Single-Molecule Fluorescence Imaging of Interfacial
DNA Hybridization Kinetics at Selective Capture Surfaces
Version 3 2017-12-16, 00:30
Version 2 2016-02-11, 19:13
Version 1 2016-02-11, 19:13
Posted on 2017-12-16 - 00:30
Accurate
knowledge of the kinetics of complementary oligonucleotide
hybridization is integral to the design and understanding of DNA-based
biosensors. In this work, single-molecule fluorescence imaging is
applied to measuring rates of hybridization between fluorescently
labeled target ssDNA and unlabeled probe ssDNA immobilized on glass
surfaces. In the absence of probe site labeling, the capture surface
must be highly selective to avoid the influence of nonspecific adsorption
on the interpretation of single-molecule imaging results. This is
accomplished by increasing the probe molecule site densities by a
factor of ∼100 compared to optically resolvable sites so that
nonspecific interactions compete with a much greater number of capture
sites and by immobilizing sulfonate groups to passivate the surface
between probe strands. The resulting substrates exhibit very low nonspecific
adsorption, and the selectivity for binding a complementary target
sequence exceeds that of a scrambled sequence by nearly 3 orders of
magnitude. The population of immobilized DNA probe sites is quantified
by counting individual DNA duplexes at low target concentrations,
and those results are used to calibrate fluorescence intensities on
the same sample at much higher target concentrations to measure a
full binding isotherm. Dissociation rates are determined from interfacial
residence times of individual DNA duplexes. Equilibrium and rate constants
of hybridization, Ka = 38 (±1) μM–1, kon = 1.64 (±0.06)
× 106 M–1 s–1,
and koff = 4.3 (±0.1) × 10–2 s–1, were found not to change with
surface density of immobilized probe DNA, indicating that hybridization
events at neighboring probe sites are independent. To test the influence
of probe-strand immobilization on hybridization, the kinetics of the
probe target reaction at the surface were compared with the same reaction
in free solution, and the equilibrium constants and dissociation and
association rates were found to be nearly equivalent. The selectivity
of these capture surfaces should facilitate sensitive investigations
of DNA hybridization at the limit of counting molecules. Because the
immobilized probe DNA on these surfaces is unlabeled, photobleaching
of a probe label is not an issue, allowing capture substrates to be
used for long periods of time or even reused in multiple experiments.
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Peterson, Eric M.; Manhart, Michael
W.; Harris, Joel M. (2016). Single-Molecule Fluorescence Imaging of Interfacial
DNA Hybridization Kinetics at Selective Capture Surfaces. ACS Publications. Collection. https://doi.org/10.1021/acs.analchem.5b03832
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AUTHORS (3)
EP
Eric M. Peterson
MM
Michael
W. Manhart
JH
Joel M. Harris
KEYWORDS
probe DNAprobe molecule site densitieshybridizationInterfacial DNA Hybridization Kineticssingle-molecule fluorescence imagingprobe target reactiontarget concentrationsSingle-Molecule Fluorescence ImagingDNA probe sitesimmobilizing sulfonate groupscalibrate fluorescence intensitiessingle-molecule imaging resultssurfaceDNA duplexesSelective Capture Surfaces