A novel class of polymeric fluorescent dyes assembled using a DNA synthesizer.

In the pursuit of a novel class of fluorescent dyes we have developed a programmable polymer system that enables the rational design and control of macromolecular constructs through simple control of polymer primary sequence. These polymers are assembled using standard phosphoramidite chemistry on a DNA synthesizer which allows for extremely rapid prototyping and enables many permutations due to the large selection of phosphoramidite monomers presently available on the market. This programmability to some extent allows us to control the interactions/spacing of payload molecules distributed along the designed polymeric backbone.

PI3K is critical for the nuclear translocation of IRF-7 and type I IFN production by human plasmacytoid predendritic cells in response to TLR activation.

Plasmacytoid predendritic cells (pDCs) are the main producers of type I interferon (IFN) in response to Toll-like receptor (TLR) stimulation. Phosphatidylinositol-3 kinase (PI3K) has been shown to be activated by TLR triggering in multiple cell types; however, its role in pDC function is not known. We show that PI3K is activated by TLR stimulation in primary human pDCs and demonstrate, using specific inhibitors, that PI3K is required for type I IFN production by pDCs, both at the transcriptional and protein levels.

Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation.

Human plasmacytoid dendritic cells (PDCs) can produce interferon (IFN)-alpha and/or mature and participate in the adaptive immune response. Three classes of CpG oligonucleotide ligands for Toll-like receptor (TLR)9 can be distinguished by different sequence motifs and different abilities to stimulate IFN-alpha production and maturation of PDCs. We show that the nature of the PDC response is determined by the higher order structure and endosomal location of the CpG oligonucleotide.

Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis.

We describe a novel multiplexing technology using a library of small fluorescent molecules, termed eTag molecules, to code and quantify mRNA targets. eTag molecules, which have the same fluorometric property, but distinct charge-to-mass ratios possess pre-defined electrophoretic characteristics and can be resolved using capillary electrophoresis. Coupled with primary Invader mRNA assay, eTag molecules were applied to simultaneously quantify up to 44 mRNA targets.

Integrity of duplex structures without hydrogen bonding: DNA with pyrene paired at abasic sites.

DNA polymerases specifically insert the hydrophobic pyrene deoxynucleotide (P) opposite tetrahydrofuran (F), an stable abasic site analog, and DNA duplexes containing this non-hydrogen-bonded pair possess a high degree of thermodynamic stability. These observations support the hypothesis that steric complementarity and stacking interactions may be sufficient for maintaining stability of DNA structure and specificity of DNA replication, even in the absence of hydrogen bonds across the base pair. Here we report the NMR characterization and structure determination of two DNA molecules containing pyrene residues.