Ma Qian, Akiyama Yoshitsugu, Xu Zhidong, Konishi Kazuhide, Hecht Sidney M
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
J Am Chem Soc. 2009 Feb 11;131(5):2013-22. doi: 10.1021/ja808629s.
A hairpin DNA library containing an 8-base pair sequence-randomized region was employed in a SELEX-type procedure to identify DNAs that bound strongly to bleomycin A(5), the latter of which was immobilized on a solid support. Ten hairpin DNAs that bound BLM A(5) strongly were identified and sequenced, and used to characterize DNA binding by the antitumor antibiotic. While all 10 selected hairpin DNAs bound to BLM strongly, they did exhibit a range of binding specificities. Further, while the binding specificity was generally the greatest for hairpin DNAs that contained at least one of the sequences (5'-GC-3' and 5'-GT-3') cleaved most frequently by Fe(II).bleomycin, the hairpin DNA exhibiting the poorest binding specificity also contained a 5'-GT-3' site. Four of the hairpin DNA substrates were 5'-(32)P end-labeled and used to assess the preferred sites of cleavage by Fe(II).BLM. The substrate DNAs included two lacking any 5'-GT-3' or 5'-GC-3' site; these were cleaved at 5'-AA-3' and (more strongly) at 5'-AT-3' and 5'-GA-3' sequences. For two hairpin DNAs containing 5'-GT-3' or 5'-GC-3' sequences, cleavage was observed at these sequences as well, but the three aforementioned sequences were also cleaved efficiently. For hairpin DNA 3, which was bound the least well of the 10 DNAs studied, a 5'-TA-3' site was also cleaved efficiently. Thus, the pattern and intensities of cleavage of the four DNAs studied were not entirely consistent with the preferred pattern of DNA cleavage reported for Fe(II).BLM in numerous published studies that have employed arbitrarily chosen DNA substrates. Also studied were the chemistry of DNA cleavage for one of the hairpin DNAs, and competition experiments in which the diminution of cleavage was measured following admixture of a molar excess of a smaller hairpin DNA shown to be an exceptionally good substrate for cleavage by Fe(II).BLM. In the aggregate, the data indicate that the relationship between DNA binding and degradation by Fe.BLM, as well as the chemistry leading to DNA degradation, are more complex than suggested by earlier studies that employed only DNA degradation product analysis as an end point.
一个包含8个碱基对序列随机区域的发夹DNA文库被用于一种类似SELEX的程序中，以鉴定与博来霉素A(5)紧密结合的DNA，博来霉素A(5)被固定在固体支持物上。鉴定并测序了10个与博来霉素A(5)紧密结合的发夹DNA，并用于表征这种抗肿瘤抗生素与DNA的结合。虽然所有10个选定的发夹DNA都与博来霉素紧密结合，但它们确实表现出一系列的结合特异性。此外，虽然对于含有至少一个被Fe(II).博来霉素最频繁切割的序列（5'-GC-3'和5'-GT-3'）之一的发夹DNA，结合特异性通常最大，但结合特异性最差的发夹DNA也含有一个5'-GT-3'位点。4个发夹DNA底物进行了5'-（32）P末端标记，并用于评估Fe(II).博来霉素的优先切割位点。底物DNA包括两个没有任何5'-GT-3'或5'-GC-3'位点的；它们在5'-AA-3'处被切割，并且在5'-AT-3'和5'-GA-3'序列处（切割更强）被切割。对于两个含有5'-GT-3'或5'-GC-3'序列的发夹DNA，在这些序列处也观察到了切割，但上述三个序列也被有效切割。对于所研究的10个DNA中结合最差的发夹DNA 3，一个5'-TA-3'位点也被有效切割。因此，所研究的4个DNA的切割模式和强度与在许多使用任意选择的DNA底物的已发表研究中报道的Fe(II).博来霉素的DNA切割优先模式并不完全一致。还研究了其中一个发夹DNA的DNA切割化学，以及竞争实验，在竞争实验中，在加入摩尔过量的较小发夹DNA（已证明是Fe(II).博来霉素切割的异常良好底物）后，测量切割的减少情况。总体而言，数据表明DNA结合与Fe.博来霉素介导的降解之间的关系，以及导致DNA降解的化学过程，比早期仅以DNA降解产物分析作为终点的研究所表明的更为复杂。
