Label-free detection of the transcription initiation factor assembly and specific inhibition by aptamers

Shuo Ren, Yuanyuan Jiang, Hye Rim Yoon, Sun Woo Hong, Donghyuk Shin, Sangho Lee, Dong Ki Lee, Moonsoo M. Jin, Irene Min, Soyoun Kim

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1279-1284
Number of pages6
JournalBulletin of the Korean Chemical Society
Volume35
Issue number5
DOIs
StatePublished - May 20 2014

Keywords

  • Aptamer
  • BLI
  • SELEX
  • Transcription factor

ASJC Scopus subject areas

  • Chemistry(all)

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