Capture of unstable protein complex on the streptavidin-coated single-walled carbon nanotubes

Purification of unstable protein complexes is a bottleneck for investigation of their 3D structure and in protein-protein interaction studies. In this paper, we demonstrate that streptavidin-coated single-walled carbon nanotubes (Strep center dot SWNT) can be used to capture the biotinylated DNA-EcoRI complexes on a 2D surface and in solution using atomic force microscopy and electrophoresis analysis, respectively. The restriction enzyme EcoRI forms unstable complexes with DNA in the absence of Mg2+ Capturing the EcoRI-DNA complexes on the Strep center dot SWNT succeeded in the absence of Mg2+, demonstrating that the Strep center dot SWNT can be used for purifying unstable protein complexes

Selective coordination of three transition metal ions within a coiled-coil peptide scaffold

Designing peptides that fold and assemble in response to metal ions tests our understanding of how peptide folding and metal binding influence one another. Here, histidine residues are introduced into the hydrophobic core of a coiled-coil trimer, generating a peptide that self-assembles upon the addition of metal ions. HisAD, the resulting peptide, is unstructured in the absence of metal and folds selectively to form an α-helical construct upon complexation with Cu(II) and Ni(II) but not Co(II) or Zn(II). The structure, and metal-binding ability, of HisAD is probed using a combination of circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. These show the peptide is trimeric and binds to both Cu(II) and Ni(II) in a 1 : 1 ratio with the histidine residues involved in the metal coordination, as designed. The X-ray crystal structure of the HisAD-Cu(II) complex reveals the trimeric HisAD peptide coordinates three Cu(II) ions; this is the first example of such a structure. Additionally, HisAD demonstrates an unprecedented discrimination between transition metal ions, the basis of which is likely to be related to the stability of the peptide-metal complexes formed.The work was supported by the NWO via a VENI grant (722.015.006) to ALB. MR acknowledges funding from the European Union’s Horizon 2020 research and innovation programme through a Marie Skłodowska-Curie Grant (Agreement No. 705857). A VICI grant (724.014.001) awarded to AK funded NSAC. GGR is supported by a European Research Council Advanced Grant (340764). NSP acknowledges the support of an NWO ZonMW grant (91116025). NS acknowledges grant number BIO2015-64216-P (MINECO/AEI/FEDER/UE)

Ultra-small graphene oxide functionalized with polyethylenimine (PEI) for very efficient gene delivery in cell and zebrafish embryos

Efficient DNA delivery is essential for introducing new genes into living cells. However, effective virus-based systems carry risks and efficient synthetic systems that are non-toxic remain to be discovered. The bottle-neck in synthetic systems is cytotoxicity, caused by the high concentration of DNA-condensing compounds required for efficient uptake of DNA. Here we report a polyethyleneimine (PEI) grafted ultra-small graphene oxide (PEI-g-USGO) for transfection. By removing the free PEI and ensuring a high PEI density on small sized graphene, we obtained very high transfection efficiencies combined with very low cytotoxicity. Plasmid DNA could be transfected into mammalian cell lines with up to 95% efficiency and 90% viability. Transfection in zebrafish embryos was 90%, with high viability, compared to efficiencies of 30% or lower for established transfection technologies. This result suggests a novel approach to the design of synthetic gene delivery vehicles for research and therapy

Stable Single-Walled Carbon Nanotube-Streptavidin Complex for Biorecognition

A novel method is described for preparing single walled carbon nanotube (SWNT)-streptavidin complexes via the biotin-streptavidin recognition. The complex shows stability in 18 days, strong biotin recognition capability, and excellent loading capacity (about I streptavidin tetramer per 20 nm of SWNT). capturing biotinylated DNA, fluorophores, and Au nanoparticles (NPs) on the SWNT-streptavidin complexes demonstrates their usefulness as a docking matrix, for instance for electron microscopy Studies, a technique requiring if virtually electron transparent Support