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  • 1. Alizadehheidari, M.
    et al.
    Frykholm, K.
    Fritzsche, J.
    Wigenius, J.
    Modesti, M.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Westerlund, F.
    Probing the Physical Properties of a DNA-Protein Complex Using Nanofluidic Channels2013In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 42, p. S134-S134Article in journal (Other academic)
  • 2. Alizadehheidari, Mohammadreza
    et al.
    Werner, Erik
    Noble, Charleston
    Nyberg, Lena
    Fritzsche, Joachim
    Mehlig, Bernhard
    Tegenfeldt, Jonas
    Ambjoernsson, Tobias
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Westerlund, Fredrik
    Nanoconfined Circular DNA2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 274A-274AArticle in journal (Other academic)
    Abstract [en]

    Nanofluidic channels have become a versatile tool to manipulate single DNA molecules. They allow investigation of confined single DNA molecules from a fundamental polymer physics perspective as well as for example in DNA barcoding techniques.

  • 3. Fornander, Louise Helena
    et al.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Fritzsche, Joachim
    Araya, Joshua
    Nevin, Philip
    Beuning, Penny
    Modesti, Mauro
    Frykholm, Karolin
    Westerlund, Fredrik
    Using Nanofluidic Channels to Probe the Dynamics of Rad51-DNA Filaments2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 692A-693AArticle in journal (Other academic)
    Abstract [en]

    Rad51 is a key protein involved in the strand exchange reaction, a reaction where genetic material is transferred between two homologous DNA strands. Strand exchange is initiated by Rad51 forming a helical filament around single-stranded DNA (ssDNA), and the strand exchange is thereafter executed with a homologous double-stranded DNA (dsDNA). The structure of Rad51-DNA filaments, and also the activity of the strand exchange reaction, is dependent on the presence of ATP and dications, where Ca2+ has been shown to promote a higher degree of strand exchange than Mg2+.

  • 4. Frykholm, Karolin
    et al.
    Alizadehheidari, Mohammadreza
    Fritzsche, Joachim
    Wigenius, Jens
    Modesti, Mauro
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Westerlund, Fredrik
    Probing Physical Properties of a DNA- Protein Complex Using Nanofluidic Channels2014In: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 10, no 5, p. 884-887Article in journal (Refereed)
    Abstract [en]

    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.

  • 5. Frykholm, Karolin
    et al.
    Alizadehheidari, Mohammadreza
    Fritzsche, Joachim
    Wigenius, Jens
    Nevin, Philip
    Araya, Joshua
    Beuning, Penny
    Modesti, Mauro
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Westerlund, Fredrik
    Probing Physical Properties of a DNA-Protein Complex Using Nanofluidic Channels2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 2, p. 428A-429AArticle in journal (Other academic)
    Abstract [en]

    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.

  • 6. Frykholm, Karolin
    et al.
    Freitag, Camilla
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Tegenfeldt, Jonas O.
    Graneli, Annette
    Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad512013In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 443, no 2, p. 261-268Article in journal (Refereed)
    Abstract [en]

    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.

  • 7.
    Hammar, Petter
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Walldén, Mats
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Fange, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Baltekin, Özden
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Ullman, Gustaf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Leroy, Prune
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Transcription factor dissociation measurements using single molecule chase in living cellsManuscript (preprint) (Other academic)
  • 8.
    Hammar, Petter
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Walldén, Mats
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fange, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Baltekin, Özden
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ullman, Gustaf
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Leroy, Prune
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Direct measurement of transcription factor dissociation excludes a simple operator occupancy model for gene regulation2014In: Nature Genetics, ISSN 1061-4036, E-ISSN 1546-1718, Vol. 46, no 4, p. 405-+Article in journal (Refereed)
    Abstract [en]

    Transcription factors mediate gene regulation by site-specific binding to chromosomal operators. It is commonly assumed that the level of repression is determined solely by the equilibrium binding of a repressor to its operator. However, this assumption has not been possible to test in living cells. Here we have developed a single-molecule chase assay to measure how long an individual transcription factor molecule remains bound at a specific chromosomal operator site. We find that the lac repressor dimer stays bound on average 5 min at the native lac operator in Escherichia coli and that a stronger operator results in a slower dissociation rate but a similar association rate. Our findings do not support the simple equilibrium model. The discrepancy with this model can, for example, be accounted for by considering that transcription initiation drives the system out of equilibrium. Such effects need to be considered when predicting gene activity from transcription factor binding strengths.

  • 9. Nyberg, L. K.
    et al.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Akerman, B.
    Westerlund, F.
    Assessing how cationic intercalators affect DNA using nanofluidic channels2013In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 42, p. S202-S202Article in journal (Other academic)
  • 10. Nyberg, Lena K.
    et al.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Berg, Johan
    Bergstrom, Johanna
    Fransson, Emelie
    Olsson, Linnea
    Persson, Moa
    Stalnacke, Antti
    Wigenius, Jens
    Tegenfeldt, Jonas O.
    Westerlund, Fredrik
    A single-step competitive binding assay for mapping of single DNA molecules2012In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 417, no 1, p. 404-408Article in journal (Refereed)
    Abstract [en]

    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.

  • 11. Nyberg, Lena
    et al.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Akerman, Bjorn
    Westerlund, Fredrik
    Assessing How Cationic Intercalators Affect DNA using Nanofluidic Channels2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 2, p. 165A-165AArticle in journal (Other academic)
  • 12. Nyberg, Lena
    et al.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Akerman, Bjorn
    Westerlund, Fredrik
    Heterogeneous staining: a tool for studies of how fluorescent dyes affect the physical properties of DNA2013In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 19, p. e184-Article in journal (Refereed)
    Abstract [en]

    The commonly used fluorescent dye YOYO-1 (YOYO) has, using bulk techniques, been demonstrated to stain DNA heterogeneously at substoichiometric concentrations. We here, using nanofluidic channels and fluorescence microscopy, investigate the heterogeneous staining on the single DNA molecule level and demonstrate that the dye distribution is continuous. The equilibration of YOYO on DNA is extremely slow but can be accelerated by increasing the ionic strength and/or the temperature. Furthermore, we demonstrate how to use the heterogeneous staining as a tool for detailed and time-efficient studies of how fluorescent dyes affect the physical properties of DNA. We show that the relative increase in extension of DNA with increasing amount of YOYO bound is higher at low ionic strengths and also extrapolate the extension of native DNA. Our study reveals important information on how YOYO affects the physical properties of DNA, but it also has broader applications. First, it reveals how cationic intercalators, such as potential DNA drugs, affect DNA under strong confinement. Second, the strategy of using heterogeneous staining is of general use for single molecule studies of DNA interacting with proteins or ligands.

  • 13.
    Persson, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Barkefors, Irmeli
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Single molecule methods with applications in living cells2013In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 24, no 4, p. 737-744Article, review/survey (Refereed)
    Abstract [en]

    Our knowledge about dynamic processes in biological cells systems has been obtained roughly on two levels of detail; molecular level experiments with purified components in test tubes and system wide experiments with indirect readouts in living cells. However, with the development of single molecule methods for application in living cells, this partition has started to dissolve. It is now possible to perform detailed biophysical experiments at high temporal resolution and to directly observe processes at the level of molecules in living cells. In this review we present single molecule methods that can easily be implemented by readers interested to venture into this exciting and expanding field. We also review some recent studies where single molecule methods have been used successfully to answer biological questions as well as some of the most common pitfalls associated with these methods.

  • 14.
    Persson, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fritzsche, Joachim
    Mir, Kalim U.
    Modesti, Mauro
    Westerlund, Fredrik
    Tegenfeldt, Jonas O.
    Lipid-Based Passivation in Nanofluidics2012In: Nano letters (Print), ISSN 1530-6984, E-ISSN 1530-6992, Vol. 12, no 5, p. 2260-2265Article in journal (Refereed)
    Abstract [en]

    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.

  • 15.
    Persson, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Linden, Martin
    Unoson, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    A Bayesian Approach to Single Particle Tracking Analysis2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 2, p. 177A-177AArticle in journal (Other academic)
  • 16.
    Persson, Fredrik
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Linden, Martin
    Unoson, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Extracting intracellular diffusive states and transition rates from single-molecule tracking data2013In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 10, no 3, p. 265-269Article in journal (Refereed)
    Abstract [en]

    We provide an analytical tool based on a variational Bayesian treatment of hidden Markov models to combine the information from thousands of short single-molecule trajectories of intracellularly diffusing proteins. The method identifies the number of diffusive states and the state transition rates. Using this method we have created an objective interaction map for Hfq, a protein that mediates interactions between small regulatory RNAs and their mRNA targets.

  • 17.
    Sanamrad, Arash
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Persson, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lundius, Ebba G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fange, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gynnå, Arvid H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid2014In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 31, p. 11413-11418Article in journal (Refereed)
    Abstract [en]

    Biochemical and genetic data show that ribosomes closely follow RNA polymerases that are transcribing protein-coding genes in bacteria. At the same time, electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can be reconciled if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery. However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with cotranscriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

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