Electrochemical probe for the monitoring of DNA-protein interactions.
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.
Meunier-Prest
Rita
R
Institut de Chimie Moléculaire de l'Université de Bourgogne, UMR CNRS 5260, Dijon, France. rita.meunier-prest@u-bourgogne.fr
Bouyon
Alice
A
Rampazzi
Eve
E
Raveau
Suzanne
S
Andreoletti
Pierre
P
Cherkaoui-Malki
Mustapha
M
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D002417:Cattle; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D055664:Electrochemical Techniques / Q000379:methods*; D006046:Gold; D066298:In Vitro Techniques; D008751:Methylene Blue; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000737:chemistry*; D012710:Serum Albumin, Bovine; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D004279:DNA, Viral; D011506:Proteins; D014764:Viral Proteins; D012710:Serum Albumin, Bovine; D006046:Gold; D004247:DNA; C081591:bacteriophage T3 RNA polymerase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D008751:Methylene Blue