Noll D M, Noronha A M, Miller P S
Department of Biochemistry and Molecular Biology, School of Hygiene and Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205, USA.
J Am Chem Soc. 2001 Apr 18;123(15):3405-11. doi: 10.1021/ja003340t.
Short DNA duplexes containing an N(4)C-ethyl-N(4)C interstrand cross-link, C-C, were synthesized on controlled pore glass supports. Duplexes having two, three, or four A/T base pairs on either side of the C-C cross-link and terminating with a C(4) overhang at their 5'-ends were prepared. The cross-link was introduced using a convertible nucleoside approach. Thus, an oligonucleotide terminating at its 5'-end with O(4)-triazoyl-2'-deoxyuridine was first prepared on the support. The triazole group of support-bound oligomer was displaced by the aminoethyl group of 5'-dimethoxytrityl-3'-O-tert-butyldimethylsilyl-N(4)-(2-aminoethyl)deoxycytidine to give the cross-link. The dimethoxytrityl group was removed, and the upper and lower strands of the duplex were extended from two 5'-hydroxyl groups of the cross-link using protected nucleoside 3'-phosphoramidites. The tert-butyldimethylsilyl group of the resulting partial duplex was then removed, and the chain was extended in the 3'-direction from the resulting 3'-hydroxyl of the cross-link using protected nucleoside 5'-phosphoramidites. The cross-linked duplexes were purified by HPLC and characterized by enzymatic digestion and MALDI-TOF mass spectrometry. Duplexes with three or four A/T base pairs on either side of the C-C cross-link gave sigmoidal shaped A(260) profiles when heated, a behavior consistent with cooperative denaturation of the A/T base pairs. Each cross-linked duplex could be ligated to an acceptor duplex using T4 DNA ligase, a result that suggests that the C-C cross-link does not interfere with the ligation reaction, even when it is located only two base pairs from the site of ligation. The ability to synthesize duplexes with a defined interstrand cross-link and to incorporate these duplexes into longer pieces of DNA should enable preparation of substrates that can be used for a variety of biophysical and biochemical experiments, including studies of DNA repair.
在可控孔径玻璃载体上合成了含有N(4)C-乙基-N(4)C链间交联（C-C）的短DNA双链体。制备了在C-C交联两侧具有两个、三个或四个A/T碱基对并在其5'-端以C(4)突出端终止的双链体。使用可转化核苷方法引入交联。因此，首先在载体上制备一个在其5'-端以O(4)-三唑基-2'-脱氧尿苷终止的寡核苷酸。载体结合的寡聚物的三唑基团被5'-二甲氧基三苯甲基-3'-O-叔丁基二甲基甲硅烷基-N(4)-(2-氨基乙基)脱氧胞苷的氨基乙基取代，得到交联。去除二甲氧基三苯甲基基团，使用受保护的核苷3'-亚磷酰胺从交联的两个5'-羟基延伸双链体上链和下链。然后去除所得部分双链体的叔丁基二甲基甲硅烷基基团，并使用受保护的核苷5'-亚磷酰胺从交联所得的3'-羟基在3'-方向上延伸链。通过高效液相色谱法纯化交联的双链体，并通过酶切和基质辅助激光解吸电离飞行时间质谱法进行表征。在C-C交联两侧具有三个或四个A/T碱基对的双链体在加热时给出S形的A(260)谱，这种行为与A/T碱基对的协同解链一致。每个交联的双链体可以使用T4 DNA连接酶连接到受体双链体上，这一结果表明C-C交联即使位于距连接位点仅两个碱基对处也不会干扰连接反应。合成具有确定链间交联的双链体并将这些双链体掺入更长的DNA片段中的能力应该能够制备可用于各种生物物理和生化实验（包括DNA修复研究）的底物。
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