Recently, Steitz and colleagues have proposed that protein-induced bending helps facilitate the DNA proofreading process by DNA polymerases. The applicants reinterpret that observation to hypothesize that DNA base pairing selectivity is actually greater out of the bent form of a DNA duplex than in its linear (unstressed) form. They propose to design and reduce to practice a chemical approach to DNA hybridization analysis which mimics the natural process of selectivity enhancement by bending. They propose to design and synthesize nucleic acid probes which are modified with a pair of side chains which, after binding their cognate DNA or RNA target and crosslinking of the side chains, will trap the duplex into its bent helix form. In a two step binding assay, probe will be allowed to bind to the target-modified microtitre surface, followed by a chemical crosslinking step to link the probe side chain pair. Crosslinking causes the probe to be linked to the surface (through the target) as a pseudoknot and therefore confers resistance to extreme washing conditions. In that way, labeled probe binding to the surface will be detected after stringent washing only if both duplex formation and probe-mediated crosslinking had occurred. Based upon the proposed work, a Phase II application will be formulated to develop this chemistry into a set of tools for nucleic acid hybridization enhancement, especially as part of DNA microarray technology for human diagnostics and population based screening.
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