| 1. |
- Alizadehheidari, Mohammadreza, 1987, et al.
(författare)
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Unfolding of nanoconfined circular DNA
- 2015
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Ingår i: BIOPHYSICAL JOURNAL. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 108:2 Supplement 1
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 2. |
- Frykholm, Karolin, 1977, et al.
(författare)
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Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51
- 2013
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 443:2, s. 261-268
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Tidskriftsartikel (refereegranskat)abstract
- Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity. (C) 2013 Elsevier Inc. All rights reserved.
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| 3. |
- Lin, Jun, et al.
(författare)
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Bandpass filtering of DNA elastic modes using confinement and tension
- 2012
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 102, s. 96-100
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Tidskriftsartikel (refereegranskat)abstract
- During a variety of biological and technological processes, biopolymers are simultaneously subject to both confinement and external forces. Although significant efforts have gone into understanding the physics of polymers that are only confined, or only under tension, little work has been done to explore the effects of the interplay of force and confinement. Here, we study the combined effects of stretching and confinement on a polymer's configurational freedom. We measure the elastic response of long double-stranded DNA molecules that are partially confined to thin, nanofabricated slits. We account for the data through a model in which the DNA's short-wavelength transverse elastic modes are cut off by applied force and the DNA's bending stiffness, whereas long-wavelength modes are cut off by confinement. Thus, we show that confinement and stretching combine to permit tunable bandpass filtering of the elastic modes of long polymers. © 2012 Biophysical Society.
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| 4. |
- Nyberg, Lena, 1979, et al.
(författare)
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A single-step competitive binding assay for mapping of single DNA molecules
- 2012
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 417:1, s. 404-408
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Tidskriftsartikel (refereegranskat)abstract
- Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification cif pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.
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| 5. |
- Persson, Fredrik, 1979, et al.
(författare)
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DNA in nanochannels—directly visualizing genomic information
- 2010
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Ingår i: Chemical Society Reviews. - 0306-0012. ; 39, s. 985-999
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Tidskriftsartikel (refereegranskat)abstract
- The power of nanofluidic channels to analyze DNA is described along with practical experimental hints. As an introduction, a general overview is given on conventional DNA analysis tools, as well as tools under development towards the $1000 genome. The focus of this tutorial review is the stretching of DNA in nanoscale channels for coarse-grained mapping of DNA. To understand the behavior of the DNA, basic theory is discussed. Experimental details are revealed so that the reader, with the proper equipment, should be able to perform experiments. Basic approaches to the analysis of the data are discussed. Finally, potential future directions are discussed including the application of melting mapping as a simple barcode for the DNA.
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| 6. |
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| 7. |
- Persson, Fredrik, 1979, et al.
(författare)
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Local conformation of confined DNA studied using emission polarization anisotropy
- 2010
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Ingår i: Biophysical Society 54th Annual Meeting.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- When confined in nanochannels with dimensions smaller than the DNA radius of gyration, DNA will extend along the channel. We investigate long DNA confined in nanochannels, using fluorescence microscopy and intercalated dyes. Studies of the dynamics and statics of DNA in such nanoscale confinements as a function of e.g. degree of confinement and ionic strength have yielded new insights into the physical properties of DNA with relevance for applications in genomics as well as fundamental understanding of DNA packaging in vivo. Our work extends the field by not only studying the location of the emitting dyes along a confined DNA molecule but also monitoring the polarization of the emitted light. By measuring the emission polarized parallel and perpendicular to the extension axis of the stretched DNA, information on the local spatial distribution of the DNA backbone can be obtained. Comparing polarizations in two directions for DNA confined in channels of effective diameters of 85 nm and 170 nm reveals a striking difference. Whereas the DNA in the larger channels shows an isotropic polarization of the emitted light, the light is to a large extent polarized perpendicular to the elongation of the DNA in the smaller channels. We expect this technique to have a large impact on the studies of changes in DNA conformation induced by protein binding or during DNA compactation as well as in fundamental polymer physics studies of DNA in confined environments, for example in bacterial spores and viruses.
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| 8. |
- Persson, Fredrik, 1979, et al.
(författare)
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Local conformation of confined DNA studied using emission polarization anisotropy
- 2010
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Ingår i: NanoBioTech-Montreux 2009.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- In nanochannels with dimensions smaller than the DNA radius of gyration, DNA will extend along the channel. We investigate long DNA confined in nanochannels using fluorescence microscopy and intercalated dyes. Studies of the dynamics and statics of the DNA extension or position in such nanoscale confinements as a function of e.g. DNA contour length, degree and shape of confinement as well as ionic strength have yielded new insights in the physical properties of DNA with relevance for applications in genomics as well as fundamental understanding of DNA packaging in vivo. Our work extends the field by not only studying the location of the emitting dyes along a confined DNA molecule but also monitoring the polarization of the emitted light. We use intercalating dyes (YOYO-1) whose emission is polarized perpendicular to the DNA extension axis, and by measuring the emission polarized parallel and perpendicular to the extension axis of the stretched DNA, information on the local spatial distribution of the DNA backbone can be obtained. The results obtained are analogous to linear dichroism (LD) but on a single-molecule level, and obtained in a highly parallel fashion. We will discuss results in shallow (60 nm) and deep (180 nm) channels and describe an example of how the technique can be used to investigate non-uniform stretching of DNA on the single molecule level. Comparing polarizations in two directions for DNA confined in channels of effective diameters of 85 nm and 170 nm reveals a striking difference. Whereas the DNA in the larger channels shows an isotropic polarization of the emitted light, the light is to a large extent polarized perpendicular to the elongation of the DNA in the smaller channels. The ratio of the polarization parallel and perpendicular to the elongation direction, I|| / I⊥, is a measure of the relative local orientation of the DNA backbone. We believe that this technique will have a large impact on the studies of changes in DNA conformation induced by protein binding or during DNA compactation as well as in fundamental polymer physics studies of DNA in confined environments, for example in bacterial spores and viruses.
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| 9. |
- Werner, Erik, et al.
(författare)
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Orientational correlations in confined DNA
- 2012
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Ingår i: Physical Review E. - 1539-3755 .- 2470-0045 .- 2470-0053. ; 86:4
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Tidskriftsartikel (refereegranskat)abstract
- We study how the orientational correlations of DNA confined to nanochannels depend on the channel diameter D by means of Monte Carlo simulations and a mean-field theory. This theory describes DNA conformations in the experimentally relevant regime where Flory-de Gennes theory does not apply. We show how local correlations determine the dependence of the end-to-end distance of the DNA molecule upon D. Tapered nanochannels provide the necessary resolution in D to study experimentally how the extension of confined DNA molecules depends upon D. Our experimental and theoretical results are in qualitative agreement.
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| 10. |
- Westerlund, Fredrik, 1978, et al.
(författare)
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Fluorescence Enhancement From Single DNA Molecules Confined In Si/SiO2 Nanochannels
- 2010
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Ingår i: Biophysical Society, 54th Annual Meeting.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- A large challenge in biophysics when studying single molecules using fluorescence microscopy is to obtain a signal that is clearly detectable above the background noise. Ways to improve or optimize the fluorescence signal is therefore of great interest. We here study DNA extended in 320 nm deep funnel-shaped SiO2 nanochannels with a width ranging from 40nm to 600nm. The DNA is stained with a fluorescent dye (YOYO-1) and we show that the total emission from the DNA varies significantly with the dimensions of the channels (Figure) and has a peak intensity at half the wavelength of the emitted light. Measurements at varying salt concentrations, where the same confinement leads to different extension of the DNA, confirm that it is solely the geometry of the channel that governs the enhancement effect, ruling out alternative explanations, such as self-quenching. By using polarizers on the emission side we can investigate the light polarized parallel and perpendicular to the channel separately and we see that they show vastly different behavior with the peak in emission only detected in the light polarized parallel to the channels. We will discuss how our data may be explained by cavity-resonance effects when the lateral dimensions of the channels coincide with half the wavelength of the emitted light. Our results suggest that it is possible to fine-tune the size and shape of the nanochannels to maximize the number of photons collected from the molecule under study, for example when studying DNA interacting with single DNA-binding proteins where maximizing the photon budget is paramount.
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