Jens Wigenius

A single-step competitive binding assay for mapping of single DNA molecules

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 of 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.

Turn-On, Fluorescent Nuclear Stains with Live Cell Compatibility

DNA-binding, green and yellow fluorescent probes with excellent brightness and high on/off ratios are reported. The probes are membrane permeable, live-cell compatible, and optimally matched to 405 nm and 514 nm laser lines, making them attractive alternatives to UV-excited and blue emissive Hoechst 33342 and DAPI nuclear stains. Their electronic structure was investigated by optical spectroscopy supported by TD-DFT calculations. DNA binding is accompanied by 27- to 75-fold emission enhancements, and linear dichroism demonstrates that one dye is a groove binder while the other intercalates into DNA.

Probing Physical Properties of a DNA‐Protein Complex Using Nanofluidic Channels

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.

Interactions between a luminescent conjugated polyelectrolyte and amyloid fibrils investigated with flow linear dichroism spectroscopy

Luminescent conjugated polyelectrolytes (LCPs) have emerged as novel stains to detect and distinguish between various amyloidogenic species, including prefibrillar aggregates and mature fibril deposits, both in vitro and in histological tissue samples, offering advantages over traditional amyloid stains. We here use linear dichroism (LD) spectroscopy under shear alignment to characterize interactions between the LCP poly(3-thiophene acetic acid) (PTAA) and amyloid fibrils. The positive signature in the LD spectrum of amyloid-bound PTAA suggests that it binds in the grooves between adjacent protein side-chains in the amyloid fibril core, parallel to the fibril axis, similar to thioflavin-T and congo red. Moreover, using LD we record the absorption spectrum of amyloid-bound PTAA in isolation from free dye showing a red-shift by ca 30 nm compared to in solution. This has important implications for the use of PTAA as an amyloid probe in situ and in vitro and we demonstrate how to obtain optimal amyloid-specific fluorescence read-outs using PTAA. We use the shift in maximum absorption to estimate the fraction of bound PTAA at a given concentration. PTAA binding reaches saturation when added in 36 times excess and at this concentration the PTAA density is 4-5 monomer units per insulin monomer in the fibril. Finally, we demonstrate that changes in LD intensity can be related to alterations in persistence length of amyloid fibrils resulting from changes in solution conditions, showing that this technique is useful to assess macroscopic properties of these biopolymers.

Amyloid fibrils as dispersing agents for oligothiophenes: control of photophysical properties through nanoscale templating and flow induced fibril alignment

Herein we report that protein fibrils formed from aggregated proteins, so called amyloid fibrils, serve as an excellent dispersing agent for hydrophobic oligothiophenes such as alpha-sexithiophene (6T). Furthermore, the protein fibrils are capable of orienting 6T along the fibril long axis, as demonstrated by flow-aligned linear dichroism spectroscopy and polarized fluorescence microscopy. The materials are prepared by solid state mixing of 6T with a protein capable of self-assembly. This results in a water soluble composite material that upon heating in aqueous acid undergoes self-assembly into protein fibrils non-covalently functionalized with 6T, with a typical diameter of 5-10 nm and lengths in the micrometre range. The resulting aqueous fibril dispersions are a readily available source of oligothiophenes that can be processed from aqueous solvent, and we demonstrate the fabrication of macroscopic structures consisting of aligned 6T functionalized protein fibrils. Due to the fibril induced ordering of 6T these structures exhibit polarized light emission.