Single-molecule fluorescence imaging is a powerful method
to measure
reversible reaction kinetics, allowing one to monitor the bound state
of individual probe molecules with fluorescently labeled targets.
In the case of DNA hybridization, previous studies have shown that
the presence of a fluorescent label on a target strand can exhibit
significant influence on the stability of a DNA duplex that is formed.
In this work, we have developed a super-resolution imaging method
to measure the hybridization kinetics of unlabeled target DNA that
compete with a fluorescently labeled tracer DNA strand to hybridize
with an unlabeled probe DNA immobilized at a surface. The hybridization
of an unlabeled DNA target cannot be detected directly, but its presence
blocks the immobilized probe DNA, influencing the measured time intervals
between labeled DNA hybridization events. We derive a model for competitive
hybridization kinetics to extract the association and dissociation
rate constants of the unlabeled species from the distribution of time
intervals between hybridization events of the labeled tracer DNA at
individual localized DNA probe sites. We use this methodology to determine
the hybridization kinetics of a model 11-mer unlabeled target DNA
strand and then determine how five different fluorescent labels attached
to the same target DNA strand impact the hybridization kinetics. Compared
to the unlabeled target, these labels can slow the association and
dissociation rates by as much as a factor of 5. The super-resolution
time-interval methodology provides a unique approach to determining
fundamental (label-free) rates of DNA hybridization, revealing the
significant influence of fluorescent labels on these kinetics. This
measurement concept can be extended to studies of other reversible
reaction systems, where kinetics of unlabeled species can be determined
from their influence on the reaction of a labeled species with localized
probe molecules on a surface.
Peptide nucleic acid (PNA) is a unique synthetic nucleic acid analog that has been adopted for use in many biological applications. These applications rely upon the robust Franklin-Watson-Crick base pairing...
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