therapy. We have determined that this approach can deliver damage with great specificity to sites in the human gene for the G-protein-linked receptor rhodopsin, mutations of which can lead to the genetic disorder autosomal dominant retinitis pigmentosa. We have introduced DNA monoadducts and interstrand cross-links at multiple target sites within the gene using TFOs with a photoactivatable psoralen group at the 5‘-end. The extent of formation of photoadducts (i.e., monoadducts and cross-links) was measured at target sites with a 5‘-ApT sequence at the triplex−duplex junction and at a target site with 5‘-ApT and 5‘-TpA sequences located four and seven nucleotides away, respectively. To improve psoralen reactivity at more distant sites, psoralen moieties were attached to TFOs with nucleotide “linkers” from two to nine nucleotides in length. High-affinity binding was maintained with linkers of up to 10 nucleotides, but affinities tended to decrease somewhat with increasing linker length due to faster dissociation kinetics. DNase I footprinting indicated little, if any, interaction between linkers and the duplex. Psoralen−TFO conjugates formed DNA cross-links with high efficiency (56−65%) at 5‘-ApT sequences located at triplex junctions. At a 5‘-ApT site four nucleotides away, the efficiency varied with linker length; a four-nucleotide linker gave the highest efficiency. Duplexes with 5‘-TpA and 5‘-ApT sites two nucleotides away, in otherwise identical sequences, were cross-linked with efficiencies of 56 and 38%, respectively. These results indicate that TFO−linker−psoralen conjugates allow simultaneous, efficient targeting of multiple sites in the human rhodopsin gene.