DNA-modification of eukaryotic cells.

A novel bioorthogonal method for the modification of cells with single-stranded DNA oligomers is compared to five alternative methods with respect to labeling efficacy, specificity, and effects on cell viability. The new method is based on oxime ligation of aminooxybiotin to aldehyde groups installed by periodate cleavage of cell-surface glycans, followed by the coupling of preformed DNA-streptavidin conjugates. As compared with two literature-reported methods based on direct coupling of N-hydroxysuccinimidyl (NHS)-DNA or NHS-biotinylation as well as with techniques based on strain-promoted alkyne-azide cycloaddition, this method shows the highest labeling densities and is sufficiently mild to avoid cell damage. Functionality of the DNA tags is demonstrated by DNA-directed immobilization on solid substrates and assembly of small cell aggregates.

Toward multiprotein nanoarrays using nanografting and DNA directed immobilization of proteins.

Atomic force microscopy nanografting was utilized to prepare DNA nanopatches of different sizes (200 x 200 to 1000 x 1000 nm(2)) onto which DNA-protein conjugates can be anchored through DNA-directed immobilization. Height measurements were used to assess the binding of the proteins as well as their subsequent interaction with other components, such as antibodies. The results indicate that nanografted patch arrays are well suited for application in biosensing and could enable the fabrication of multifeature protein nanoarrays.

Tuning of peroxidase activity by covalently tethered DNA oligonucleotides.

We report on the modulation of the peroxidase activity of hybrid catalysts, comprising short DNA oligonucleotides and heme enzymes by means of sequence variation of tethered oligonucleotides. In particular, binary mixtures of native heme (protophorphyrin IX) and single-stranded DNA oligonucleotides as well as the analogous covalent heme-oligonucleotide conjugates were compared with DNA-enzyme conjugates, prepared by reconstitution of apo-myoglobin or apo-horseradisch peroxidase, using the aforementioned covalent heme-oligonucleotide conjugates. In all systems, it was clearly evident that the implemented oligonucleotides markedly influence the catalytic activity in a sequence-dependent matter. Greater than 100-fold changes in catalytic constants were observed, depending on which oligonucleotide was incorporated in the hybrid catalyst. We also observed that the tethered oligomers affect the inhibition of HRP-mediated peroxidation by means of small molecule inhibitors. On the basis of the quantitative description of this phenomenon and consideration of the current state of knowledge, we hypothesize that distinct interactions, such as hydrogen bonding or electrostatic contacts, occur between the oligonucleotides and the heme-containing catalyst, which account for the effects observed.