Investigations of the Effect of Overhang and Strand Length on DNA Hybridization at the Solid-Liquid Interface

Many biotechnologies and assays, such as sequence detection and bridge PCR, rely on interactions between mobile DNA analytes or targets and surface chemistries and features to perform their desired functions. Single-molecule techniques allow for investigations of large statistical samples of individual molecular interactions to gain a more fundamental understanding behind what is causing behaviors in a bulk system. This work looks specifically at DNA hybridization at the solid-liquid interface, where Total Internal Reflection Fluorescence (TIRF) microscopy allows us to look at the behavior of single-stranded oligonucleotides. This information elucidates how different degrees of complementarity affect hybridization and searching behaviors as indicated by Fӧrster Resonance Energy Transfer (FRET) signals, allowing us to observe when two strands are in close proximity to each other. Determining the fundamental processes via this analysis results in better sensing behavior, such as stronger signals on a detector for gene sequencing, mutation detection, and genetic mapping applications.

This study looks specifically at the effect of strand length and the appearance of overhang on hybridization mechanisms and two-dimensional surface diffusion. DNA targets in solution at 100 pM were flown across a solid interface with covalently attached complementary strands. Experiments were performed with systems designed to create short (15bp) duplexes, long (30bp) duplexes, or a partially complementary system where a 30-base target hybridizes with a 15-base probe, introducing a 15-base overhang. Analyses of association lifetimes show that a majority of short associations with long-lived distribution tails, indicative of different melting modes. The longest duplexes maintain longer lifetimes, as expected due to higher stability and more Watson-Crick base pair bonding. The presence of a toehold, or overhang, on the duplex decreases association lifetimes. This suggests exposed and unhybridized DNA nucleobases increase the target’s attraction to the surface, stabilizing the transition from the hybridized to a surface state. The long-lived events appear to have 1^st-order dissociation behavior, indicating that melting is initiated by a nucleation event before undergoing complex unzipping behavior. This gives further insight into the mechanisms within surface-mediated DNA hybridization systems, so that devices relying on these interactions in the future can be designed to give stronger signals and more stable hybridizations to provide their desired functions.