Model-free 3D localization with precision estimates for brightfield-imaged particles

Kovari, Daniel T.; Dunlap, David; Weeks, Eric R.; Finzi, Laura

doi:10.1364/oe.27.029875

Cited by 12 publications

(13 citation statements)

References 20 publications

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“…Our magnetic tweezers parameters have been previously described 39, 40 . All MTs experiments were conducted at room temperature and a protocol similar to what already described 32 was followed.…”

Section: Methodsmentioning

confidence: 99%

Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops

Xu¹,

Yan²,

Artsimovitch³

et al. 2021

Preprint

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During elongation, RNA polymerase (RNAP) must navigate through proteins that decorate genomic DNA. Several of these mediate long-distance interactions via structures, such as loops, that alter DNA topology and create torsional barriers. We used the tethered particle motion (TPM) technique and magnetic tweezers to monitor transcription of DNA templates in the presence of the lac repressor (LacI) protein which could bind at two sites, one proximal to, and one distal from, the promoter. The bivalent LacI tetramer binds recognition sites (operators) with up to nanomolar affinity depending on the sequence, and the concentration of LacI was adjusted to promote binding to either one or both operators, so as to produce unlooped or looped DNA. We observed that RNAP pausing before a LacI-securing loop was determined not by the affinity of LacI for the operator, but by the order in which the elongating RNAP encountered these operators. TPM experiments showed that, independent of affinity, LacI bound at the promoter-proximal operator became a stronger roadblock when securing a loop. In contrast, LacI bound to the distal operator was a weaker roadblock in a looped configuration suggesting that RNAP might more easily displace LacI obstacles within a torsion-constrained DNA loop. Since protein junctions can efficiently block the diffusion of DNA supercoiling, these data indicate that the positive supercoiling generated ahead of a transcribing RNAP may facilitate the dissociation of a roadblock. In support of this idea, magnetic tweezers measurements indicated that pauses are shorter when RNAP encounters obstacles on positively supercoiled than on relaxed DNA. Furthermore, at similar winding levels of the DNA template, RNAP pause duration decreased with tension. These findings are significant for our understanding of transcription within the crowded and tensed nucleoid.

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Section: Methodsmentioning

confidence: 99%

Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops

Xu¹,

Yan²,

Artsimovitch³

et al. 2021

Preprint

Self Cite

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“…A magnetic tweezer (MT) was used to supercoil DNA and to assay the dynamics of HUmediated conformational changes in the DNA tethers. Permanent magnets were translated along and rotated about the optical axis of the microscope to vary the strength and orientation of the magnetic field in the microchamber [14,15]. Intact, single DNA tethers were identified through extension vs. twist measurements [16].…”

Section: Magnetic Tweezingmentioning

confidence: 99%

Ionic strength modulates HU protein-induced DNA supercoiling

Zhang

Yan

Leng

et al. 2021

Preprint

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The histone-like protein from E. coli strain U93 (HU) is an abundant nucleoid-associated protein that contributes to the compaction of the bacterial genome as well as to the regulation of many of its transactions. Despite many years of investigations, the way and extent to which HU binding alters the DNA double helix and/or generates hierarchical structures using DNA as a scaffold is not completely understood. Here we combined single-molecule magnetic measurements with circular dichroism studies to monitor structural changes in the DNA-HU fiber as HU concentration was increased from 0 to 1000 nM under low and physiological monovalent salt conditions. We confirmed that DNA compaction correlated with HU concentration in a biphasic manner but DNA unwinding varied monotonically with HU concentration in 100 mM KCl. Instead, in more physiological 200 mM salt conditions, DNA compaction was monotonic while HU-induced DNA unwinding was negligible. Differential compaction and unwinding of DNA may be part of the response of bacteria to large variations in salt concentrations.

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“…From the previously fitted cubic polynomial, a prediction for the microswimmer out-of-plane position is thus obtained from Z(inertia) with an average prediction error of approximately +/-0.5 µm. The described methodology carries a distinct advantage over more conventional confocal approaches, in that a much higher FPS is achievable since only one Z plane is imaged with wide-field microscopy (55). Such methods also require computationally expensive point spread functions (PSFs) for accurate center finding (56).…”

Section: Supplementary Materialsmentioning

confidence: 99%

Microrobots from Toposelective Nanoparticle Attachment

Bailey,

Grillo,

Spencer

et al. 2021

Preprint

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Microrobots hold promise for applications ranging from targeted delivery to enhanced mixing at the microscale. However, current fabrication techniques suffer from limited throughput and material selection. Here, we demonstrate a versatile route enabling the synthesis of microrobots from off-the-shelf microand nano-particles. Our protocol hinges on the toposelective attachment of photocatalytic nanoparticles onto microparticles, exploiting a multi-functional polymer and a Pickering-wax emulsification step, to yield large quantities of photo-responsive active Janus particles. The polymer presents both silane and nitrocatechol groups, binding silica microspheres to a range of metal oxide nanoparticles. The Pickering-wax emulsions protect part of the microspheres' surface, enabling asymmetric functionalization, as required for selfpropulsion. The resulting photocatalytic microrobots display a characteristic

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Model-free 3D localization with precision estimates for brightfield-imaged particles

Cited by 12 publications

References 20 publications

Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops

Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops

Ionic strength modulates HU protein-induced DNA supercoiling

Microrobots from Toposelective Nanoparticle Attachment

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