Measuring DNA hybridization using fluorescent DNA-stabilized silver clusters to investigate mismatch effects on therapeutic oligonucleotides.

BACKGROUND: Short nucleic acid oligomers have found a wide range of applications in experimental physics, biology and medicine, and show potential for the treatment of acquired and genetic diseases. These applications rely heavily on the predictability of hybridization through Watson-Crick base pairing to allow positioning on a nanometer scale, as well as binding to the target transcripts, but also off-target binding to transcripts with partial homology. These effects are of particular importance in the development of therapeutic oligonucleotides, where off-target effects caused by the binding of mismatched sequences need to be avoided. RESULTS: We employ a novel method of probing DNA hybridization using optically active DNA-stabilized silver clusters (Ag-DNA) to measure binding efficiencies through a change in fluorescence intensity. In this way we can determine their location-specific sensitivity to individual mismatches in the sequence. The results reveal a strong dependence of the hybridization on the location of the mismatch, whereby mismatches close to the edges and center show a relatively minor impact. In parallel, we propose a simple model for calculating the annealing ratios of mismatched DNA sequences, which supports our experimental results. CONCLUSION: The primary result shown in this work is a demonstration of a novel technique to measure DNA hybridization using fluorescent Ag-DNA. With this technique, we investigated the effect of mismatches on the hybridization efficiency, and found a significant dependence on the location of individual mismatches. These effects are strongly influenced by the length of the used oligonucleotides. The novel probe method based on fluorescent Ag-DNA functions as a reliable tool in measuring this behavior. As a secondary result, we formulated a simple model that is consistent with the experimental data.

Dynamics of dual-fluorescent polymersomes with durable integrity in living cancer cells and zebrafish embryos.

The long-term fate of biomedical nanoparticles after endocytosis is often only sparsely addressed in vitro and in vivo, while this is a crucial parameter to conclude on their utility. In this study, dual-fluorescent polyisobutylene-polyethylene glycol (PiB-PEG) polymersomes were studied for several days in vitro and in vivo. In order to optically track the vesicles' integrity, one fluorescent probe was located in the membrane and the other in the aqueous interior compartment. These non-toxic nanovesicles were quickly endocytosed in living A549 lung carcinoma cells but unusually slowly transported to perinuclear lysosomal compartments, where they remained intact and luminescent for at least 90 h without being exocytosed. Fluorescence-assisted flow cytometry indicated that after endocytosis, the nanovesicles were eventually degraded within 7-11 days. In zebrafish embryos, the polymersomes caused no lethality and were quickly taken up by the endothelial cells, where they remained fully intact for as long as 96 h post-injection. This work represents a novel case-study of the remarkable potential of PiB-PEG polymersomes as an in vivo bio-imaging and slow drug delivery platform.

Surfactant-free Colloidal Particles with Specific Binding Affinity.

Colloidal particles with specific binding affinity are essential for in vivo and in vitro biosensing, targeted drug delivery, and micrometer-scale self-assembly. Key to these techniques are surface functionalizations that provide high affinities to specific target molecules. For stabilization in physiological environments, current particle coating methods rely on adsorbed surfactants. However, spontaneous desorption of these surfactants typically has an undesirable influence on lipid membranes. To address this issue and create particles for targeting molecules in lipid membranes, we present here a surfactant-free coating method that combines high binding affinity with stability at physiological conditions. After activating charge-stabilized polystyrene microparticles with EDC/Sulfo-NHS, we first coat the particles with a specific protein and subsequently covalently attach a dense layer of poly(ethyelene) glycol. This polymer layer provides colloidal stability at physiological conditions as well as antiadhesive properties, while the protein coating provides the specific affinity to the targeted molecule. We show that NeutrAvidin-functionalized particles bind specifically to biotinylated membranes and that Concanavalin A-functionalized particles bind specifically to the glycocortex of Dictyostelium discoideum cells. The affinity of the particles changes with protein density, which can be tuned during the coating procedure. The generic and surfactant-free coating method reported here transfers the high affinity and specificity of a protein onto colloidal polystyrene microparticles.

Expressional profile of the diacylglycerol kinase eta gene DGKH.

Bipolar disorder (BPD) is a genetically complex mental disorder, which is characterized by recurrent depressive and manic episodes, occurring with a typical cyclical course. In a recent study, we were able to identify a risk haplotype for BPD, as well as for unipolar depression and adult attention-deficit/hyperactivity disorder (ADHD), within the DGKH gene. DGKH codes for the eta (η) isoform of diacylglycerol kinase, which is involved in the phosphoinositol pathway. In the present study, we determined the expressional profile of Dgkh using quantitative real-time PCR (qPCR), in situ hybridization and immunohistological staining in the human and in the mouse brain. Expression studies showed that two different Dgkh transcripts exhibited distinct occurrence in a variety of murine tissues and also differed in their expression levels. The proteins encoded by those transcripts differ in functional protein domains suggesting distinct biochemical and cell biological properties and functions. qPCR analyses revealed an increase in Dgkh expression during mouse brain development indicating a possible role of this kinase in late developmental stages. Immunostainings revealed strong Dgkh expression in neurons of the hippocampus and the cerebellum of the murine brain, whereas highest expression levels of DGKH in the human brain were found in the striatum. Taken together, our studies revealed expressional changes during mouse brain development and occurrence of Dgkη in neurons of regions that have been linked to BPD as well as ADHD in humans providing evidence for the implication of DGKH in those disorders.

Fluorescence-tunable Ag-DNA biosensor with tailored cytotoxicity for live-cell applications.

DNA-stabilized silver clusters (Ag-DNA) show excellent promise as a multi-functional nanoagent for molecular investigations in living cells. The unique properties of these fluorescent nanomaterials allow for intracellular optical sensors with tunable cytotoxicity based on simple modifications of the DNA sequences. Three Ag-DNA nanoagent designs are investigated, exhibiting optical responses to the intracellular environments and sensing-capability of ions, functional inside living cells. Their sequence-dependent fluorescence responses inside living cells include (1) a strong splitting of the fluorescence peak for a DNA hairpin construct, (2) an excitation and emission shift of up to 120 nm for a single-stranded DNA construct, and (3) a sequence robust in fluorescence properties. Additionally, the cytotoxicity of these Ag-DNA constructs is tunable, ranging from highly cytotoxic to biocompatible Ag-DNA, independent of their optical sensing capability. Thus, Ag-DNA represents a versatile live-cell nanoagent addressable towards anti-cancer, patient-specific and anti-bacterial applications.