Preparation and characterization of self-assembled double-stranded DNA (dsDNA) microarrays for protein : dsDNA screening using atomic force microscopy

We report details on the development of a self-assembled, double-stranded D NA (dsDNA) microarray fabrication strategy suitable for protein:dsDNA scree ning using the atomic force microscope (AFM). Using disulfide-modified dsDN A (26-mer) synthesized to contain the recognition sequence for ECoR1, we ha ve created micron-sized mixed monolayer surfaces where both the spatial ori entation and packing density of the immobilized oligonucleotides, two criti cal parameters for screening protein:dsDNA interactions, are controlled. Be fore exposure to ECoR1, the topography of microarrays that were composed of 26-mers containing the recognition sequence for EcoR1 was 8.8 nm +/- 1.5 n m (n = 5), a value consistent with that predicted by X-ray diffraction stud ies. After enzyme digestion, the topography of the microarray decreased to 4.3 nm +/- 0.8 nm (n = 14), a value consistent with predictions based on th e position of the recognition sequence within the oligonucleotides. In cont rast, the topography of microarrays that were composed of 26-mers that did not contain the recognition sequence for ECoR1 remained essentially the sam e before (8.9 nm +/- 1.5 nm (n = 5)) and after (8.3 nm +/- 1.4 nm (n = 5)) exposure to ECoR1. Furthermore, because the dsDNA were synthesized to inclu de a fluorescein moiety above the recognition sequence, the loss of fluores cence after exposure to ECoR1 was also used to detect enzymatic cleavage. W e believe that this technology holds promise as a tool for the rapid and fa cile screening of multiple protein interactions using massively parallel ds DNA microarrays.