Label-free Detection of the Transcription Initiation Factor Assembly and Specific Inhibition by Aptamers

Bulletin of the Korean chemical society

Yuanyuan Jiang¹  Donghyuk Shin¹  Shuo Ren¹  Soyoun Kim¹  Hye Rim Yoon¹  Sun Woo Hong¹  Irene M. Min¹  Dong-ki Lee¹  Sangho Lee¹  Moonsoo M. Jin¹ 

关键词: SELEX;    Aptamer;    Transcription factor;    BLI;   

DOI  :  

学科分类：化学（综合）

来源: Korean Chemical Society

【 摘 要 】

The binding of TATA-binding protein (TBP) to the TATA-box containing promoter region is aided by many other transcriptional factors including TFIIA and TFIIB. The mechanistic insight into the assembly of RNA polymerase II preinitation complex (PIC) has been gained by either directly altering a function of target protein or perturbing molecular interactions using drugs, RNAi, or aptamers. Aptamers have been found particularly useful for studying a role of a subset of PIC on transcription for their ability to inhibit specific molecular interactions. One major hurdle to the wide use of aptamers as specific inhibitors arises from the difficulty with traditional assays to validate and determine specificity, affinity, and binding epitopes for aptamers against targets. Here, using a technique called the bio-layer interferometry (BLI) designed for a label-free, real-time, and multiplexed detection of molecular interactions, we studied the assembly of a subset of PIC, TBP binding to TATA DNA, and two distinct classes of aptamers against TPB in regard to their ability to inhibit TBP binding to TFIIA or TATA DNA. Using BLI, we measured not only equilibrium binding constants (KD), which were overall in close agreement with those obtained by electrophoretic mobility shift assay, but also kinetic constants of binding (kon and koff), differentiating aptamers of comparable KDs by their difference in binding kinetics. The assay developed in this study can readily be adopted for high throughput validation of candidate aptamers for specificity, affinity, and epitopes, providing both equilibrium and kinetic information for aptamer interaction with targets.

【 授权许可】

Unknown