Monitoring Cooperative Binding Using Electrochemical DNA-Based Sensors
| Autor: | Ryan J. White, Richard L. Karpel, Florika C. Macazo |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Nanotechnology
Cooperativity Biosensing Techniques 010402 general chemistry 01 natural sciences Article 03 medical and health sciences Electron transfer chemistry.chemical_compound Electrochemistry Molecule General Materials Science Spectroscopy 030304 developmental biology 0303 health sciences Oligonucleotide Hybridization probe Cooperative binding DNA Surfaces and Interfaces Condensed Matter Physics Small molecule 0104 chemical sciences DNA-Binding Proteins chemistry DNA Probes |
| Zdroj: | Langmuir |
| ISSN: | 1520-5827 0743-7463 |
| Popis: | Electrochemical DNA-based (E-DNA) sensors are utilized to detect a variety of targets including complementary DNA, small molecules, and proteins. These sensors typically employ surface-bound single-stranded oligonucleotides that are modified with a redox-active molecule on the distal 3′ terminus. Target-induced flexibility changes of the DNA probe alter the efficiency of electron transfer between the redox active methylene blue and the electrode surface, allowing for quantitative detection of target concentration. While numerous studies have utilized the specific and sensitive abilities of E-DNA sensors to quantify target concentration, no studies to date have demonstrated the ability of this class of collision-based sensors to elucidate biochemical-binding mechanisms such as cooperativity. In this study, we demonstrate that E-DNA sensors fabricated with various lengths of surface-bound oligodeoxythymidylate [(dT)n] sensing probes are able to quantitatively distinguish between cooperative and noncooperative binding of a single-stranded DNA-binding protein. Specifically, we demonstrate that oligo(dT) E-DNA sensors are able to quantitatively detect nM levels (50 nM–4 μM) of gene 32 protein (g32p). Furthermore, the sensors exhibit signal that is able to distinguish between the cooperative binding of the full-length g32p and the noncooperative binding of the core domain (*III) fragment to single-stranded DNA. Finally, we demonstrate that this binding is both probe-length- and ionic-strength-dependent. This study illustrates a new quantitative property of this powerful class of biosensor and represents a rapid and simple methodology for understanding protein–DNA binding mechanisms. |
| Databáze: | OpenAIRE |
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