Synthesis and biochemical properties of cyanuric acid nucleoside-containing DNA oligomers
Synthesis and biochemical properties of cyanuric acid nucleoside-containing DNA oligomers
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DOI：
10.1021/tx980255e
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发表时间：
1999-07-01
影响因子：
4.1
通讯作者：
Cadet, J
中科院分区：
文献类型：
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作者：
Gasparutto, D;Da Cruz, S;Cadet, J
1-（2-脱氧-β-D-吡喃-戊呋喃糖基）氰尿酸（氰尿酸核苷，dY）（1）已被证明是在DNA组分暴露于电离辐射和激发的光敏剂时形成的。为了研究DNA中dY残基的生物学和结构意义，化学制备了后者修饰的2 '-脱氧核苷，然后将其位点特异性地掺入寡脱氧核糖核苷酸（ODNs）中。这是使用亚磷酰胺方法以良好的产率实现的。为此，设计了一种方便的糖基化方法，包括3，5-保护的2-脱氧呋喃核苷氯化物和氰尿酸（2，4，6-三羟基-1，3，5-三嗪）。通过硅胶纯化5 ′-O-二甲氧基三苯甲基化的衍生物来拆分修饰的2 ′-脱氧核糖核苷的异头混合物（1/2 α/β），然后，亚磷酸化得到所需的β-亚磷酰胺单体（5）。固相浓缩和最终脱保护后，使用不同的互补技术，如HPLC和聚丙烯酰胺凝胶电泳，以及电喷雾电离和MALDI-TOF质谱法检查修饰的合成DNA片段的纯度和完整性。发现在14聚体中存在三聚氰酸核苷对双链DNA片段具有不稳定作用，如从解链温度测量推断的。应用于含dY的ODNs的哌啶测试支持插入寡核苷酸链中的氰尿酸核苷的高稳定性。进行了几个酶实验，旨在确定这样的DNA损伤的生物学特征。因此，研究了核酸酶P-1、蛇毒磷酸二酯酶（SVPDE）、小牛脾磷酸二酯酶（CSPDE）和修复酶（包括大肠杆菌内切核酸酶III（endo III）和Fpg糖基化酶（Fpg））对dY的加工。最后，一个22聚体的ODN轴承氰尿酸残基被用作模板，以研究在体外核苷酸掺入相反的Klenow片段关闭的损害。大肠杆菌聚合酶I。
1-(2-Deoxy-beta-D-erythro-pentofuranosyl)cyanuric acid (cyanuric acid nucleoside, dY) (1) has been shown to be formed upon exposure of DNA components to ionizing radiation and excited photosensitizers, To investigate the biological and structural significance of dY residue in DNA, the latter modified 2'-deoxynucleoside was chemically prepared and then site-specifically incorporated into oligodeoxyribonucleotides (ODNs). This was achieved in good yields using the phosphoramidite approach. For this purpose, a convenient glycosylation method involving 3,5-protected 2-deoxyribofuranoside chloride and cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine) was devised. The anomeric mixture of modified 2'-deoxyribonucleosides (1/2 alpha/beta) was resolved by silica gel purification of the 5'-O-dimethoxytritylated derivatives, and then, phosphitylation afforded the desired beta-phosphoramidite monomer (5). After solid-phase condensation and final deprotection, the purity and the integrity of the modified synthetic DNA fragments were checked using different complementary techniques such as HPLC and polyacrylamide gel electrophoresis, together with electrospray ionization and MALDI-TOF mass spectrometry. The presence of cyanuric acid nucleoside in a 14-mer was found to have destabilizing effects on the double-stranded DNA fragment as inferred from melting temperature measurements. The piperidine test applied to dY-containing ODNs supported the high stability of cyanuric acid nucleoside inserted into the oligonucleotide chain. Several enzymatic experiments aimed at determining the biological features of such a DNA lesion were carried out. Thus, processing of dY by nuclease P-1, snake venom phosphodiesterase (SVPDE), calf spleen phosphodiesterase (CSPDE), and repair enzymes, including Escherichia coli endonuclease III (endo III) and Fapy glycosylase (Fpg), was investigated. Finally, a 22-mer ODN bearing a cyanuric acid residue was used as a template to study the in vitro nucleotide incorporation opposite the damage by the Klenow fragment off. coli polymerase I.