Preparation and Characterization of Self-Assembled Double-Stranded DNA (dsDNA) Microarrays for Protein:dsDNA Screening Using Atomic Force Microscopy

Abstract

We report details on the development of a self-assembled, double-stranded DNA (dsDNA) microarray fabrication strategy suitable for protein:dsDNA screening using the atomic force microscope (AFM). Using disulfide-modified dsDNA (26-mer) synthesized to contain the recognition sequence for ECoR1, we have created micron-sized mixed monolayer surfaces where both the spatial orientation and packing density of the immobilized oligonucleotides, two critical parameters for screening protein:dsDNA interactions, are controlled. Before 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 nm (n = 5), a value consistent with that predicted by X-ray diffraction studies. 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 the position of the recognition sequence within the oligonucleotides. In contrast, the topography of microarrays that were composed of 26-mers that did not contain the recognition sequence for ECoR1 remained essentially the same 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 include a fluorescein moiety above the recognition sequence, the loss of fluorescence after exposure to ECoR1 was also used to detect enzymatic cleavage. We believe that this technology holds promise as a tool for the rapid and facile screening of multiple protein interactions using massively parallel dsDNA microarrays.