François J C, Saison-Behmoaras T, Chassignol M, Thuong N T, Hélène C
Laboratoire de Biophysique, I.N.S.E.R.M., U. n degrees 201, Paris.
C R Acad Sci III. 1988;307(20):849-54.
A homopyrimidine oligonucleotide d(TTTCCTCCTCT) was covalently linked to 1,10-phenanthroline via a 5'-thiophosphate group. In the presence of copper ions and a reducing agent the copper-phenanthroline complex induced cleavage reactions in duplex DNA. The oligonucleotide binds to the major groove of DNA at a homopurine.homopyrimidine sequence, forming a local triple helix. It is oriented parallel to the homopurine strand. Watson-Crick A.T and G.C base pairs are recognized via Hoogsteen-type hydrogen bonding by thymine and protonated cytosine, respectively. The cleavage patterns on opposite strands of duplex DNA at the homopurine.homopyrimidine sequence are asymmetric. They are shifted toward the 3'-side indicating that cleavage takes place from the minor groove even though the oligonucleotide is bound to the major groove. It is therefore suggested that the phenanthroline ring attached to the oligonucleotide intercalates into DNA at the junction between the triple and the double helix and that the copper complex forms in the minor groove where radical reactions leading to strand cleavage occur. The homopyrimidine oligodeoxynucleotide d(TTTCCTCCTCT) tethered to phenanthroline binds to a single site on SV 40 DNA. It cleaves circular and linear SV 40 DNA at this single binding site. Cleavage requires both copper ions and a reducing agent. The unsubstituted oligonucleotide competes with the oligonucleotide-phenanthroline conjugate and prevents site-specific cleavage. These results demonstrate that oligonucleotide-phenanthroline conjugates can be used to induce sequence-specific cleavage of duplex DNA. Such artificial endonucleases could be used, among other things, to map genes on long DNA fragments, to induce site-specific mutations or to block gene expression at the transcriptional level.
同嘧啶寡核苷酸d(TTTCCTCCTCT)通过5'-硫代磷酸基团与1,10-菲咯啉共价连接。在铜离子和还原剂存在的情况下，铜-菲咯啉复合物诱导双链DNA发生切割反应。该寡核苷酸在同嘌呤-同嘧啶序列处与DNA的大沟结合，形成局部三链螺旋。它与同嘌呤链平行排列。沃森-克里克A·T和G·C碱基对分别通过胸腺嘧啶和质子化胞嘧啶的Hoogsteen型氢键被识别。双链DNA在同嘌呤-同嘧啶序列处相对链上的切割模式是不对称的。它们向3'-侧偏移，表明切割从小沟发生，尽管寡核苷酸与大沟结合。因此，有人提出连接到寡核苷酸上的菲咯啉环在三链和双链螺旋的交界处插入DNA中，并且铜复合物在小沟中形成，在那里发生导致链切割的自由基反应。连接到菲咯啉上的同嘧啶寡脱氧核苷酸d(TTTCCTCCTCT)与SV 40 DNA上的一个单一部位结合。它在这个单一结合部位切割环状和线性SV 40 DNA。切割需要铜离子和还原剂。未取代的寡核苷酸与寡核苷酸-菲咯啉缀合物竞争并阻止位点特异性切割。这些结果表明，寡核苷酸-菲咯啉缀合物可用于诱导双链DNA的序列特异性切割。这种人工内切酶可用于，除其他外，在长DNA片段上绘制基因图谱、诱导位点特异性突变或在转录水平阻断基因表达。
