A Canvas of Spatially Arranged DNA Strands that Can Produce 24-bit Color Depth

[Image: see text] Nucleic acid microarray photolithography combines density, throughput, and positional control in DNA synthesis. These surface-bound sequence libraries are conventionally used in large-scale hybridization assays against fluorescently labeled, perfect-match DNA strands. Here, we introduce another layer of control for in situ microarray synthesis—hybridization affinity—to precisely modulate fluorescence intensity upon duplex formation. Using a combination of Cy3-, Cy5-, and fluorescein-labeled targets and an ensemble of truncated DNA probes, we organize 256 shades of red, green, and blue intensities that can be superimposed and merged. In so doing, hybridization alone is able to produce a large palette of 16 million colors or 24-bit color depth. Digital images can be reproduced with high fidelity at the micrometer scale by using a simple process that assigns sequence to any RGB value. Largely automated, this approach can be seen as miniaturized DNA-based painting.