Super-Resolution Imaging of Competitive Unlabeled DNA Hybridization Reveals the Influence of Fluorescent Labels on Duplex Formation and Dissociation Kinetics

Abstract

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.

Evaluating the effect of ionic strength on PNA:DNA duplex formation kinetics

“…The sensor readout was at 1 MHz to minimize read noise, and the electron-multiplying amplifier was deactivated to avoid additional EM photon-counting noise. 57 Images were acquired as 15–60 min 16-bit monochrome FITS-image stacks using Andor SOLIS software version 4.27.30001.0.…”

“…The excitation laser exposure experienced by fluorophores on target DNA bound at the interface before dissociation has been shown to result in minimal photobleaching. 40,57 Images were collected in 300 Â 300 pixel sub-regions of the sensor, corresponding to 50 Â 50 mm in the sample. The sensor readout was at 1 MHz to minimize read noise, and the electronmultiplying amplifier was deactivated to avoid additional EM photon-counting noise.…”

Confocal Raman Microscopy Enables Label-Free, Quantitative, and Structurally Informative Detection of DNA Hybridization at Porous Silica Surfaces

“…Future applications of this methodology will allow label-free measurements of association constants for the hybridization of target DNA with immobilized probes, thereby avoiding the significant impact that fluorescent labels have on the kinetics and stability of DNA duplexes. , This technology can detect not only immobilized single-stranded DNA and hybridized DNA targets but also molecules that interact with double-stranded DNA. This capability will allow the association of small-molecule and peptide-based drugs that can modulate gene expression , to be investigated without labels.…”

“…A major limitation in characterization of these interfacial reactions is that the surface densities of duplex oligonucleotides are small, in the range of 1–10 nmol/m 2 , making the detection of these interfacial reactions challenging. Incorporation of a fluorescent label into a target DNA strand allows very low densities of duplex formation events to be detected and quantified by means of single-molecule fluorescence imaging. − Fluorescence labeling has drawbacks, however, including the required ligation and purification of a labeled product and the influence that a label can have on the energetics and kinetics of hybridization. − Label-free fluorescence detection of DNA at surfaces is not practical because the room temperature fluorescence quantum yields of DNA bases are vanishingly small, ∼10 –4 . Label-free detection of hybridization at surfaces can be accomplished by surface plasmon resonance − or micro-ring resonators, which are sensitive to small changes in the interfacial refractive index that accompany duplex formation at immobilized DNA probes.…”

Comparing Transient Oligonucleotide Hybridization Kinetics Using DNA-PAINT and Optoplasmonic Single-Molecule Sensing on Gold Nanorods

“…[18][19][20] Despite this steady progress, single-molecule techniques suffer from different drawbacks that limit the information one can extract. For example, it is well-known that fluorescent labels may affect the kinetics and dynamics of biomolecular systems, [21][22][23] while LSPR-based approaches use local field enhancements that are not uniform across a plasmonic nanostructure, and the local surface geometry and heterogeneity of plasmonic nanoparticles can play a significant role in the observed statistics. [24][25][26] In view of this, finding an accurate description of bioprocesses at the singlemolecule level will require the combination of information arising from different methods applied to the same molecular system.…”