Fabrication of Double-Stranded DNA Microarray on Solid Surface for Studying DNA-Protein Interactions

Abstract

This paper presents two novel methods for fabricating double-stranded DNA (dsDNA) microarray. In the first method, the presynthesized single-stranded DNA (ssDNA) oligonucleotides containing two reverse complementary sequences at their 3′ hydroxyl end were firstly immobilize on the surface of the aldehyde-derivatized glass slides by their 5′ end, and then the two reverse complementary sequences were annealed to form a short dsDNA hairpin structure which provided the primer for later polymerase elongation. Finally, the ssDNA microarrays were converted into the unimolecular dsDNA microarrays by an on-chip polymerase reaction. In the second method, the two kinds of ssDNA oligonucleotides named constant oligonucleotide (CO) and target oligonucleotides (TOs) were synthesized. Then the different TOs harboring the DNA-binding sites were respectively annealed and ligated with the same CO containing an internal aminated dT in tubes. The reaction products were immobilized on the surface of the aldehyde-derivatized glass slides by the aminated dT to fabricate the partial-dsDNA microarrays. Finally, the partial-dsDNA microarrays were converted into the unimolecular dsDNA microarrays by an on-chip polymerase reaction. The excellent efficiency and high accuracy of the enzymatic synthesis in two methods were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and the digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3 labeled NF-κB (p50) to dsDNA microarrays. Therefore, the dsDNA microarray containing 66 probes representing 30 all-possible single-nucleotide mutant NF-κB binding targets of Ig-κB and 36 wild-type NF-κB binding targets were fabricated to determine the binding affinities of NF-κB homodimer p50 to all probes on chip. We found the binding results were very consistent with that from x-ray crystallography studies and gel mobility-shift analysis. The unimolecular dsDNA microarray has great potentials to provide a high-throughput platform for investigating the sequence-specific DNA-protein interactions involved in gene expression regulation, restriction and so on.