Comprehensive and parallel thermodynamic characterization of DNA/DNA and LNA-DNA/DNA duplexes on microarrays

Deoxyribonucleic acid (DNA) microarrays have become a tool of choice for genomic material analyses and monitoring biomolecular interactions. Indeed, they contain hundreds to tens of thousands of DNA sequences (probes) either immobilized or synthesized at specific positions on silicon-based supports, allowing large scale parallel screenings. Despite the numerous publications related to microarrays, the factors modulating the recognition specificity are still not completely understood. Thorough characterization of these factors is essential for improving the microarray experimental designs.; In this work, arrays of probes containing both DNA and locked nucleic acid (LNA) oligonucleotides were created using a microchip synthesis technology that provides unprecedented flexibility in the sequences displayed. Based on a design allowing experiments to be performed in parallel, 640 DNA probes and 1344 LNA-DNA probes were synthesized on the microchips. Subsequently, the binding affinities of these DNA/DNA and LNA-DNA/DNA duplexes were thoroughly characterized by monitoring real time associations/dissociations of fluorescently labeled DNA targets as a function of temperature. Programs were written in Visual Basic to automate the data treatment. Melting temperatures and thermodynamic parameters were derived based on a two-state transition model. These results provide the first comprehensive set of microarrays DNA/DNA and LNA-DNA/DNA duplexes thermodynamic parameters, for all perfect matched and mismatched base pairs, with all nearest-neighbors (XYZ), at three positions (5 ', middle, 3').; This investigation revealed that for DNA/DNA duplexes, mismatches located toward the ends of the probes were less destabilized than those located in the middle. As observed in solution, the "gx" and "cx" mismatches were the most and least stable, respectively. However, the "gx" mismatches were more destabilized than expected, which, along with the lack of nearest-neighbors effect, was attributed to a loss of stacking interactions. The presence of LNA significantly stabilized perfect matches, but not mismatches, regardless of the substitution position. Therefore, the discrimination between perfect match and mismatches was improved, especially in presence of LNA pyrimidines. This improvement was not proportional to the number of LNA and tends to saturate. The results also showed that alternate substitutions were as effective as consecutive substitutions. This knowledge will be useful to improve the design of specific probes for microarrays applications.