Single-Molecule Study of<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mi>G</mml:mi></mml:math>-Quadruplex Disruption Using Dynamic Force Spectroscopy

Messieres, Michel de; Chang, Jen-Chien; Brawn-Cinani, Barbara; Porta, A. La

doi:10.1103/physrevlett.109.058101

Cited by 40 publications

(51 citation statements)

References 30 publications

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“…2 f), or by clamping the position of the bead relative to the optical trap (x bead ¼ const.) and increasing the optical power at a constant rate (dF/dt ¼ À(dk trap /dt) , x bead ) (54). Data collected with DFS can be converted, using appropriate models, to get the kinetics of bond detachment under constant forces (47,55).…”

Section: Dynamic Geometriesmentioning

confidence: 99%

Interrogating Biology with Force: Single Molecule High-Resolution Measurements with Optical Tweezers

Capitanio

Pavone

2013

Biophysical Journal

129

108

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Single molecule force spectroscopy methods, such as optical and magnetic tweezers and atomic force microscopy, have opened up the possibility to study biological processes regulated by force, dynamics of structural conformations of proteins and nucleic acids, and load-dependent kinetics of molecular interactions. Among the various tools available today, optical tweezers have recently seen great progress in terms of spatial resolution, which now allows the measurement of atomic-scale conformational changes, and temporal resolution, which has reached the limit of the microsecond-scale relaxation times of biological molecules bound to a force probe. Here, we review different strategies and experimental configurations recently developed to apply and measure force using optical tweezers. We present the latest progress that has pushed optical tweezers' spatial and temporal resolution down to today's values, discussing the experimental variables and constraints that are influencing measurement resolution and how these can be optimized depending on the biological molecule under study.

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Section: Dynamic Geometriesmentioning

confidence: 99%

Interrogating Biology with Force: Single Molecule High-Resolution Measurements with Optical Tweezers

Capitanio

Pavone

2013

Biophysical Journal

129

108

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“…27,30 Weaker stacking interactions between adjacent G-tetrad planes in RNA quadruplexes with respect to those of DNA may be responsible for the lower force needed to unfold TERRA with respect to comparable DNA quadruplex motifs.…”

mentioning

confidence: 99%

Mechanical unfolding of long human telomeric RNA (TERRA)

et al. 2013

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We report the first single molecule investigation of TERRA molecules.By using optical-tweezers and other biophysical techniques, we have found that long RNA constructions of up to 25 GGGUUA repeats form higher order structures comprised of single parallel G-quadruplex blocks, which unfold at lower forces than their DNA counterparts.Telomeres are nucleoprotein structures that protect chromosome ends from being recognized as DNA breaks.1 Mammalian telomere DNA consists of long tandem arrays of double-stranded TTAGGG repeats that are maintained by the telomerase. 2 The 3 0 end of each telomere terminates in a G-rich single-stranded overhang (150-200 nucleotides) that is able to self-fold into G-quadruplexes. The demonstration of the in vivo presence of DNA G-quadruplexes at telomeres suggests that these non-canonical secondary structures have a role in telomere end-protection, and therefore in chromosome stability. 3,4 It has also been found that telomere end-binding proteins control the folding and unfolding of DNA G-quadruplexes in vitro 5 and in vivo. 4Telomeres are transcribed from the subtelomeric regions towards chromosome ends into telomeric repeat-containing RNA (TERRA). 6,7 These non-coding RNA molecules contain subtelomere-derived sequences and an average of 34 GGGUUA repeats at their 3 0 end.8 TERRA acts as a scaffold for the assembly of telomeric proteins involved in telomere maintenance and telomeric heterochromatin formation. 9,10 Importantly, there is also evidence for the presence of TERRA RNA G-quadruplexes in living cells. Here, we report the synthesis and purification of TERRA molecules up to 96nt (twice the largest molecule previously analyzed by NMR), a size similar to the endogenous nuclear TERRA 8 (see ESI †).We have also used optical-tweezers (OT) to study RNA molecules with different numbers of possible quadruplexes ranging from 1 (Q1) to 6 (Q6). CD spectra of the RNA sequence Q4 in 25 mM potassium phosphate pH 7 buffer showed a positive band at 260 nm and a negative band at 240 nm (Fig. 1a), the characteristic signature of parallel-stranded G-quadruplex conformations. This result is in agreement with previously reported observations showing that other telomeric RNA sequences are folded into parallel G-quadruplex structures both in sodium and potassium solutions. 12,13,[16][17][18][19][20] It is also important to mention that no band at 300 nm was observed for this long telomeric RNA as it was reported for 22-mer and 45-mer telomeric RNA in ammonium acetate buffer. 15 On the other hand, the normalized CD spectra of the bimolecular G-quadruplex formed by r(UAGGGUUAGGGU) and Q4 showed a similar shape and amplitude, which indicates that most of the Q4 molecules form four G-quadruplexes (Fig. 1b). 21Thermal stability of Q4 was also measured by recording CD melting curves. To test the influence of the potassium concentration on the thermal stability we performed the experiments in two different potassium-containing buffers (Fig. 1c). Melting temperatures of 65.5 1C and 72.5 1C were obtained in 2...

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“…Both AFM and optical tweezers force spectroscopy showed that, when applying a linearly increasing force over time, G-quadruplex structures are disrupted when the force reaches ∼50 pN [71, 72]. In one AFM study, the gold-coated Si 3 N 4 probe as well as the gold surface were functionalized with a self-assembled monolayer of DNA containing G-rich domains [71].…”

Section: Results From Single-molecule Studiesmentioning

confidence: 99%

“…When the tip approached the surface, intermolecular G-quadruplexes were formed between the tip and surface. Optical tweezers based dynamic force spectroscopy, in which the distribution of rupture forces was measured for different loading rates, was used to characterize the transition state barrier for unfolding of the G-quadruplex structure [72]. The high precision of the optical tweezers allowed de Messieres et al .…”

Section: Results From Single-molecule Studiesmentioning

confidence: 99%

Unraveling secrets of telomeres: One molecule at a time

et al. 2014

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Telomeres play important roles in maintaining the stability of linear chromosomes. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as G-quadruplexes, T-loops and t-circles. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure-function relationships that govern telomere maintenance. In this review, we present a brief overview of the principles of single-molecule imaging and manipulation techniques. We then highlight results obtained from applying these single-molecule techniques for studying structure, dynamics and functions of G-quadruplexes, telomerase, and shelterin proteins.

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Single-Molecule Study ofG-Quadruplex Disruption Using Dynamic Force Spectroscopy

Cited by 40 publications

References 30 publications

Interrogating Biology with Force: Single Molecule High-Resolution Measurements with Optical Tweezers

Interrogating Biology with Force: Single Molecule High-Resolution Measurements with Optical Tweezers

Mechanical unfolding of long human telomeric RNA (TERRA)

Unraveling secrets of telomeres: One molecule at a time

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