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While designing oligonucleotide-based microarrays, cross-hybridizationbetween surface-bound oligos and non-intended labeled targets is probably themost difficult parameter to predict. Although literature describesrules-of-thumb concerning oligo length, overall similarity, and continuousstretches, the final behavior is difficult to predict. The aim of this studywas to investigate the effect of well-defined mismatches on hybridizationspecificity using CodeLink Activated Slides, and to study quantitatively therelation between hybridization intensity and Gibbs free energy (Delta G),taking the mismatches into account. Our data clearly showed a correlationbetween the hybridization intensity and Delta G of the oligos over three ordersof magnitude for the hybridization intensity, which could be described by theLangmuir model. As Delta G was calculated according to the nearest-neighbormodel, using values related to DNA hybridizations in solution, this studyclearly shows that target-probe hybridizations on microarrays with athree-dimensional coating are in quantitative agreement with the correspondingreaction in solution. These results can be interesting for some practicalapplications. The correlation between intensity and Delta G can be used inquality control of microarray hybridizations by designing probes andcorresponding RNA spikes with a range of Delta G values. Furthermore, thiscorrelation might be of use to fine-tune oligonucleotide design algorithms in away to improve the prediction of the influence of mismatching targets onmicroarray hybridizations.

Thermodynamic behavior of short oligonucleotides in microarray hybridizations can be described using Gibbs free energy in a nearest-neighbor model