Biofunctional Medical Engineering Research Area, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa, Japan.
Biofunctional Medical Engineering Research Area, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa, Japan.
Bioorg Med Chem Lett. 2024 Jan 15;98:129597. doi: 10.1016/j.bmcl.2023.129597. Epub 2023 Dec 26.
Natural DNA restriction enzymes bind duplex DNA with high affinity at multiple sites; however, for some of the artificial chemical-based restriction moieties, invasion of the double-strand for efficient cleavage is an obstacle. We have previously reported photo-induced double-duplex invasion (pDDI) using 3-cyanovinylcarbazole (K)-containing probes for both the target strands that photo-crosslink with pyrimidine bases in a sequence-specific manner on both the strands, stabilizing the opened double-strand for cleavage. The drawback of the pDDI was low efficiency due to inter-probe cross-linking, solved by the inclusion of 5-cyano-uridine at -1 position on the complimentary strand with respect to K in both probes. Although this led to reduced inter-probe cross-linking, the pDDI efficiency was still low.
Here, we report that inter-probe cross-linking and intra-probe cross-linking of a single probe is also leading to reduced pDDI efficiency. We addressed this problem by designing DDI probes to inhibit both inter-probe and intra-probe cross-linking.
Based on the new design of pDDI probe with 5-cyano uridine led to a drastic increase in the efficiency of pDDI in (400-mer) double-stranded DNA with only 1 s of photo-irradiation.
天然 DNA 限制酶以高亲和力在多个位点结合双链 DNA；然而，对于一些基于人工化学的限制部分，双链体的入侵对于有效的切割是一个障碍。我们之前报道了使用含有 3-氰基乙烯基咔唑 (K) 的探针进行光诱导双链体入侵 (pDDI)，这些探针可以与双链体上的嘧啶碱基以序列特异性的方式光交联，稳定双链体以进行切割。pDDI 的缺点是由于探针之间的交联导致效率低，通过在互补链上相对于 K 在 -1 位置包含 5-氰基尿嘧啶来解决。尽管这导致探针之间的交联减少，但 pDDI 的效率仍然很低。
在这里，我们报告说单个探针的探针之间交联和探针内交联也会导致 pDDI 效率降低。我们通过设计 DDI 探针来抑制探针之间和探针内的交联来解决这个问题。
基于新设计的 pDDI 探针，带有 5-氰基尿嘧啶，在仅 1 秒的光照射下，（400 -mer）双链 DNA 的 pDDI 效率大大提高。
