An integrated laser-tweezers instrument for microanalysis of individual protein aggregates

Mao, Hanbin; Luchette, Paul

doi:10.1016/j.snb.2007.09.052

Cited by 53 publications

(65 citation statements)

References 61 publications

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“…The beads attached with DNA construct and the beads coated with streptavidin (diameter: 1.87 μm) were injected into the top and bottom channels of a three-channel microfluidic chamber, respectively. The two types of beads were trapped by laser tweezers in the middle channel43, to which 10 mM Tris-HCl buffer (pH 7.0) containing different metal ions was injected. The two trapped beads were brought close to each other to tether the other end of the DNA construct to the streptavidin-coated bead via biotin/streptavidin linkage.…”

Section: Methodsmentioning

confidence: 99%

Divalent cations and molecular crowding buffers stabilize G-triplex at physiologically relevant temperatures

Jiang

Cui

Zhao

et al. 2015

Sci Rep

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G-triplexes are non-canonical DNA structures formed by G-rich sequences with three G-tracts. Putative G-triplex-forming sequences are expected to be more prevalent than putative G-quadruplex-forming sequences. However, the research on G-triplexes is rare. In this work, the effects of molecular crowding and several physiologically important metal ions on the formation and stability of G-triplexes were examined using a combination of circular dichroism, thermodynamics, optical tweezers and calorimetry techniques. We determined that molecular crowding conditions and cations, such as Na+, K+, Mg2+ and Ca2+, promote the formation of G-triplexes and stabilize these structures. Of these four metal cations, Ca2+ has the strongest stabilizing effect, followed by K+, Mg2+, and Na+ in a decreasing order. The binding of K+ to G-triplexes is accompanied by exothermic heats, and the binding of Ca2+ with G-triplexes is characterized by endothermic heats. G-triplexes formed from two G-triad layers are not stable at physiological temperatures; however, G-triplexes formed from three G-triads exhibit melting temperatures higher than 37°C, especially under the molecular crowding conditions and in the presence of K+ or Ca2+. These observations imply that stable G-triplexes may be formed under physiological conditions.

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Section: Methodsmentioning

confidence: 99%

Divalent cations and molecular crowding buffers stabilize G-triplex at physiologically relevant temperatures

Jiang

Cui

Zhao

et al. 2015

Sci Rep

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“…A detailed description of the laser tweezers instrument has been reported elsewhere 38 . In brief, P - and S -polarized laser beams from the same source (a diode pumped solid-state (DPSS) laser with a 1,064 nm wavelength, 4 W, continuous-wave (c.w.)…”

Section: Methodsmentioning

confidence: 99%

A single-molecule platform for investigation of interactions between G-quadruplexes and small-molecule ligands

et al. 2011

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Ligands that stabilize the formation of telomeric DNA G-quadruplexes have potential as cancer treatments, because the G-quadruplex structure cannot be extended by telomerase, an enzyme over-expressed in many cancer cells. Understanding the kinetic, thermodynamic and mechanical properties of small-molecule binding to these structures is therefore important, but classical ensemble assays are unable to measure these simultaneously. Here, we have used a laser tweezers method to investigate such interactions. With a force jump approach, we observe that pyridostatin promotes the folding of telomeric G-quadruplexes. The increased mechanical stability of pyridostatin-bound G-quadruplex permits the determination of a dissociation constant Kd of 490 ± 80 nM. The free-energy change of binding obtained from a Hess-like process provides an identical Kd for pyridostatin and a Kd of 42 ± 3 μM for a weaker ligand RR110. We anticipate that this single-molecule platform can provide detailed insights into the mechanical, kinetic and thermodynamic properties of liganded bio-macromolecules, which have biological relevance.

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“…The mechanical unfolding of G-quadruplex in hTel4G was performed in a home-built dual-trap 1064 nm laser tweezers (29) at room temperature under different buffer conditions (10 mM Tris buffer that contains 100 mM of either NaCl or KCl species and 40% w/v DMSO, ACN or BSA at pH 7.4). Before the unfolding, the DNA construct was immobilized onto a 2.10 µm bead through the digoxigenin–anti-digoxigenin–antibody complex.…”

Section: Methodsmentioning

confidence: 99%

Structural and mechanical properties of individual human telomeric G-quadruplexes in molecularly crowded solutions

Dhakal

Cui

Koirala

et al. 2013

Nucleic Acids Research

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108

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Recent experiments provided controversial observations that either parallel or non-parallel G-quadruplex exists in molecularly crowded buffers that mimic cellular environment. Here, we used laser tweezers to mechanically unfold structures in a human telomeric DNA fragment, 5′-(TTAGGG)4TTA, along three different trajectories. After the end-to-end distance of each unfolding geometry was measured, it was compared with PDB structures to identify the best-matching G-quadruplex conformation. This method is well-suited to identify biomolecular structures in complex settings not amenable to conventional approaches, such as in a solution with mixed species or at physiologically significant concentrations. With this approach, we found that parallel G-quadruplex coexists with non-parallel species (1:1 ratio) in crowded buffers with dehydrating cosolutes [40% w/v dimethyl sulfoxide (DMSO) or acetonitrile (ACN)]. In crowded solutions with steric cosolutes [40% w/v bovine serum albumin (BSA)], the parallel G-quadruplex constitutes only 10% of the population. This difference unequivocally supports the notion that dehydration promotes the formation of parallel G-quadruplexes. Compared with DNA hairpins that have decreased unfolding forces in crowded (9 pN) versus diluted (15 pN) buffers, those of G-quadruplexes remain the same (20 pN). Such a result implies that in a cellular environment, DNA G-quadruplexes, instead of hairpins, can stop DNA/RNA polymerases with stall forces often <20 pN.

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An integrated laser-tweezers instrument for microanalysis of individual protein aggregates

Cited by 53 publications

References 61 publications

Divalent cations and molecular crowding buffers stabilize G-triplex at physiologically relevant temperatures

Divalent cations and molecular crowding buffers stabilize G-triplex at physiologically relevant temperatures

A single-molecule platform for investigation of interactions between G-quadruplexes and small-molecule ligands

Structural and mechanical properties of individual human telomeric G-quadruplexes in molecularly crowded solutions

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