Warren M A, Murray J B, Connolly B A
Department of Biochemistry and Genetics, University of Newcastle, Newcastle upon Tyne, UK.
J Mol Biol. 1998 May 29;279(1):89-100. doi: 10.1006/jmbi.1998.1719.
A method for the preparation of an oligodeoxynucleotide, 20 bases in length, containing centrally located thio analogues of (6-4) pyrimidine-pyrimidinone thymine photo-dimers is reported. The approach is based on the selective irradiation, at 350 nm, of a Tp4ST (4ST = 4-thiothymidine) step within a 20-mer having the sequence: d(ACTCGGACCT(4sT)CGCTGTGAT). Conversion of the S5-(6-4)/S5-thietane pyrimidine-pyrimidinone, initially formed, to its S5-Dewar isomer is by a subsequent irradiation at 300 nm. Both of the photo-dimer-containing oligonucleotides were purified by HPLC (ion exchange and reverse phase) and characterised by base composition analysis. The S5-(6-4)/S5-thietane pyrimidine-pyrimidinone containing 20-mer has a characteristic UV absorbance at 320 nm and exhibits strong fluorescence when excited at this wavelength. As expected, conversion to the S5-Dewar isomer abolished both the 320 nm absorbance and the fluorescence emission. The lengths of the oligonucleotides produced allowed the formation of stable double-stranded DNA, by hybridisation to a complementary sequence. Examination of these duplexes by circular dichroism spectroscopy showed that they formed B-DNA, with little changes to their gross structure as compared to the parent duplex. However, local structural perturbations in the region of the photo-dimer cannot be excluded. The S5-(6-4)/S5-thietane photoproduct lowered the tm by 10.5 deg. C and the Dewar isomer by 12 deg. C. The degree of curvature induced in the DNA sequence by the introduction of the photo-dimers was assessed by analysing the migration of modified and unmodified multimer ladders on polyacrylamide gels. Both photoproducts induced considerable bending into the DNA. A comparison with a six-base-pair T tract, a bending standard that has a known bend angle of 19 degrees, gave values of around 47 degrees for the S5-(6-4)/S5-thietane product and about 28 degrees for the S5-Dewar isomer.
报道了一种制备长度为20个碱基的寡脱氧核苷酸的方法，该寡核苷酸在中心位置含有（6-4）嘧啶-嘧啶酮胸腺嘧啶光二聚体的硫代类似物。该方法基于对具有序列d(ACTCGGACCT(4sT)CGCTGTGAT)的20聚体中Tp4ST（4ST = 4-硫代胸苷）步骤在350 nm处进行选择性照射。最初形成的S5-(6-4)/S5-硫杂环丙烷嘧啶-嘧啶酮通过随后在300 nm处照射转化为其S5-杜瓦异构体。两种含光二聚体的寡核苷酸均通过HPLC（离子交换和反相）纯化，并通过碱基组成分析进行表征。含有S5-(6-4)/S5-硫杂环丙烷嘧啶-嘧啶酮的20聚体在320 nm处具有特征性紫外吸收，并且在该波长激发时表现出强荧光。正如预期的那样，转化为S5-杜瓦异构体消除了320 nm处的吸收和荧光发射。所产生的寡核苷酸长度允许通过与互补序列杂交形成稳定的双链DNA。通过圆二色光谱法对这些双链体进行检查表明，它们形成了B-DNA，与亲本双链体相比，其总体结构变化很小。然而，不能排除光二聚体区域的局部结构扰动。S5-(6-4)/S5-硫杂环丙烷光产物使熔解温度降低了10.5℃，杜瓦异构体使熔解温度降低了12℃。通过分析修饰和未修饰的多聚体梯在聚丙烯酰胺凝胶上的迁移来评估光二聚体引入对DNA序列诱导的弯曲程度。两种光产物均使DNA产生相当大的弯曲。与已知弯曲角度为19度的弯曲标准六碱基对T序列进行比较，S5-(6-4)/S5-硫杂环丙烷产物的值约为47度，S5-杜瓦异构体的值约为28度。
