Monitoring cooperative binding using electrochemical DNA-based sensors

Abstract

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.

Scheme 1. E-DNA Sensors with Oligodeoxythymidylate [(dT)_n] DNA Probes Exhibit a Decrease in the Current Signal When a Target g32 Protein Is Bound

Oligo(dT) E-DNA sensors comprise a sensing electrode modified with single-stranded, unstructured oligo(dT), [(dT)_n], DNA strands of various lengths (7, 14, and 21 nucleotides) modified at their distal ends with a redox-active methylene blue. Protein binding to the oligo(dT) E-DNA sensor causes a change in the flexibility of the (dT)_n probe; thus, the efficiency with which electrons can be transferred results in a decrease in the measured current (signal-off).

Figure 1

Oligo(dT) E-DNA sensor specifically responds to the presence of gene 32 protein (g32p) in solution. E-DNA sensors modified with (dT)₂₁ probe are employed to detect the presence of g32p. Upon binding of g32p, a decrease in the voltammetric peak current is observed, which is readily measured using square wave voltammetry.

Figure 2

(dT)₂₁ E-DNA sensor does not show any significant nonspecific interactions with the gold electrode surface, double-strand DNA (dsDNA), and non-DNA-binding protein. (Top) A double-strand E-DNA sensor architecture is achieved by adding the complement, (dA)₂₁, and used to test the selectivity and specificity of the single-strand E-DNA sensor. No significant changes in the current signals are observed, which indicates that the sensors are specific to single strand DNA binding. (Bottom) To further test specificity, E-DNA sensors modified with (dT)₂₁ DNA probes are tested against a similarly sized protein biomolecule, thrombin (∼37 kDa), using the same experimental conditions. Again, no significant changes in the current signals are obtained, which strongly demonstrates a highly specific DNA-binding protein binding sensor response.

Figure 3

E-DNA sensors quantitatively detect varying concentrations of gene 32 protein. Sensors modified with (dT)₂₁ probe are used to detect increasing concentrations of g32p. As g32p binds to the (dT)₂₁ E-DNA sensor, a decrease in the voltammetric peak current, expressed as percent signal change, is observed at low salt conditions (20 mM NaCl). Titration at low ionic conditions leads to tight binding of g32p to the DNA-based electrochemical sensor, with an observed binding affinity (K_Dapp) estimated to be about ∼100 nM.

Figure 4

Protein binding and consequently sensor signaling are dependent on both ionic strength and DNA probe length. (Top) Oligo(dT) E-DNA sensors modified with 7, 14, and 21 thymidylate residues are used to evaluate the ionic-strength- and probe-length- dependence of g32p binding. Under low salt conditions, the binding curves for all probe lengths are not significantly different from each other, resulting from electrostatic DNA–protein binding interactions (K_Dapp = ∼100 nM). (Bottom) Under high salt conditions, the binding curves show dependence on DNA probe length because more binding sites per probe exist (2 and 3 for (dT)₁₄ and (dT)₂₁ probe, respectively). A decrease in the apparent affinity is calculated and expected with the onset of cooperative binding. Lines are drawn to guide the reader’s eye.

Figure 5

Oligo(dT) E-DNA sensors distinguish between cooperative binding of full-length g32p and noncooperative binding of the core domain (*III) fragment. Sensors modified with (dT)₇, (dT)₁₄, (dT)₂₁, and (dT)₈₀ DNA probes are utilized to differentiate between cooperative and noncooperative binding, with the full-length g32p and truncated core domain (*III) protein used as targets. At high ionic strength, the full-length g32p cooperatively binds single-strand DNA in a probe-length-dependent manner (black lines), while the *III protein binds tightly and noncooperatively to DNA (gray lines). As seen in the binding curves, the full-length g32p generally exhibits weaker DNA–protein binding interactions relative to *III protein, which is characteristic of cooperativity between protein molecules. Except in the plots showing binding to (dT)₇, which is fit to the Langmuir expression, lines are drawn to guide the reader’s eye.