A novel method to synthesize versatile multiple-branched DNA (MB-DNA) by reversible photochemical ligation
A novel method to synthesize versatile multiple-branched DNA (MB-DNA) by reversible photochemical ligation
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DOI：
10.1002/cbic.200500188
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发表时间：
2005-10-01
期刊：
影响因子：
3.2
通讯作者：
Fujimoto, K
中科院分区：
文献类型：
--
作者：
Ogasawara, S;Fujimoto, K
为了证明这种利用支链ODN构建MB-ODN的新合成方法的可行性，我们研究了双支链ODN （DB-ODN）的构建对含有两个cvU （Scheme3）的碱基DNA （ODN 1c）的影响。图2a (b)显示了模板ODN 3c存在下ODNs 1c和2c的光照射混合物的CGE谱，显示了预期的32-mer连接DBODN的清洁和有效形成，ODNs 1c和2c完全消失。分离的新产物通过MALDI-TOF质谱和酶切进行了表征（见支持信息）。通过MALDI-TOF质谱测定，进一步在312 nm照射DB-DNA导致ODNs 1c和2c完全还原。图2b表明，由于DBODN是在上游cvU或下游cvU通过SB-ODN合成的，因此与ODN 1c的下降相比，DB-ODN产率的上升时间有所延迟。我们在366nm照射开始时清晰地观察到这些峰（见辅助信息）。在图2a (b)中，这些峰的余辉在大约80分钟时被确认为一个宽峰。我们在312nm处观察到类似的模式。DB-ODN通过SB-ODN还原为原始ODN，如图3B所示，其中DB-ODN在312nm处辐照后转化为ODN 1c。综上所述，我们证明了中游含有cvU的ODN可以在模板ODN存在的情况下，通过366nm辐照，高效且无副反应地合成支链ODN。在312 nm的辐照下，光固化的支链ODN恢复到原来的ODN。我们还展示了通过创建DB-ODN构建MB-DNA的可行性。这个演示表明我们可以概念化
To demonstrate the feasibility of this novel synthetic method of using branched ODN to construct MB-ODN, we examined the effects of the construction of doubly branched ODN (DB-ODN) on base DNA (ODN 1c) containing two cvU (Scheme3). Figure2A (b) presents a CGE profile of the photoirradiated mixture of ODNs 1c and 2c in the presence of template ODN 3c, and shows the clean and efficient formation of the expected ligated 32-mer DBODN and the complete disappearance of ODNs 1c and 2c. The isolated new product was characterized by MALDI-TOF MS and enzymatic digestion (see Supporting Information). Further irradiation of DB-DNA at 312 nm resulted in a complete reversion to ODNs 1c and 2c, as determined by MALDI-TOF MS (see Supporting Information). Figure2B shoes that rising time for the yield of DB-ODN was delayed compared with the decline of ODN 1c because DBODN was synthesized through the SB-ODN at the upstream cvU or downstream cvU. We observed these peaks clearly at the beginning of the irradiation at 366nm (see Supporting Information). The afterglow of these peaks is confirmed in Figure2A (b) as a broad peak at around 80 min. We observed a similar pattern for irradiation at 312nm. The DB-ODN reverted to the original ODN through the SB-ODN as shown in Figure 3B, in which re-formation of ODN 1c from DB-ODN irradiated at 312nm occurred after conversion of DB-ODN. In conclusion, we have demonstrated that ODN containing cvU at midstream can be used to synthesize branched ODNs in the presence of a template ODN by irradiation at 366nm with high efficiency and without any side reaction. Irradiation at 312 nm caused the photoligated branched ODN to revert to the original ODNs. We have also shown the feasibility of constructing MB-DNA by creating a DB-ODN. This demonstration indicates that we can conceptual-