Mechanisms of surface-mediated DNA hybridization.
Single-molecule total internal reflection fluorescence microscopy was employed in conjunction with resonance energy transfer (RET) to observe the dynamic behavior of donor-labeled ssDNA at the interface between aqueous solution and a solid surface decorated with complementary acceptor-labeled ssDNA. At least 100,000 molecular trajectories were determined for both complementary strands and negative control ssDNA. RET was used to identify trajectory segments corresponding to the hybridized state. The vast majority of molecules from solution adsorbed nonspecifically to the surface, where a brief two-dimensional search was performed with a 7% chance of hybridization. Successful hybridization events occurred with a characteristic search time of ∼0.1 s, and unsuccessful searches resulted in desorption from the surface, ultimately repeating the adsorption and search process. Hybridization was reversible, and two distinct modes of melting (i.e., dehybridization) were observed, corresponding to long-lived (∼15 s) and short-lived (∼1.4 s) hybridized time intervals. A strand that melted back onto the surface could rehybridize after a brief search or desorb from the interface. These mechanistic observations provide guidance for technologies that involve DNA interactions in the near-surface region, suggesting a need to design surfaces that both enhance the complex multidimensional search process and stabilize the hybridized state.
Department of Chemical and Biological Engineering, University of Colorado Boulder , Boulder, Colorado 80309, United States.
Jon H
JH
Monserud
Daniel K
DK
Schwartz
D000327:Adsorption; D004247:DNA / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004735:Energy Transfer; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009693:Nucleic Acid Hybridization; D013499:Surface Properties; D014732:Vibration
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D004247:DNA