Xiao Sai Jin, Hu Ping Ping, Li Yuan Fang, Huang Cheng Zhi, Huang Tao, Xiao Geng Fu
Education Ministry Key Laboratory on Luminescence and Real-Time Analysis, College of Chemistry and Chemical Engineering, Southwest University, Chongqing, China.
Talanta. 2009 Oct 15;79(5):1283-6. doi: 10.1016/j.talanta.2009.05.040. Epub 2009 Jun 6.
An aptamer-participated haprin structure was designed by employing cellular prion protein (PrP(C)) as a model protein, and thus an aptamer-mediated turn-on fluorescence assay for proteins was developed in this contribution. The designed aptamer-participated haprin structure consists of three segments. Namely, an aptamer sequence located in the loop, three guanine bases at 3'-terminal, and a fluophor modified at 5'-terminal. It was found that the guanine bases at the 3'-terminal could quench the fluorescence of the fluophor such as tetramethyl-6-carboxyrhodamine (TAMRA) at the 5'-terminal about 76.6% via electron transfer if the guanine bases are close enough to the fluophor, and the quenched fluorescence could get restored when the target protein is present since the interaction, which could be confirmed by measuring fluorescence lifetime, between TAMRA-aptamer and the target protein forces the guanines away from TAMRA so that TAMRA-modified aptamer changes into turn-on state. A linear relationship was then constructed between the turn-on fluorescence intensity and the concentration of PrP(C) in the range from 1.1 to 44.7 microg/mL with a limit of detection of 0.3 microg/mL (3sigma).
以细胞朊蛋白（PrP(C)）作为模型蛋白，设计了一种适体参与的发夹结构，在此基础上开发了一种适体介导的蛋白质开启型荧光检测方法。所设计的适体参与的发夹结构由三个部分组成。即，位于环中的适体序列、3'-末端的三个鸟嘌呤碱基以及5'-末端修饰的荧光团。研究发现，如果鸟嘌呤碱基与荧光团足够接近，3'-末端的鸟嘌呤碱基可通过电子转移使5'-末端的荧光团如四甲基-6-羧基罗丹明（TAMRA）的荧光猝灭约76.6%，当目标蛋白存在时，猝灭的荧光会恢复，这是因为TAMRA-适体与目标蛋白之间的相互作用（可通过测量荧光寿命来证实）会使鸟嘌呤远离TAMRA，从而使TAMRA修饰的适体转变为开启状态。然后在开启型荧光强度与PrP(C)浓度在1.1至44.7μg/mL范围内建立了线性关系，检测限为0.3μg/mL（3σ）。
