(659d) Specific and Nonspecific Oligonucleotide Interfacial Dynamics On Modified Silica Surfaces

Single-molecule total internal reflection fluorescence microscopy was used to observe the dynamic behavior of ssDNA oligonucleotides on modified silica surfaces.  High throughput molecular tracking was used to determine >75,000 molecular trajectories. Nonspecific interactions occur where the oligonucleotides interact with a surface lacking the oligonucleotide’s compliment strand covalently bound to the surface. These interactions were monitored using total internal reflection fluorescence microscopy (TIRF) and labeled probe oligonucleotides in solution. Specific oligonucleotide interactions occur when target molecules in solution hybridize to probe molecules attached to the surface. These interactions were monitored using labeled probe and target oligonucleotides, where the labels are a resonance energy transfer (RET) pair and two-channel TIRF. The use of RET provides a signal when a molecule transitions to a hybridized state. Using the molecular trajectories acquired through RET TIRF experiments, the rate constants of molecules transitioning between hybridized and melted states, as well as the fraction of molecules in a specific state that transition to each potential state, can be extracted.