Nucleic Acids Research, Vol 27, Issue 20 4100-4105, Copyright © 1999 by Oxford University Press
AL Drobyshev, AS Zasedatelev, GM Yershov and AD Mirzabekov
A generic oligodeoxyribonucleotide microchip was used to determine the
sequence specificity of Hoechst 33258 binding to double-stranded DNA. The
generic microchip contained 4096 oxctadeoxynucleo-tides in which all
possible 4(6)= 4096 hexadeoxy-nucleotide sequences are flanked on both the
3'- and 5'-ends with equimolar mixtures of four bases. The microchip was
manufactured by chemical immobilization of presynthesized 8mers within
polyacrylamide gel pads. A selected set of immobilized 8mers was converted
to double-stranded form by hybridization with a mixture of fluorescently
labeled complementary 8mers. Massive parallel measurements of melting
curves were carried out for the majority of 2080 6mer duplexes, in both the
absence and presence of the Hoechst dye. The sequence-specific affinity for
Hoechst 33258 was calculated as the increase in melting temperature caused
by ligand binding. The dye exhibited specificity for A:T but not G:C base
pairs. The affinity is low for two A:T base pairs, increases significantly
for three, and reaches a plateau for four A:T base pairs. The relative
ligand affinity for all trinucleotide and tetranucleotide sequences
(A/T)(3)and (A/T)(4)was estimated. The free energy of dye binding to
several duplexes was calculated from the equilibrium melting curves of the
duplexes formed on the oligonucleotide microchips. This method can be used
as a general approach for massive screening of the sequence specificity of
DNA-binding compounds.
ARTICLES
Massive parallel analysis of DNA-Hoechst 33258 binding specificity with a generic oligodeoxyribonucleotide microchip
Joint Human Genome Program, Russian Academy of Sciences, Moscow, Russia.
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