Three-Dimensional Characterization of Tethered Microspheres by Total Internal Reflection Fluorescence Microscopy

Blumberg, Seth; Gajraj, Arivalagan; Pennington, Matthew W.; Meiners, Jens-Christian

doi:10.1529/biophysj.105.061242

Cited by 52 publications

(82 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Inspired by the literature (25,27) and the results from Fig. S3, we imposed the following selection criteria to minimize the occurrence of unspecific tethers in the analyzed datasets: (i) Results from beads that probably were attached directly to the surface, or by unspecific tethers, and hence had a small RMSD were discarded, and (ii) if the positions visited by the bead displayed a low degree of symmetry (s) as defined in SI Text, the bead might be attached to the surface by more than one DNA tether and the time series was discarded.…”

Section: Resultsmentioning

confidence: 99%

DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch

Nørregaard

Andersson

Sneppen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Bacteriophage λ stably maintains its dormant prophage state but efficiently enters lytic development in response to DNA damage. The mediator of these processes is the λ repressor protein, CI, and its interactions with λ operator DNA. This λ switch is a model on the basis of which epigenetic switch regulation is understood. Using single molecule analysis, we directly examined the stability of the CI-operator structure in its natural, supercoiled state. We marked positions adjacent to the λ operators with peptide nucleic acids and monitored their movement by tethered particle tracking. Compared with relaxed DNA, the presence of supercoils greatly enhances juxtaposition probability. Also, the efficiency and cooperativity of the λ switch is significantly increased in the supercoiled system compared with a linear assay, increasing the Hill coefficient. The decision between the dormant state (the lysogenic state) and the vegetative state (the lytic state) made by bacteriophage λ upon infection of an Escherichia coli was the first epigenetic switch to be deciphered (1) and continues to provide insights into biological processes (2, 3). Once the lysogenic state is established, it is exceptionally stable and departure in the absence of the DNA damage sensing system is nearly always caused by mutation (4-6). Maintenance of lysogeny is mediated by the λ repressor protein, CI, and its formation of a complex with phage DNA. The CI protein binds at two regulatory regions called operator right (OR) and operator left (OL), located about 2.3 kbp apart on the phage genome. Each operator is a constellation of three adjacent subsites that each bind dimers of CI in a hierarchical manner (1, 7). In a lysogenic cell, CI prevents lytic growth by directly repressing the lytic promoters pR and pL. This repression is enhanced by longrange cooperative interactions between CI dimers bound to the OL1-OL2 and OR1-OR2 regions, thus looping the DNA that lies between them (8) (Fig. 1 A and B). CI-mediated DNA looping brings OR3 in proximity to OL3. This juxtaposition allows a CI dimer bound to the strong OL3 to facilitate another CI dimer to bind to the intrinsically weak OR3, thus reducing the concentration of CI necessary to occupy OR3. In this manner CI can autoregulate its own expression by binding to OR2 (activation) or by binding to OR3 (repression). This autoregulation is crucial for the phage to maintain repression while preventing excessive accumulation of CI. When DNA in the lysogen is damaged, the bacterial DNA-damage-sensing system is triggered, leading to CI inactivation by self-cleavage (9) and to an efficient switch to the lytic state, eventually leading to production of progeny phage and cell death (1).The switch from the lysogenic to the lytic state must be decisive. Partial entry into vegetative growth may kill the host without producing a full burst of progeny phage. Because the in vivo state of DNA is supercoiled (10), we sought to examine protein-mediated DNA looping on native, supercoiled DNA. In vitro single molecule exper...

show abstract

Section: Resultsmentioning

confidence: 99%

DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch

Nørregaard

Andersson

Sneppen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Multiply tethered particles are identified by asymmetry of the position distribution. 19,23 For this reason we visually inspected 2D histograms ͓see, e.g., Fig. 7͑a͔͒ of the positions of the moving nanoparticles to verify an isotropic distribution.…”

Section: Discussionmentioning

confidence: 99%

“…7͑a͔͒ of the positions of the moving nanoparticles to verify an isotropic distribution. Blumberg et al 19 and Pouget et al 23 discarded any position distribution with an anisotropy above 20% and 10%, respectively. Visual inspection shows that an anisotropy of 10% is already clearly visible by eye, therefore we can safely assume that we kept only singly tethered particles.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

The persistence length of double stranded DNA determined using dark field tethered particle motion

Brinkers

Dietrich

Groote

et al. 2009

The Journal of Chemical Physics

113

114

View full text Add to dashboard Cite

The wormlike chain model describes the micromechanics of semiflexible polymers by introducing the persistence length. We propose a method of measuring the persistence length of DNA in a controllable near-native environment. Using a dark field microscope, the projected positions of a gold nanoparticle undergoing constrained Brownian motion are captured. The nanoparticle is tethered to a substrate using a single double stranded DNA ͑dsDNA͒ molecule and immersed in buffer. No force is exerted on the DNA. We carried out Monte Carlo simulations of the experiment, which give insight into the micromechanics of the DNA and can be used to interpret the motion of the nanoparticle. Our simulations and experiments demonstrate that, unlike other similar experiments, the use of nanometer instead of micrometer sized particles causes particle-substrate and particle-DNA interactions to be of negligible effect on the position distribution of the particle. We also show that the persistence length of the tethering DNA can be estimated with a statistical error of 2 nm, by comparing the statistics of the projected position distribution of the nanoparticle to the Monte Carlo simulations. The persistence lengths of 45 single molecules of four different lengths of dsDNA were measured under the same environmental conditions at high salt concentration. The persistence lengths we found had a mean value of 35 nm ͑standard error of 2.8 nm͒, which compares well to previously found values using similar salt concentrations. Our method can be used to directly study the effect of the environmental conditions ͑e.g., buffer and temperature͒ on the persistence length.

show abstract

“…It should be noted that at the start of an experiment we selected the best particles ͑ϳ20%͒ based on symmetry of the intensity-position plots to exclude cases of ͑additional͒ nonspecific sticking. 17,23 Particles with slightly less symmetry in the intensity-position plots showed lower height displacements but still the same trend in the histograms for diluted buffers ͑narrowing and shifting of the peak͒. Finally, we note that buffer replacement in the scattered intensity detection setup ͑using a cover glass instead of an injection-molded polystyrene cover͒ generates higher fluidic forces and, therefore, leads to a substantial increase in the loss of bound particles in the fluidic wash steps ͑more than half of the particles over several wash steps͒.…”

Section: Fig 2 ͑A͒mentioning

confidence: 99%

Electrostatic height modulation of bound particle labels by buffer exchange in an evanescent wave biosensor

Ommering

Paesen

Zon

et al. 2010

Journal of Applied Physics

View full text Add to dashboard Cite

We introduce a technique to electrostatically modulate the height of bound particle labels on a biosensor surface by exchanging the buffer. In an evanescent wave biosensor, height modulation leads to a modulation of the scattered and reflected light intensity. We measured a lower scattering and, therefore, a higher reflection for a decreasing ionic strength, which can be explained by an increasing electrostatic force repelling bound particles from the surface. By comparing bonds with troponin, 105 base pair ͑bp͒ DNA and 290 bp DNA, we found that the signal change for an ensemble of bound particles was related to the length of the analyte. Additionally, we observed for individual particles that the thermal fluctuations of scattered light intensity became smaller for decreasing ionic strength and that the average intensity shifted toward lower values, corresponding to larger particle heights. A quantitative model comprising electrostatic repulsion and van der Waals interaction could fit the measured height displacements for four different DNA lengths ͑105 bp to 590 bp͒ as analytes. Height manipulation of bound particle labels thus reflects analyte-specific properties and may lead to biosensors with enhanced specificity.

show abstract

Three-Dimensional Characterization of Tethered Microspheres by Total Internal Reflection Fluorescence Microscopy

Cited by 52 publications

References 23 publications

DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch

DNA supercoiling enhances cooperativity and efficiency of an epigenetic switch

The persistence length of double stranded DNA determined using dark field tethered particle motion

Electrostatic height modulation of bound particle labels by buffer exchange in an evanescent wave biosensor

Contact Info

Product

Resources

About