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| 2. |
- Fornander, Louise, 1984, et al.
(author)
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Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels
- 2016
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In: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 32:33, s. 8403-8412
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Journal article (peer-reviewed)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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| 3. |
- Fritzsche, Joachim, 1977, et al.
(author)
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A lipid-based passivation scheme for nanofluidics
- 2012
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In: 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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Conference paper (peer-reviewed)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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| 4. |
- Persson, Fredrik, 1979, et al.
(author)
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Lipid-Based Passivation in Nanofluidics
- 2012
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In: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 12:5, s. 2260-2265
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Journal article (peer-reviewed)abstract
- Stretching DNA in nanochannels is a useful tool for direct, visual studies of genomic DNA at the single molecule level. 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 demonstrate virtually complete long-term passivation of nanochannel surfaces to a range of relevant reagents, including streptavidin-coated quantum dots, RecA proteins, and RecA-DNA complexes. We show that the performance of the lipid bilayer is significantly better than that of standard bovine serum albumin-based passivation. Finally, we show how the passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation by DNase I. We expect that our approach will open up for detailed, systematic studies of a wide range of protein-DNA interactions with high spatial and temporal resolution.
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| 5. |
- Fornander, Louise, 1984, et al.
(author)
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Using nanofluidic channels to probe dynamics of RAD51-Filaments
- 2015
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In: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014. - 9780979806476 ; , s. 1826-1828
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Conference paper (peer-reviewed)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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| 7. |
- Frykholm, Karolin, 1977, et al.
(author)
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Probing physical properties of DNA-protein complexes using nanofluidic channels
- 2013
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In: 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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Conference paper (peer-reviewed)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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