Synthetic nucleic acid mimics provide opportunity for redesigning the specificity and affinity of hybridization with natural DNA or RNA. Such redesign is of great interest for diagnostic applications where it can enhance the desired signal against a background of competing interactions. This report compares hybridization of DNA analyte strands with morpholinos (MOs), which are uncharged nucleic acid mimics, to the corresponding DNA-DNA case in solution and on surfaces. In solution, MO-DNA hybridization is found to be independent of counterion concentration, in contrast to DNA-DNA hybridization. On surfaces, when immobilized MO or DNA “probe” strands hybridize with complementary DNA “targets” from solution, both the MO-DNA and DNA-DNA processes depend on ionic strength but exhibit qualitatively different behaviors. At lower ionic strengths, MO-DNA surface hybridization exhibits hallmarks of kinetic limitations when separation between hybridized probe sites becomes comparable to target dimensions, whereas extents of DNA-DNA surface hybridization are instead consistent with limits imposed by buildup of surface (Donnan) potential. The two processes also fundamentally differ at high ionic strength, under conditions when electrostatic effects are weak. Here, variations in probe coverage have a much diminished impact on MO-DNA than on DNA-DNA hybridization for similarly crowded surface conditions. These various observations agree with a structural model of MO monolayers in which MO-DNA duplexes segregate to the buffer interface while unhybridized probes localize near the solid support. A general perspective is presented on using uncharged DNA analogues, which also include compounds such as peptide nucleic acids (PNA), in surface hybridization applications.
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