The advancement of nonviral gene therapy hinges on the ability to exert highly specific spatial and temporal control of gene delivery systems to enable localized release of DNA. In this work, we have developed a system capable of promoting localized delivery of a plasmid by utilizing peptide nucleic acid (PNA) technology to bind DNA to a substrate via an enzymatically labile peptide sequence. The successful immobilization of the DNA to the model substrate as well as the specificity of the binding was confirmed with atomic force microscopy (AFM) and AFM-confocal overlay imaging. Fluorescence-based quantification revealed that surfaces treated with the conjugates had 49 +/- 22 ng of DNA/cm(2), while there were 4.2 +/- 2.1 ng of DNA/cm(2) on surfaces treated with unfunctionalized DNA. When NIH/3T3 cells were grown on the modified substrates, a significantly higher percentage of cells were transfected when the peptide tether was protease sensitive as compared with when it was not labile. These results indicated that the peptide must be cleaved to release the DNA In addition to providing cell-triggered release, this system decouples the properties of the complexation agent and the substrate from the method of immobilization/release to provide a model system that can be tailored to specific applications.