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Nucleic Acids Research Advance Access published online on May 12, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn246
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Gel-based oligonucleotide microarray approach to analyze protein–ssDNA binding specificity

Olga A. Zasedateleva*, Andrey L. Mikheikin, Alexander Y. Turygin, Dmitry V. Prokopenko, Alexander V. Chudinov, Elena E. Belobritskaya, Vladimir R. Chechetkin and Alexander S. Zasedatelev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, 119991 Moscow, Russian Federation

*To whom correspondence should be addressed. Tel: +7 495 1359980; Fax: +7 495 1351405; Email: lana{at}biochip.ru

Received August 6, 2007. Revised March 7, 2008. Accepted April 16, 2008.

Gel-based oligonucleotide microarray approach was developed for quantitative profiling of binding affinity of a protein to single-stranded DNA (ssDNA). To demonstrate additional capabilities of this method, we analyzed the binding specificity of ribonuclease (RNase) binase from Bacillus intermedius (EC 3.1.27.3) to ssDNA using generic hexamer oligodeoxyribonucleotide microchip. Single-stranded octamer oligonucleotides were immobilized within 3D hemispherical gel pads. The octanucleotides in individual pads 5'-{N}N1N2N3N4N5N6{N}-3' consisted of a fixed hexamer motif N1N2N3N4N5N6 in the middle and variable parts {N} at the ends, where {N} represent A, C, G and T in equal proportions. The chip has 4096 pads with a complete set of hexamer sequences. The affinity was determined by measuring dissociation of the RNase–ssDNA complexes with the temperature increasing from 0°C to 50°C in quasi-equilibrium conditions. RNase binase showed the highest sequence-specificity of binding to motifs 5'-NNG(A/T/C)GNN-3' with the order of preference: GAG > GTG > GCG. High specificity towards G(A/T/C)G triplets was also confirmed by measuring fluorescent anisotropy of complexes of binase with selected oligodeoxyribonucleotides in solution. The affinity of RNase binase to other 3-nt sequences was also ranked. These results demonstrate the applicability of the method and provide the ground for further investigations of nonenzymatic functions of RNases.


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