(180f) Effects of Inhibited Oligonucleotide Transport on Interfacial Hybridization and Molecular Searching

Multiple biotechnologies rely on the hybridization of mobile DNA oligonucleotides in solution with complementary DNA strands covalently attached to a solid surface. The surface grafting density of immobilized strands has been shown to have strong and complex effects on hybridization kinetics, affecting the function of DNA sensing. Single-molecule fluorescence microscopy is ideal for observing molecular adsorption and anomalous transport on a DNA surface, allowing us to identify heterogeneous searching behaviors that lead to hybridization. We use FÓ§rster Resonance Energy Transfer (FRET) to identify association events between complementary strands, which allows us to observe how transport and molecular searching behaviors influence surface-mediated hybridization. We introduced mobile DNA in aqueous solution to a surface of immobilized complementary strands across a wide range of surface grafting densities. We observed two regimes of distinctly different transport and hybridization behavior across these grafting density ranges. An increase in grafting density resulted in an increase in the rate of hybridization on the surface, where an estimated grafting density of 10⁴ µm^-2 represented a threshold value dividing the two grafting density regimes. Successful molecular search distances comprised of heterogeneous short- and long-range populations at low grafting density, while increases in grafting density resulted in a systematic decrease in this long-range population. Transport for both desorption-mediated diffusion and short-range “crawling” diffusion was also inhibited at high grafting density, suggesting that immobilized DNA on the surface acts as an obstacle to mobile oligonucleotides. These findings suggest that oligonucleotide transport is an important factor for hybridization on a solid-liquid interface.