Engelen Wouter, Merkx Maarten
Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Den Dolech 2, 5612, MB, Eindhoven, The Netherlands.
Methods Mol Biol. 2017;1596:179-194. doi: 10.1007/978-1-4939-6940-1_12.
Synthetic protein switches that sequence-specifically respond to oligonucleotide-based input triggers provide valuable tools for the readout of oligonucleotide-based biomolecular systems and networks. Here, we discuss a highly modular approach to reversibly control the DNA-directed assembly and disassembly of a complex between TEM1-β-lactamase and its inhibitor protein BLIP. By conjugating each protein to a unique handle oligonucleotide, the enzyme-inhibitor pair is noncovalently assembled upon the addition of a complementary ssDNA template strand, resulting in inhibition of enzyme activity. Hybridization of an input-oligonucleotide that is complementary to a target recognition sequence in the ssDNA template strand results in the formation of a rigid dsDNA helix that mechanically disrupts the enzyme-inhibitor complex, hereby restoring enzyme activity. Following this noncovalent approach allowed straightforward tuning of the ssDNA template recognition sequence and target oligonucleotide lengths with only a single set of oligonucleotide-functionalized enzyme and inhibitor domains. Using a fluorescent substrate, as little as 10 pM target oligonucleotide resulted in a distinguishable increase in enzyme activity.
能够对基于寡核苷酸的输入触发物进行序列特异性响应的合成蛋白质开关，为基于寡核苷酸的生物分子系统和网络的读出提供了有价值的工具。在此，我们讨论一种高度模块化的方法，用于可逆地控制TEM1-β-内酰胺酶与其抑制剂蛋白BLIP之间复合物的DNA定向组装和解聚。通过将每种蛋白质与独特的手柄寡核苷酸偶联，在添加互补的单链DNA模板链时，酶-抑制剂对非共价组装，导致酶活性受到抑制。与单链DNA模板链中的靶标识别序列互补的输入寡核苷酸的杂交导致形成刚性双链DNA螺旋，该螺旋机械地破坏酶-抑制剂复合物，从而恢复酶活性。遵循这种非共价方法，仅使用一组寡核苷酸功能化的酶和抑制剂结构域，就可以直接调节单链DNA模板识别序列和靶标寡核苷酸的长度。使用荧光底物，低至10 pM的靶标寡核苷酸就会导致酶活性出现明显增加。
