Etchable plasmonic nanoparticle probes to image and quantify cellular internalization.

There is considerable interest in using nanoparticles as labels or to deliver drugs and other bioactive compounds to cells in vitro and in vivo. Fluorescent imaging, commonly used to study internalization and subcellular localization of nanoparticles, does not allow unequivocal distinction between cell surface-bound and internalized particles, as there is no methodology to turn particles 'off'. We have developed a simple technique to rapidly remove silver nanoparticles outside living cells, leaving only the internalized pool for imaging or quantification. The silver nanoparticle (AgNP) etching is based on the sensitivity of Ag to a hexacyanoferrate-thiosulphate redox-based destain solution. In demonstration of the technique we present a class of multicoloured plasmonic nanoprobes comprising dye-labelled AgNPs that are exceptionally bright and photostable, carry peptides as model targeting ligands, can be etched rapidly and with minimal toxicity in mice, and that show tumour uptake in vivo.

Identification of a second DNA binding site in human DNA methyltransferase 3A by substrate inhibition and domain deletion.

The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with muM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.

Near-IR mediated intracellular uncaging of NO from cell targeted hollow gold nanoparticles.

We demonstrate modulation of nitric oxide release in solution and in human prostate cancer cells from a thiol functionalized cupferron (TCF) absorbed on hollow gold nanoshells (HGNs) using near-infrared (NIR) light. NO release from the TCF-HGN conjugates occurs through localized surface heating due to NIR excitation of the surface plasmon. Specific HGN targeting is achieved through cell surface directed peptides, and excitation with tissue penetrating NIR light provides unprecedented spatio-temporal control of NO delivery to biological targets.