Results: 5

1.
Fig. 5.

Fig. 5. From: Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging.

The DNA overstretching transition at 110 pN is a melting transition. (A) Images of two different 3′5′-5′3′ attached DNA molecules labeled with the ssDNA marker eGFP-RPA, one held at a tension of 95 pN (before the onset of the OS transition) and the other at 135 pN (beyond the transition). At force below the transition, no eGFP-RPA binding is observed. Beyond the transition, eGFP-RPA covers the DNA homogeneously and completely. (B) Images of two different 3′5′-5′3′ attached DNA molecules at a tension of 110 pN labeled sequentially with eGFP-RPA (Top), and dsDNA-intercalator POPO (Bottom). eGFP-RPA binds throughout the DNA molecules and no unique nucleation point can be observed.

Joost van Mameren, et al. Proc Natl Acad Sci U S A. 2009 October 27;106(43):18231-18236.
2.
Fig. 3.

Fig. 3. From: Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging.

The DNA overstretching transition at 65 pN is a melting transition. (A) Three consecutive fluorescence images of overstretched 3′-3′ attached DNA exposed to mitochondrial single-stranded binding protein (mtSSB) labeled with Alexa-555. MtSSB accumulates in two spots (white arrows) that brighten and translocate upon further extension. (B) The intensity in the moving spot scales linearly with the distance moved from the bead, showing that ssDNA accumulates during overstretching. The spot to the right remains stationary relative to the right bead and does not brighten. The inset shows the fraction of base-paired nucleotides as a function of the relative extension, L/L0. The fraction is obtained from the distance between the mtSSB spots, assuming dsDNA in between. (C) Interpretation of images of fluorescent mtSSB bound to overstretched DNA (A). Overstretching initiates on fraying DNA extremities near the beads. MtSSB binds to the melted DNA strand that is not connected to the bead and consequently not under tension.

Joost van Mameren, et al. Proc Natl Acad Sci U S A. 2009 October 27;106(43):18231-18236.
3.
Fig. 4.

Fig. 4. From: Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging.

Concomitant labeling of dsDNA and ssDNA segments for the 3′-3′ attached DNA shows that, during the overstretching transition at 65 pN, dsDNA is converted into ssDNA. (A and B) Images of overstretched 3′-3′ attached DNA labeled with Alexa-555-mtSSB and YOYO. (Top) Separate emission channels. (Bottom) Merged image (green: mtSSB, red: YOYO). The images show that mtSSB dots coincide with the edges of YOYO-segments (white arrows). In (A) two melting fronts are observed, leading to two relaxed ssDNA segments forming dots, in line with conversion from dsDNA to ssDNA being nucleation limited. In (B) two additional melting fronts can be discerned, originating from a nick. (C) Images of overstretched DNA labeled sequentially with eGFP-RPA (green), and dsDNA-intercalator POPO (red). Three segments can be readily identified: (i) a homogeneous dsDNA segment, (ii) two dots caused by relaxed ssDNA at the intersection between ssDNA and dsDNA, (iii) two stretched ssDNA segments connected to the beads. (D) Image of the same DNA molecule shown in (C), with the application of a gentle buffer flow perpendicular to the DNA, extending the relaxed ssDNA segments.

Joost van Mameren, et al. Proc Natl Acad Sci U S A. 2009 October 27;106(43):18231-18236.
4.
Fig. 1.

Fig. 1. From: Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging.

The OS transition of dsDNA under tension. (A) Typical force-extension curve of a 3′-3′ attached DNA, with free 5′ ends (schematically represented in the inset). The elastic properties of DNA below the OS force of 65 pN are well described by the extensible worm-like chain model (WLC, gray line). At 65 pN, the DNA molecule undergoes the OS transition, during which the intrinsic contour length of the DNA increases from 100% to about 170%. (B) In a 3′5′-5′3′ attachment geometry, where all four strand ends are linked to the microspheres (schematically represented in the inset), the OS force raises to 110 pN. The elastic properties of this DNA construct is well described by the extensible worm-like chain model (WLC, gray line) up to forces of 60 pN. (C) Schematic representation of two models explaining the nature of the OS transition. In the first model, the transition is a result of gradual cooperative unwinding of the DNA double helix resulting in a base-paired structure, “S-DNA,” resembling a parallel ladder. In the second model, force-induced melting of the two strands causes the transition.

Joost van Mameren, et al. Proc Natl Acad Sci U S A. 2009 October 27;106(43):18231-18236.
5.
Fig. 2.

Fig. 2. From: Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging.

Specific staining of 3′-3′ attached dsDNA stretches with the intercalating dye YOYO shows that the DNA overstretching transition at 65 pN is a nucleation-limited, first-order phase transition. (A) Fluorescence images of different, extended dsDNA molecules exposed to YOYO show a binary subdivision of the DNA in labeled and unlabeled segments. The vertical dashed lines highlight the locations of the optically trapped beads. Image 1 is taken before the OS transition (L/L0 < 1) at a force of 50 pN, where the DNA is labeled along its full length. At higher extensions (L/L0 > 1), where the DNA undergoes the OS transition at a force of 65 pN, discrete unlabeled segments appear at the expense of labeled, double-stranded segments. This subdivision in large labeled and unlabeled segments reveals that the OS transition is nucleation limited. Image 3 shows an unlabeled segment halfway, suggesting that another OS nucleation took place at a nick. Image 6 is taken beyond the OS transition at a force of 80 pN, yet shows that the two strands are still connected by a short YOYO-labeled segment. (B) The fraction of dsDNA plotted as a function of DNA extension. This fraction was obtained from the length of YOYO-labeled segments in fluorescence images such as in (A), assuming them to have the same length as B-form dsDNA at this low degree of labeling (see Fig. S1). The gray dashed line connecting the two gray points indicates the behavior expected for a first-order phase transition from 100% dsDNA at the start of the OS transition (L/L0 = 1) to no dsDNA at the end (L/L0 = 1.7).

Joost van Mameren, et al. Proc Natl Acad Sci U S A. 2009 October 27;106(43):18231-18236.

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