Destabilizing universal linkers for signal amplification in self-ligating probes for RNA
Destabilizing universal linkers for signal amplification in self-ligating probes for RNA
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DOI：
10.1021/ja046791c
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发表时间：
2004-11-03
影响因子：
15
通讯作者：
Kool, ET
中科院分区：
文献类型：
--
作者：
Abe, H;Kool, ET
最近的研究已经确立了寡核苷酸连接方法在溶液中和细胞成像中 DNA 和 RNA 检测中的实用性。值得注意的是，连接的全长寡核苷酸产物通常比两个起始半探针与靶核酸的结合更紧密，这有效地将所得信号限制为每个靶标一个信号。在这里，我们报告了一种在模板促进的自连接反应中使连接产物不稳定的分子策略，从而每个目标产生多个信号。描述了一种新的通用接头设计，其中将 dabsyl 离去基团置于短烷烃链上。与早期的连接策略相比，这允许在任何 DNA 序列的末端放置亲电子试剂，并且还将反应速率加快 4-5 倍。这种新型分子接头/激活剂在 DNA 和 RNA 检测中可将信号放大多达 92 倍，并且无需酶、添加试剂或热循环。该接头显示出会破坏连接产物的稳定性，但不会破坏连接过渡态的稳定性。这降低了产物抑制，因此目标 DNA 或 RNA 成为等温生成多个检测信号的催化剂。这种增强的信号生成在溶液实验和固体支持的测定中得到了证明。
Recent studies have established the utility of oligonucleotide ligation methods in the detection of DNAs and RNAs in solution and in cellular imaging. Notably, the ligated full-length oligonucleotide products commonly bind to the target nucleic acid much more tightly than do the two starting half-probes, which effectively limits the resulting signals to one per target. Here, we report on a molecular strategy for destabilizing ligated products in template-promoted self-ligation reactions, thus yielding multiple signals per target. A new universal linker design is described in which a dabsyl leaving group is placed on a short alkane tether. This allows the placement of an electrophile at the end of any DNA sequence, in contrast to earlier ligation strategies, and it also speeds reaction rates by a factor of 4-5. This new class of molecular linker/activator yields as much as 92-fold amplification of signals in DNA and RNA detection, and proceeds without enzymes, added reagents, or thermal cycling. The linker is shown to destabilize the ligation product without destabilizing the transition state for ligation. This lowers product inhibition, and the target DNA or RNA thus becomes a catalyst for isothermally generating multiple signals for its detection. This enhanced signal generation is demonstrated in solution experiments and in solid supported assays.