Thériault N Y, Krueger W C, Prairie M D
Biopolymer Chemistry, Upjohn Company, Kalamazoo, MI 49001.
Chem Biol Interact. 1988;65(2):187-201. doi: 10.1016/0009-2797(88)90054-3.
In this paper, we report on the base pair binding specificity of CC-1065 to oligomer duplexes of several lengths and base composition as determined by circular dichroism (CD) methods. The oligomers are synthesized using the phosphoramidite triester coupling approach and purified by either polyacrylamide gel electrophoresis or anion-exchange HPLC. CC-1065 binds by two different mechanisms to form a reversibly bound species and an irreversibly bound species, both of which show intense induced CD bands. The reversible to irreversible binding transition is characterized by a shift of the CD band to shorter wavelength (392----371 nm) characteristic of the reaction between the cyclopropyl group of CC-1065 and the N-3 of adenine. The induced CD acquired by the CC-1065 chromophore increases with increasing oligomer chain length and with an increase in the number of bases to the 5' end of the site of attachment whether a purine or a pyrimidine is at position 5 (or 4) from the site of attachment at the 3' end is not crucial for binding. The binding sequences 5'-GATAT and 5'-GTATA show a slower conversion to an irreversibly bound species relative to the preferred sequences 5'-AAA and 5'-TTA. A G base pair at position 3 in 5'-AAGAA hinders the formation of the irreversibly bound species relative to the 5'-GAAAA and 5'-AGAAA sequences. Very stable reversible binding is observed with the sequences 5'-GAATT and 5'-AAGAA. The sequences 5'-GCGAA and 5'-AGAG also show reversible binding but are characterized by a relatively small induced molar ellipticity which decreases with time. These binding characteristics signify weaker binding for these sequences. Overall, these binding studies agree with earlier sequence studies which found two preferred binding sequences, 5'-AAAAA and 5'-PuNTTA, with CC-1065 attached to the 3' end of the binding site. Furthermore, according to studies of the oligomer 5'-CGCGAATTCGCG-3' under different experimental conditions, the annealing conditions of this work produced oligomer duplex structures, not hairpin structures. In these studies, we found that CC-1065 binds very little or not at all to hairpin structures.
在本文中，我们报告了通过圆二色性（CD）方法测定的CC - 1065与几种长度和碱基组成的寡聚双链体的碱基对结合特异性。这些寡聚物采用亚磷酰胺三酯偶联方法合成，并通过聚丙烯酰胺凝胶电泳或阴离子交换高效液相色谱进行纯化。CC - 1065通过两种不同机制结合，形成可逆结合物种和不可逆结合物种，两者均显示出强烈的诱导CD带。可逆到不可逆结合转变的特征是CD带向较短波长（392----371 nm）移动，这是CC - 1065的环丙基与腺嘌呤的N - 3之间反应的特征。CC - 1065发色团获得的诱导CD随着寡聚物链长的增加以及在附着位点5'端碱基数量的增加而增加，无论在3'端附着位点的第5（或4）位是嘌呤还是嘧啶对于结合都不是关键的。与优选序列5'-AAA和5'-TTA相比，结合序列5'-GATAT和5'-GTATA向不可逆结合物种的转化较慢。5'-AAGAA中第3位的G碱基对相对于5'-GAAAA和5'-AGAAA序列阻碍了不可逆结合物种的形成。用序列5'-GAATT和5'-AAGAA观察到非常稳定的可逆结合。序列5'-GCGAA和5'-AGAG也显示可逆结合，但其特征是诱导摩尔椭圆率相对较小，且随时间降低。这些结合特征表明这些序列的结合较弱。总体而言，这些结合研究与早期的序列研究一致，早期研究发现两个优选的结合序列，5'-AAAAA和5'-PuNTTA，CC - 1065附着在结合位点的3'端。此外，根据在不同实验条件下对寡聚物5'-CGCGAATTCGCG-3'的研究，本工作的退火条件产生了寡聚双链体结构，而不是发夹结构。在这些研究中，我们发现CC - 1065与发夹结构的结合很少或根本不结合。
