Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia.
Skolkovo Institute of Science and Technology, Moscow 121205, Russia.
Sensors (Basel). 2020 Feb 9;20(3):915. doi: 10.3390/s20030915.
Aptasensors became popular instruments in bioanalytical chemistry and molecular biology. To increase specificity, perspective signaling elements in aptasensors can be separated into a G-quadruplex (G4) part and a free fluorescent dye that lights up upon binding to the G4 part. However, current systems are limited by relatively low enhancement of fluorescence upon dye binding. Here, we added duplex modules to G4 structures, which supposedly cause the formation of a dye-binding cavity between two modules. Screening of multiple synthetic GFP chromophore analogues and variation of the duplex module resulted in the selection of dyes that light up after complex formation with two-module structures and their RNA analogues by up to 20 times compared to parent G4s. We demonstrated that the short duplex part in TBA25 is preferable for fluorescence light up in comparison to parent TBA15 molecule as well as TBA31 and TBA63 stabilized by longer duplexes. Duplex part of TBA25 may be partially unfolded and has reduced rigidity, which might facilitate optimal dye positioning in the joint between G4 and the duplex. We demonstrated dye enhancement after binding to modified TBA, LTR-III, and Tel23a G4 structures and propose that such architecture of short duplex-G4 signaling elements will enforce the development of improved aptasensors.
适体传感器已成为生物分析化学和分子生物学中流行的仪器。为了提高特异性，可以将适体传感器中的潜在信号元件分离成 G-四链体 (G4) 部分和与 G4 部分结合后发光的游离荧光染料。然而，当前的系统受到染料结合后荧光增强相对较低的限制。在这里，我们在 G4 结构中添加了双链模块，这应该会在两个模块之间形成一个染料结合腔。通过筛选多种合成 GFP 发色团类似物并改变双链模块，选择了与双链结构及其 RNA 类似物形成复合物后发光的染料，与亲本 G4 相比，荧光增强高达 20 倍。我们证明与亲本 TBA15 分子以及由更长双链稳定的 TBA31 和 TBA63 相比，TBA25 中的短双链部分更有利于荧光点亮。TBA25 的双链部分可能部分展开且刚性降低，这可能有助于将染料最佳定位在 G4 和双链之间的连接处。我们证明了结合修饰后的 TBA、LTR-III 和 Tel23a G4 结构后染料增强，并提出这种短双链-G4 信号元件的结构将促进改进型适体传感器的发展。
