| 21. |
- Fornander, Louise, 1984, et al.
(författare)
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Using nanofluidic channels to probe dynamics of RAD51-Filaments
- 2015
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Ingår i: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014. - 9780979806476 ; , s. 1826-1828
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Konferensbidrag (refereegranskat)abstract
- Using nanochannels, passivated with a lipid bilayer to avoid sticking of proteins, we study Rad51 filaments bound to single- and double stranded DNA. We demonstrate how we can discern different properties of the filaments by studying them at different degrees of confinement. Unlike the bacterial homologue RecA, that forms homogeneous filaments along DNA, Rad51 forms heterogeneous filaments containing both rigid kinks as well as flexible regions. Varying the counterion, the DNA substrate as well as the initial protein concentration, we try to understand the factors governing the structure of the filaments.
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| 22. |
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| 23. |
- Fornander, Louise, 1984, et al.
(författare)
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Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels
- 2016
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 32:33, s. 8403-8412
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Tidskriftsartikel (refereegranskat)abstract
- RAD51 is the key component of the homologous recombination pathway in eukaryotic cells and performs its task by forming filaments on DNA. In this study we investigate the physical properties of RAD51 filaments formed on DNA using nanofluidic channels and fluorescence microscopy. Contrary to the bacterial ortholog RecA, RAD51 forms inhomogeneous filaments on long DNA in vitro, consisting of several protein patches. We demonstrate that a permanent "kink" in the filament is formed where two patches meet if the stretch of naked DNA between the patches is short. The kinks are readily seen in the present microscopy approach but would be hard to identify using conventional single DNA molecule techniques where the DNA is more stretched. We also demonstrate that protein patches separated by longer stretches of bare DNA roll up on each other and this is visualized as transiently overlapping filaments. RAD51 filaments can be formed at several different conditions, varying the cation (Mg2+ or Ca2+), the DNA substrate (single-stranded or double-stranded), and the RAD51 concentration during filament nucleation, and we compare the properties of the different filaments formed. The results provide important information regarding the physical properties of RAD51 filaments but also demonstrate that nanofluidic channels are perfectly suited to study protein-DNA complexes.
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| 24. |
- Freitag, C., et al.
(författare)
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Visualizing the entire DNA from a chromosome in a single frame
- 2015
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Ingår i: Biomicrofluidics. - : AIP Publishing. - 1932-1058. ; 9:4
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Tidskriftsartikel (refereegranskat)abstract
- The contiguity and phase of sequence information are intrinsic to obtain complete understanding of the genome and its relationship to phenotype. We report the fabrication and application of a novel nanochannel design that folds megabase lengths of genomic DNA into a systematic back-and-forth meandering path. Such meandering nanochannels enabled us to visualize the complete 5.7 Mbp (1mm) stained DNA length of a Schizosaccharomyces pombe chromosome in a single frame of a CCD. We were able to hold the DNA in situ while implementing partial denaturation to obtain a barcode pattern that we could match to a reference map using the Poland-Scheraga model for DNA melting. The facility to compose such long linear lengths of genomic DNA in one field of view enabled us to directly visualize a repeat motif, count the repeat unit number, and chart its location in the genome by reference to unique barcode motifs found at measurable distances from the repeat. Meandering nanochannel dimensions can easily be tailored to human chromosome scales, which would enable the whole genome to be visualized in seconds. (C) 2015 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License.
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| 25. |
- Friedrich, R., et al.
(författare)
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A nano flow cytometer for single lipid vesicle analysis
- 2017
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Ingår i: Lab on a Chip - Miniaturisation for Chemistry and Biology. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 17:5, s. 830-841
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Tidskriftsartikel (refereegranskat)abstract
- We present a nanofluidic device for fluorescence-based detection and characterization of small lipid vesicles on a single particle basis. The device works like a nano flow cytometer where individual vesicles are visualized by fluorescence microscopy while passing through parallel nanochannels in a pressure-driven flow. An experiment requires less than 20 mu l sample volume to quantify both the vesicle content and the fluorescence signals emitted by individual vesicles. We show that the device can be used to accurately count the number of fluorescent synthetic lipid vesicles down to a vesicle concentration of 170 fM. We also show that the size-distribution of the vesicles can be resolved from their fluorescence intensity distribution after calibration. We demonstrate the applicability of the assay in two different examples. In the first, we use the nanofluidic device to determine the particle concentration in a sample containing cell-derived extracellular vesicles labelled with a lipophilic dye. In the second, we demonstrate that dual-color detection can be used to probe peptide binding to synthetic lipid vesicles; we identify a positive membrane-curvature sensing behavior of an arginine enriched version of the Antennapedia homeodomain peptide penetratin. Altogether, these results illustrate the potential of this nanofluidic-based methodology for characterization and quantification of small biological vesicles and their interactors without ensemble averaging. The device is therefore likely to find use as a quantitative analytical tool in a variety of fields ranging from diagnostics to fundamental biology research. Moreover, our results have potential to facilitate further development of automated lab-on-a-chip devices for vesicle analysis.
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| 26. |
- Fritzsche, Joachim, 1977, et al.
(författare)
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A lipid-based passivation scheme for nanofluidics
- 2012
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Ingår i: 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012; Okinawa; Japan; 28 October 2012 through 1 November 2012. - 9780979806452 ; , s. 1876-1878
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Konferensbidrag (refereegranskat)abstract
- Stretching DNA in nanochannels allows for direct, visual studies of genomic DNA at the single molecule level. In order to facilitate the study of the interaction of linear DNA with proteins in nanochannels, we have implemented a highly effective passivation scheme based on lipid bilayers. We show long-term passivation of nanochannel surfaces to several relevant reagents and demonstrate that the performance of the lipid bilayer is significantly better compared to standard bovine serum albumin-based passivation. Moreover, we demonstrate how the passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation.
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| 27. |
- Frykholm, Karolin, 1977, et al.
(författare)
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Fast size-determination of intact bacterial plasmids using nanofluidic channels
- 2015
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 15:13, s. 2739-2743
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate how nanofluidic channels can be used as a tool to rapidly determine the number and sizes of plasmids in bacterial isolates. Each step can be automated at low cost, opening up opportunities for general use in microbiology labs.
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| 28. |
- Frykholm, Karolin, et al.
(författare)
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Probing Physical Properties of a DNA-Protein Complex Using Nanofluidic Channels
- 2014
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 106:2, s. 428A-429A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Nanofluidic channels have become an important tool to investigate single DNA molecules both from a fundamental polymer physics perspective as well as in e.g. optical mapping techniques. However, less effort has been made to study DNA-protein complexes. A main reason is that the extreme surface-to-volume ratio in the nanochannels causes most proteins to stick to the channel walls. We have recently overcome this problem by coating the channels with a lipid bilayer, thereby eliminating sticking.
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| 29. |
- Frykholm, Karolin, 1977, et al.
(författare)
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Probing Physical Properties of a DNA- Protein Complex Using Nanofluidic Channels
- 2014
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Ingår i: Small. - : Wiley. - 1613-6810 .- 1613-6829. ; 10:5, s. 884-887
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Tidskriftsartikel (refereegranskat)abstract
- A method to investigate physical properties of a DNA-protein complex in solution is demonstrated. By using tapered nanochannels and lipid passivation the persistence length of a RecA filament formed on double-stranded DNA is determined to 1.15 μm, in agreement with the literature, without attaching protein or DNA to any handles or surfaces.
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| 30. |
- Frykholm, Karolin, 1977, et al.
(författare)
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Probing physical properties of DNA-protein complexes using nanofluidic channels
- 2013
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Ingår i: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013; Freiburg; Germany; 27 October 2013 through 31 October 2013. - 9781632666246 ; 2, s. 1311-1313
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Konferensbidrag (refereegranskat)abstract
- We present the use of nanofluidic channels as a tool for determining physical properties of single DNA-protein complexes. By coating the nanochannels with a lipid bilayer we avoid sticking of proteins to the channel walls. RecA is a prokaryotic protein involved in recombination and DNA repair. We study filaments of RecA, bound to both double stranded (ds) and single stranded (ss) DNA. We determine the persistence length of RecA filaments on both dsDNA and ssDNA and obtain values in agreement with the literature. Neither the DNA nor the protein has to be attached to handles or surfaces, and the technique is directly transferable to Lab-on-a-Chip technologies for high throughput measurements in solution.
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