Using photons to manipulate enzyme inhibition by an azobenzene-modified nucleic acid probe
Using photons to manipulate enzyme inhibition by an azobenzene-modified nucleic acid probe
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DOI：
10.1073/pnas.0812402106
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发表时间：
2009-04-21
影响因子：
11.1
通讯作者：
Tan, Weihong
中科院分区：
文献类型：
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作者：
Kim, Youngmi;Phillips, Joseph A.;Tan, Weihong
以高空间分辨率抑制特定组织中的酶的能力与容易获得的解毒剂相结合，应该会发现许多生物医学应用。我们利用偶氮苯分子的顺-反光异构化反应完成了这一工作。具体而言，我们定位偶氮苯部分互补的DNA序列内的15个碱基长的凝血酶适体，然后连接偶氮苯修饰的cDNA的适体的聚乙二醇（PEG）接头，使单分子共轭。在偶氮苯通过可见光的光异构化过程中，凝血酶的抑制被禁用，因为探针与反式偶氮苯构象的cDNA杂交，使得适体不能结合其靶凝血酶。然而，当施加UV光时，发夹结构（双链体）的解链经由反式至顺式转化而被诱导，从而改变适体的构象并使适体自由地结合并抑制其靶凝血酶。通过使用标准的凝血试验，我们测量了两种状态下各种探针设计的IC 200，并得出结论，使用光子能量在时间和空间上调节这些酶促反应的可行性。因此，我们可以报道以光子可控（凝血酶）抑制剂（称为PCI）的形式开发DNA探针，我们预计这种方法在未来的生物医学和制药应用中将非常有益。
The ability to inhibit an enzyme in a specific tissue with high spatial resolution combined with a readily available antidote should find many biomedical applications. We have accomplished this by taking advantage of the cis-trans photoisomerization of azobenzene molecules. Specifically, we positioned azobenzene moieties within the DNA sequence complementary to a 15-base-long thrombin aptamer and then linked the azobenzene-modified cDNA to the aptamer by a polyethylene glycol (PEG) linker to make a unimolecular conjugate. During the photoisomerization of azobenzene by visible light, the inhibition of thrombin is disabled because the probe hybridizes with the cDNA in the trans-azobenzene conformation so that the aptamer cannot bind its target thrombin. However, when UV light is applied, melting of the hairpin structure (duplex) is induced via trans-to-cis conversion, thereby changing conformation of the aptamer and making the aptamer free to bind to and inhibit its target thrombin. By using standard clotting assays, we measured the IC200 of various probe designs in both states and concluded the feasibility of using photon energy to temporally and spatially regulate these enzymatic reactions. Thus, we can report the development of DNA probes in the form of photon-controllable (thrombin) inhibitors, termed PCIs, and we expect that this approach will be highly beneficial in future biomedical and pharmaceutical applications.