Rebalancing Surface Hybridization Thermodynamics

Nucleic acid hybridization on solid supports is often less favorable than that in solution. An unfavorable ranking of surface relative to solution affinities is a complicating factor in applications where the surface reaction must compete with double-stranded structure in a solution analyte. Here, a thermodynamic comparison is undertaken to identify factors that govern selectivity for surface relative to solution hybridization. Free energies of hybridization, on solid supports and in solution, are derived from melting data and compared as a function of temperature, salt concentration, and type of immobilized probe - either DNA or a morpholino nucleic acid mimic with an uncharged backbone. Selectivity for the surface reaction improves at elevated temperatures for both probe types. In addition, ionic strength provides useful control over selectivity for the surface reaction when morpholino probes are used. While surface environments also impose penalties on duplex formation not encountered in solution, it is shown that proper choice of experimental conditions can overcome this disadvantage.

Acknowledgments

This work was supported by the National Institutes of Health (NHGRI  R01HG004512), the National Science Foundation (DMR 12-06754), and by New York University.