Parallel multiplex thermodynamic analysis of coaxial base stacking in DNA duplexes by oligodeoxyribonucleotide microchips

Parallel thermodynamic analysis of the coaxial stacking effect of two bases localized in one strand of DNA duplexes has been performed. Oligonucleotid es were immobilized in an array of three-dimensional polyacrylamide gel pad s of microchips (MAGIChips (TM)). The stacking effect was studied for all c ombinations of two bases and assessed by measuring the increase in melting temperature and in the free energy of duplexes formed by 5mers stacked to m icrochip-immobilized 10mers, For any given interface, the effect was studie d for perfectly paired bases, as well as terminal mismatches, single base o verlaps, single and double gaps, and modified terminal bases, Thermodynamic parameters of contiguous stacking determined by using microchips closely c orrelated with data obtained in solution, The extension of immobilized olig onucleotides with 5,6-dihydroxyuridine, a urea derivative of deoxyribose, o r by phosphate, decreased the stacking effect moderately, while extension w ith FITC or Texas Red virtually eliminated stacking. The extension of the i mmobilized oligonucleotides with either acridine or 5-nitroindole increased stacking to mispaired bases and in some GC-rich interfaces. The measuremen ts of stacking parameters were performed in different melting buffers. Alth ough melting temperatures of AT- and CC-rich oligonucleotides in 5 M tetram ethylammonium chloride were equalized, the energy of stacking interaction w as significantly diminished.