Ghosh Souradyuti, Greenberg Marc M
Department of Chemistry, Johns Hopkins University , 3400 North Charles Street, Baltimore, Maryland 21218, United States.
J Org Chem. 2014 Jul 3;79(13):5948-57. doi: 10.1021/jo500944g. Epub 2014 Jun 20.
DNA interstrand cross-links are an important family of DNA damage that block replication and transcription. Recently, it was discovered that oxidized abasic sites react with the opposing strand of DNA to produce interstrand cross-links. Some of the cross-links between 2'-deoxyadenosine and the oxidized abasic sites, 5'-(2-phosphoryl-1,4-dioxobutane) (DOB) and the C4-hydroxylated abasic site (C4-AP), are formed reversibly. Chemical instability hinders biochemical, structural, and physicochemical characterization of these cross-linked duplexes. To overcome these limitations, we developed methods for preparing stabilized analogues of DOB and C4-AP cross-links via solid-phase oligonucleotide synthesis. Oligonucleotides of any sequence are attainable by synthesizing phosphoramidites in which the hydroxyl groups of the cross-linked product were orthogonally protected using photochemically labile and hydrazine labile groups. Selective unmasking of a single hydroxyl group precedes solid-phase synthesis of one arm of the cross-linked DNA. The method is compatible with commercially available phosphoramidites and other oligonucleotide synthesis reagents. Cross-linked duplexes containing as many as 54 nt were synthesized on solid-phase supports. Subsequent enzyme ligation of one cross-link product provided a 60 bp duplex, which is suitable for nucleotide excision repair studies.
DNA链间交联是一类重要的DNA损伤，会阻碍复制和转录。最近，人们发现氧化脱碱基位点会与DNA的互补链发生反应，从而产生链间交联。2'-脱氧腺苷与氧化脱碱基位点之间的一些交联，即5'-(2-磷酰基-1,4-二氧丁烷)(DOB)与C4-羟基化脱碱基位点(C4-AP)之间的交联，是可逆形成的。化学不稳定性阻碍了这些交联双链体的生化、结构和物理化学特性表征。为克服这些限制，我们开发了通过固相寡核苷酸合成制备DOB和C4-AP交联稳定类似物的方法。通过合成亚磷酰胺可以获得任何序列的寡核苷酸，其中交联产物的羟基使用光化学不稳定和肼不稳定基团进行正交保护。在交联DNA的一条臂进行固相合成之前，先对单个羟基进行选择性脱保护。该方法与市售亚磷酰胺和其他寡核苷酸合成试剂兼容。在固相支持物上合成了含有多达54个核苷酸的交联双链体。随后对一种交联产物进行酶连接，得到了一个60 bp的双链体，适用于核苷酸切除修复研究。
