DNA microarrays have become an invaluable high throughput biotechnology method, which allows a parallel investigation of thousands of cellular events in a single experiment. The principle behind the technology is very simple: fluorescently labeled single stranded target molecules bind to their specific probes deposited on the microarray surface. However, the microarray data rarely represent a yes or no answer to a biological community, but rather provide a direction for further investigation. There is a complicated quantitative relationship between a detected spot signal and the amount of target present in the unknown mixture. We hypothesize that physical characteristics of probe and target molecules complicate the binding reaction between target and probe. To test this hypothesis, we designed a controlled microarray experiment in which the amount and stability of the secondary structure present in the probe-binding regions of target as biophysical properties of nucleic acids varies in a known way. Based on computational simulations of hybridization, we hypothesize that secondary structure formation in the target can result in considerable interference with the process of probe-target binding. This interference will have the effect of lowering the spot signal intensity. We simulated hybridization between probe and target and analyzed the simulation data to predict how much the microarray signal is affected by folding of the target molecule, for the purpose of developing a new generation of microarray design and analysis software.
