Artificial Restriction Enzymes for Future Nucleic Acids Chemistry

利用我们的人工系统，我们已经实现了DNA和RNA的序列选择性切割。DNA裂解我们发现GAP位点上的磷酸二酯键优先被Ce(IV)/EDTA络合物水解。尽管该配合物没有共价结合到任何序列识别部分，但DNA断裂选择性地发生在间隙位置，因为其中的连接比双链部分的连接更容易被Ce(IV)配合物催化。此外，通过在GAP位引入单磷酸基团和在GAP位引入Ce(IV)络合物，极大地促进了这种GAP选择性DNA水解。通过Ce(IV)/EDTA与两个伪互补多肽核酸(PCPNAs)结合，双链DNA中的两条链都被选择性地在靶位上水解。当两个pcPNA侵入双链DNA时，只有两条链中的指定部分是单链结构。在用Ce(IV)/EDTA处理这种入侵络合物时，两个单链部分都被选择性地水解。使用两个含有氨基羧酸盐或磷酸盐的pcPNAs，大大促进了靶部位的DNA断裂。此外，利用T4连接酶将裂解产物成功连接到外源双链DNA上。RNA裂解，当含有两个吖啶基团的寡核苷酸与底物RNA形成异双链时，吖啶前面的两个磷酸二酯键被选择性地激活，并优先被稀土离子水解。利用这些体系，从长RNA底物中获得预定长度的RNA片段，并用MALDI-TOF MS分析纯合子和杂合子样品中的单核苷酸多态，根据质量数的差异准确和容易地检测到单核苷酸多态。

We have achieved the sequence selective scissions of both DNA and RNA by using our artificial systems.1. DNA cleavageWe found that the phosphodiester linkages in gap sites were preferentially hydrolyzed by a Ce(IV)/EDTA complex. Even though the complex was not covalently bound to any sequence-recognizing moiety, the DNA scission selectively occurs at the gap sites, since the linkages therein is more susceptible to catalysis by the Ce(IV) complex than are those in double-stranded portions. Furthermore, this gap-selective DNA hydrolysis was greatly promoted by introducing monophosphate groups at the gap sites and recruiting the Ce(IV) complex to the gap site.By combining Ce(IV)/EDTA with, two pseudocomplementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were selectively hydrolyzed at the target site. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was single-stranded like structure. On the treatment of this invasion complex with Ce(IV)/EDTA, both of the single-stranded portions are selectively hydrolyzed. With the use of two pcPNAs bearing either aminocarboxylates or phosphates, the DNA scission at target site was greatly promoted. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using T4 ligase.2. RNA cleavage,When oligonucleotides bearing two acridine groups form heteroduplexes with substrate RNA, the two phosphodiester linkages in front of the acridines were selectively activated and preferentially hydrolyzed by lanthanide ion. By using these systems, RNA fragments of predetermined length were obtained from long RNA substrates and analyzed by MALDI-TOF MS. Single nucleotide polymorphisms in homozygous and heterozygous samples were accurately and easily detected in terms of difference in mass number.