Quantitative dose-response curves from subcellular lipid multilayer microarrays.

The dose-dependent bioactivity of small molecules on cells is a crucial factor in drug discovery and personalized medicine. Although small-molecule microarrays are a promising platform for miniaturized screening, it has been a challenge to use them to obtain quantitative dose-response curves in vitro, especially for lipophilic compounds. Here we establish a small-molecule microarray assay capable of controlling the dosage of small lipophilic molecules delivered to cells by varying the sub-cellular volumes of surface supported lipid micro- and nanostructure arrays fabricated with nanointaglio. Features with sub-cellular lateral dimensions were found necessary to obtain normal cell adhesion with HeLa cells. The volumes of the lipophilic drug-containing nanostructures were determined using a fluorescence microscope calibrated by atomic-force microscopy. We used the surface supported lipid volume information to obtain EC-50 values for the response of HeLa cells to three FDA-approved lipophilic anticancer drugs, docetaxel, imiquimod and triethylenemelamine, which were found to be significantly different from neat lipid controls. No significant toxicity was observed on the control cells surrounding the drug/lipid patterns, indicating lack of interference or leakage from the arrays. Comparison of the microarray data to dose-response curves for the same drugs delivered liposomally from solution revealed quantitative differences in the efficacy values, which we explain in terms of cell-adhesion playing a more important role in the surface-based assay. The assay should be scalable to a density of at least 10,000 dose response curves on the area of a standard microtiter plate.

Nucleic acid supercoiling as a means for ionic switching of DNA--nanoparticle networks.

Oligomeric nanoparticle networks, generated by the self-assembly of bis-biotinylated double-stranded DNA fragments and streptavidin, have been studied by scanning force microscopy (SFM). SFM imaging revealed the presence within the networks of irregular thick DNA molecules, which were often associated with distinct, Y-shaped structural elements. Closer analysis revealed that the Y structures are formed by condensation (thickening and shortening) of two DNA fragments, most likely through the supercoiling of two DNA molecules bound to adjacent binding sites of the streptavidin particle. The frequency of supercoiling was found to be dependent on the ionic strength applied during the immobilization of the oligomeric networks on mica surfaces. Potential applications of the structural changes as a means for constructing ion-dependent molecular switches in nanomaterials are discussed.

High-quality mapping of DNA-protein complexes by dynamic scanning force microscopy.

Biomolecular complexes, nanocircles and aggregates of DNA and streptavidin (STV) are studied by dynamic scanning force microscopy. More structural details are observed with an improved dynamic mode assisted with a special feedback circuit (Q-control), as shown in the picture. Under otherwise identical conditions, these improvements indicate that the well cited models relating enlarged lateral size to the finite geometry of the SFM tip have to be modified for soft samples susceptible to tip-sample interactions.

Self-assembly of DNA-streptavidin nanostructures and their use as reagents in immuno-PCR.

The self-assembly of bis-biotinylated double-stranded DNA and the tetravalent biotin-binding protein streptavidin (STV) have been studied by non-denaturing gel electrophoresis and atomic force microscopy (AFM). The rapid self-assembly reproducibly generated populations of individual oligomeric complexes. Most strikingly, the oligomers predominantly contained bivalent STV molecules bridging two adjacent DNA fragments to form linear nanostructures. Trivalent STV branch points occurred with a lower frequency and the presence of tetravalent STV was scarce. However, valency distribution, size and the exchange dynamics of the supramolecular aggregates were highly sensitive to stoichiometric variations in the relative molar coupling ratio of bis-biotinylated DNA and STV. The largest aggregates were obtained from equimolar amounts while excess STV led to the formation of smaller oligomers appearing as fingerprint-like band patterns in electrophoresis. Excess DNA, however, induces a complete breakdown of the oligomers, likely a consequence of the instability of STV conjugates containing more than two biotinylated DNA fragments. It was demonstrated that the oligomers can further be functionalized, for instance by the coupling of biotinylated immunoglobulins. Both pure and also antibody-modified DNA-STV oligomers were used as reagents in immuno-PCR (IPCR), a highly sensitive detection method for proteins and other antigens. Employment of the supramolecular reagents led to an approximately 100-fold enhanced sensitivity compared to the conventional IPCR procedure.