Abstract
We describe a microarray format that can detect double-stranded DNA sequences with a high degree of sequence selectivity. Cyclooctyne-derivatized pyrrole-imidazole polyamides were immobilized on azide-modified glass substrates using microcontact printing and a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. These polyamide-immobilized substrates selectively detected a seven-base-pair binding site incorporated within a double-stranded oligodeoxyribonucleotide sequence even in the presence of an excess of a sequence with a single-base-pair mismatch.
Highlights
Recent innovations in microarray technology have extended their present capability to include the assessment of recognition profiles of transcription factors[9,10] and small molecules[11−13] in a parallel and high-throughput fashion using double-stranded DNA probe sequences immobilized on a solid substrate.[11−15] Detection of a dsDNA binding event is achieved either by a reporter directly attached to the DNA-binding molecule[12,13] or via a fluorescently tagged antibody specific for the transcription factor of interest.[9]
A strain-promoted azide−alkyne cycloaddition (SPAAC) protocol[20,27−29] was adopted for the immobilization of PA (1) ink onto an azide substrate, as it dispels the need for copper(I) catalysis as required in conventional copper-catalyzed Huisgen [3 + 2] azide−alkyne cycloaddition (CuAAC).[30,31]
A tetraethylene glycol tether was installed between the cyclooctyne unit and the PA core to impart water solubility and endow PA (1) with sufficient flexibility to access optimal binding conformations with its corresponding dsDNA target binding site
Summary
Recent innovations in microarray technology have extended their present capability to include the assessment of recognition profiles of transcription factors[9,10] and small molecules[11−13] in a parallel and high-throughput fashion using double-stranded DNA (dsDNA) probe sequences immobilized on a solid substrate.[11−15] Detection of a dsDNA binding event is achieved either by a reporter directly attached to the DNA-binding molecule[12,13] or via a fluorescently tagged antibody specific for the transcription factor of interest.[9] Both of these microarray formats utilize immobilized dsDNA oligodeoxyribonucleotides (ODNs) to probe the dsDNA binding characteristics of the DNA binder present in solution. To the best of our knowledge, a microarray setup that examines the binding affinity and selectivity of dsDNA-binding molecules where dsDNA is the analyte present in solution has not been reported
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