Interactions in Oligonucleotide Hybrid Duplexes on Microarrays

We investigated Affymetrix GeneChip intensity data in terms of chip-averaged sensitivities over all perfect match (PM) and mismatch (MM) probes possessing a common triple of neighboring bases in the middle of their sequence. This approach provides a model-independent estimation of base-specific contributions to the probe sensitivities. We found that fluorescent labels attached to nucleotide bases forming Watson−Crick (WC) pairs in most cases decrease their binding affinity and, thus, decrease the sensitivity of the probe. Single-base-related mean sensitivity values rank in ascending order according to C > G ≈ T > A. The central base of PM and MM probes mainly forms WC pairings in duplexes with nonspecific transcripts, which obviously dominate the chip-averaged sensitivity values. Linear combinations of the triple-averaged probe sensitivities provide nearest-neighbor (NN) sensitivity terms, which rank in a similar order as the respective NN free-energy terms obtained from previous thermodynamic studies on the stability of RNA/DNA duplexes in solution. Systematic deviations between both data sets can be mostly attributed to the labeling of the target RNA in the chip experiments. Our results provide a set of molecular NN and single-base-related interaction parameters which consider specific properties of duplex formation in microarray hybridization experiments.