Nucleic Acids Research, 2001, Vol. 29, No. 11 2303-2313
© 2001 Oxford University Press
Parallel multiplex thermodynamic analysis of coaxial base stacking in DNA duplexes by oligodeoxyribonucleotide microchips
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia, 2Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Russia and 3Biochip Technology Center, Argonne National Laboratory, Argonne, 9700, south Cass Avenue, IL 60439-4833, USA
Parallel thermodynamic analysis of the coaxial stacking effect of two bases localized in one strand of DNA duplexes has been performed. Oligonucleotides were immobilized in an array of three-dimensional polyacrylamide gel pads of microchips (MAGIChips‘). The stacking effect was studied for all combinations of two bases and assessed by measuring the increase in melting temperature and in the free energy of duplexes formed by 5mers stacked to microchip-immobilized 10mers. For any given interface, the effect was studied for perfectly paired bases, as well as terminal mismatches, single base overlaps, single and double gaps, and modified terminal bases. Thermodynamic parameters of contiguous stacking determined by using microchips closely correlated with data obtained in solution. The extension of immobilized oligonucleotides with 5,6-dihydroxyuridine, a urea derivative of deoxyribose, or by phosphate, decreased the stacking effect moderately, while extension with FITC or Texas Red virtually eliminated stacking. The extension of the immobilized oligonucleotides with either acridine or 5-nitroindole increased stacking to mispaired bases and in some GC-rich interfaces. The measurements of stacking parameters were performed in different melting buffers. Although melting temperatures of AT- and GC-rich oligonucleotides in 5 M tetramethylammonium chloride were equalized, the energy of stacking interaction was significantly diminished.
* To whom correspondence should be addressed at: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov strasse 32, 119991 Moscow, Russia. Tel: +7 095 1350559; Fax: +7 095 1351405; Email: amir{at}genome.eimb.relarn.ru
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