ABSTRACT
Self-assembly of thiol-terminated oligonucleotides on gold substrates provides a convenient way for DNA-functionalized surfaces. Here we describe the development of an electrochemical assay for the detection of DNA-protein interactions based on the modification of the electrochemical response of methylene blue (MB) intercalated in the DNA strands. Using a functionalized electrode with double stranded DNA carrying T3 RNA polymerase binding sequence, we show a substantial attenuation of the current upon the DNA-protein interaction. Moreover, a Langmuir binding isotherm for T3 RNA polymerase (T3 Pol) gives a dissociation constant K(D) equal to 0.46+/-0.23 microM. Such value is 100 times lower than the calculated K(D) for the non-specific interaction of bovine serum albumin (BSA) with T3 Pol promoter. In addition, the use of the T7 RNA polymerase (T7 Pol) promoter instead of the T3 Pol promoter induces an increase of K(D) from 0.46 microM to more than 25 microM. Accordingly, this strong decrease in the affinity of T3 Pol towards an off-target DNA promoter reveals an electrochemical sequence-specific discrimination of DNA-protein interactions. In conclusion, our results show that the developed electrochemical test allows the monitoring of DNA-protein interactions with high specificity and with an in situ protein detection threshold at a nanomolar range.
Subject(s)
Biosensing Techniques/methods , DNA/chemistry , Electrochemical Techniques/methods , Proteins/chemistry , Animals , Base Sequence , Binding Sites/genetics , Cattle , DNA/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Gold , In Vitro Techniques , Methylene Blue , Promoter Regions, Genetic , Serum Albumin, Bovine , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
The development of biosensors based on DNA hybridization requires a more precise knowledge of the thermodynamics of the hybridization at a solid interface. In particular, the selectivity of hybridization can be affected by a lot of parameters such as the single-strand (ss)DNA density, the pH, the ionic strength or the temperature. The melting temperature, T(m), is in part a function of the ionic strength and of the temperature and therefore provides a useful variable in the control of the selectivity and sensitivity of a DNA chip. The electrochemical technique has been used to determine the T(m) values when the probe is tethered by a DNA self-assembled monolayer (SAM). We have built a special thin layer cell, which allows the recording of the cyclic voltammogram under controlled temperature conditions. T(m) has been determined by recording the thermogram (current versus temperature) of a redox indicator on a double-stranded hybrid (dsDNA) modified electrode and comparison with the corresponding ssDNA response. T(m) of supported DNA varies linearly with the ionic strength. The stability of the SAMs has been considered and comparison between T(m) in solution and on a solid support has been discussed.
