Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
Biosens Bioelectron. 2011 Apr 15;26(8):3596-601. doi: 10.1016/j.bios.2011.02.009. Epub 2011 Feb 16.
Molecular beacons (MBs) have been extensively used for real-time monitoring of RNA/DNA and protein molecules. However, such versatility also brings about multiple sources of positive signals. Moreover, the covalently attached quencher or fluorophore may even be cleaved from the strand by the exonucleases, followed by complete degradation of the probe. These undesirable false-positive signals (FPSs) have seriously limited the application of MBs to detect real world samples. In this paper, we propose a novel and efficient approach for discrimination of FPSs of MBs due to non-specific MB-protein interactions and nuclease degradation by combination of heat inactivation and using single walled carbon nanotubes (SWNTs). The mechanisms of different DNA-protein interactions that are responsible for the generation of FPSs of MBs were investigated in detail. The proposed strategy can quickly identify the possible sources of FPSs caused by mechanisms other than hybridization in detecting real samples, which would be very helpful in choosing a proper way to modify the structure of the MBs or using a specific inhibitor. The established method was successfully applied to verify the FPSs in the measurement of a plant tissue sample.
分子信标（MBs）已被广泛用于实时监测 RNA/DNA 和蛋白质分子。然而，这种多功能性也带来了多个来源的正信号。此外，共价连接的猝灭剂或荧光团甚至可能被核酸外切酶从链上切割下来，随后探针被完全降解。这些不理想的假阳性信号（FPS）严重限制了 MBs 在检测实际样本中的应用。在本文中，我们提出了一种新颖有效的方法，通过热失活和使用单壁碳纳米管（SWNTs）的组合，来区分由于非特异性 MB-蛋白相互作用和核酸酶降解而产生的 MBs 的 FPS。详细研究了导致 MBs 的 FPS 产生的不同 DNA-蛋白相互作用的机制。该策略可以快速识别在检测实际样品时由于杂交以外的机制产生 FPS 的可能来源，这对于选择适当的方法来修饰 MBs 的结构或使用特定抑制剂非常有帮助。所建立的方法成功地应用于验证植物组织样品测量中的 FPS。
