Recognition of DNA by thymine dimers specific antibodies

紫外线诱导的DNA损伤被认为是导致突变、肿瘤细胞转化和细胞死亡的主要原因。紫外光诱导的光产物主要有顺式环丁烷嘧啶二聚体(CPDS)、嘧啶(6-4)嘧啶酮光产物[(6-4)光产物]和DNA中相邻的嘧啶类化合物之间形成的杜瓦异构体。我们一直在研究一系列能识别CPDS或(6-4)光产物的单抗。为了了解这些抗体对光产物的分子识别作用，我们克隆了可变区基因并进行了测序。比较这些序列显示，所有四个抗(6-4)光产物抗体彼此高度相似，尽管在潜在的抗原接触区有一些差异。为了评估这些序列差异…的影响在结构水平上更多的CES，为这些抗体构建了计算机模型。大多数序列差异发生在潜在的抗原接触区，这表明特定的位置可能解释了观察到的结合亲和力和选择性的差异。因此，构建了这些抗体的单链抗体衍生物并对其进行了表征，以提供一个可以测试结构-功能关系的实验系统。这些衍生物可以通过金属亲和层析步骤从大肠杆菌中分离出来，并具有与亲本单抗相同的结合特异性。用Biacore生物传感器比较了含有CPDS或(6-4)光产物的八脱氧核苷酸与单链抗体和相应纤维的结合常数。单链抗体对抗原类似物的解离速率常数(Kdiss)与Fab的解离速率常数(Kdiss)几乎相同，而2scFv的结合速率常数(Kass)比Fab的结合速率常数(Kass)高一倍，这些结果表明scFv的抗原结合区似乎具有完整抗体的功能。此外，升高的氯化钠浓度显著降低了Fab和ScFv的KASS值，这表明这些抗体通过静电相互作用与含有寡核苷酸的光产物结合。较少

DNA damage induced by ultraviolet (UV) light is considered to be a principal cause of mutation, neoplastic cellualr transformation and cell death. The major classes of UV-induced photoproducts are cis-syn cyclobutane pyrimidine dimers (CPDs) , pyrimidine (6-4) pyrimidone photoproducts [ (6-4) photoproducts] and the Dewar isomers formed between adjacent pyrimidines in DNA.In order to detect and quantitate these DNA photoproducts, several monoclonal antibodies have been established. We have been studying a series of monoclonal antibodies that specifically recognize either CPDs or (6-4) photoproducts.In order to understand the molecular recognitions of the antibodies for the photoproducts, we have cloned and sequenced the variable region genes. Comparing these sequences revealed that all four anti- (6-4) photoproducts antibodies were highly similar to one another, although there were some differences in potential antigen-contact regions. To assess the influences of these sequence differen … More ces at the structural level, computer models were constructed for these antibodies. Most of the sequence differences occurred in potential antigen contact regions, suggesting specific positions that might account for the observed differences in binding affinities and selectivities. Single-chain Fv derivatives of these antibodies were therefore constructed and characterized to provide an experimental system in which structure-function relationships can be tested. These derivatives could be isolated from E.coli using metal affinity chromatographic steps and possessed the same binding specificities as the parent monoclonal antibodies. The binding constants for octadeoxynucleotide containing either CPDs or (6-4) photoproducts as to both scFvs and corresponding Fabs were compared by means of BIAcore biosensor. The dissociation rate constants (kdiss) of scFv for the antigen analog was found to be almost the same as that of Fab, while the association rate constant (kass) of 2scFv was two-fold higher than that of Fab, These results indicate that the antigen binding region of the scFv seems to function as that of the intact antibody. Furthermore, elevated concentrations of NaCl decreased kass values of both Fab and scFv drastically, suggesting that these antibodies bind to oligonucleotide containing photoproducts through electrostatic interactions. Less