(241g) Molecular Interactions in DNA and Morpholino Surface Hybridization

Surface hybridization is widely encountered in diagnostic technologies for genotyping and gene expression analysis. In order to better understand the underlying fundamental processes, we compare two situations: (1) immobilized DNA oligonucleotides hybridizing to ones free in solution and (2) immobilized morpholino oligonucleotides hybridizing to free DNA oligonucleotides. Morpholinos, which obey standard base-pairing behavior, have a redesigned backbone chemistry that is charge-neutral. The influence of backbone charge is assessed from "two-color" hybridization electrochemical experiments in which surface populations of immobilized "probe" and hybridizing "target" species are simultaneously monitored. Hybridization thermodynamics in solution are analyzed for comparison. Qualitatively different behavior is encountered for the two scenarios of DNA or morpholino probes. For morpholino-DNA surface hybridization, probe coverage is a weak variable. As probe coverage increases, after an initial rise in target coverage due to elevation in surface binding capacity, hybridization becomes nearly independent of probe coverage. In contrast, DNA-DNA hybridization is highly sensitive to the immobilized probe, with strong suppression at high probe coverages. Both types of surface hybridization exhibit strong dependence on salt concentration; in the morpholino-DNA case this sensitivity is attributed to accumulation of repulsive interactions between hybridized target strands. Interestingly, morpholino-DNA hybridization in solution is independent of salt concentration in the range from deionized water to 1 M buffer. Comparison of experimental data with physical models incorporating electrostatic and associative strand interactions suggests influence of attractive strand-strand and strand-surface interactions.