Intended transcriptional silencing with siRNA results in gene repression through sequence-specific off-targeting.

Transcriptional gene silencing has been reported with siRNA targeting the promoter region of genes. We tested several siRNAs directed against the human VEGF promoter. Of these, siVFp(-992) exhibited > or =50% suppression of VEGF production in two human cell lines.

Sequence determinants of innate immune activation by short interfering RNAs.

Background: Short interfering RNAs (siRNAs) have been shown to induce immune stimulation through a number of different receptors in a range of cell types. In primary cells, both TLR7 and TLR8 have been shown to recognise siRNAs however, despite the identification of a number of TLR7/8 stimulatory RNA motifs, the complete and definitive sequence determinants of TLR7 and TLR8 are yet to be elucidated.

Results: A total of 207 siRNA sequences were screened for TLR7/8 stimulation in human PBMCs.

Interfering ribonucleic acids that suppress expression of multiple unrelated genes.

Background: Short interfering RNAs (siRNAs) have become the research tool of choice for gene suppression, with human clinical trials ongoing. The emphasis so far in siRNA therapeutics has been the design of one siRNA with complete complementarity to the intended target. However, there is a need for multi-targeting interfering RNA in diseases in which multiple gene products are of importance.

Primary leukocyte screens for innate immune agonists.

The innate immune system of mammals is a key defense mechanism against invading foreign pathogens. Innate immune stimulants may have applications as vaccine adjuvants as well as in the treatment of cancer and some viral diseases, and clinical studies have been performed using agonists of Toll-like receptors (TLRs) 7, 8, and 9. The high-throughput screens for such agonists have typically relied on the overexpression of a single TLR gene in an immortalized cell line and are inherently artificial systems that are restricted to the identification of agonists for a single receptor.

Dz13, a DNAzyme targeting c-jun, induces off-target cytotoxicity in endothelial cells with features of nonapoptotic programmed cell death.

We have previously shown that Dz13, a catalytic DNA molecule (DNAzyme) designed against c-jun, is cytotoxic to nonquiescent cells by a mechanism independent of c-jun mRNA cleavage. In this report, we evaluated programmed cell death (PCD) pathways in order to gain further insight into the mechanism of action of Dz13. Using human dermal microvascular endothelial cells (HMEC-1), we found that Dz13-mediated cell death is characterized by mitochondrial depolarization, caspase-8 activation, lysosomal increase, and autophagosome formation.

Cytotoxic G-rich oligodeoxynucleotides: putative protein targets and required sequence motif.

It has recently been shown that certain oligodeoxynucleotides (ODNs) designed as catalytic DNA molecules (DNAzymes) exhibit potent cytotoxicity independent of RNA-cleavage activity in a number of cell lines. These cytotoxic ODNs all featured a 5' G-rich sequence and induced cell death by a TLR9-independent mechanism. In this study, we examined the sequence and length dependence of ODNs for cytotoxicity.

Campylobacter jejuni response to ox-bile stress.

Campylobacter jejuni is a pathogen that colonizes the intestinal tract of humans and some animals. The in vitro responses of the bacterium to ox-bile were studied using proteomics to understand the molecular mechanisms employed by C. jejuni to survive bile stress.

The DNAzymes Rs6, Dz13, and DzF have potent biologic effects independent of catalytic activity.

DNAzymes are catalytic DNA molecules capable of cleaving RNA substrates and therefore constitute a possible gene-suppression technology. We examined whether the previously reported potency of a DNAzyme targeting c-jun (Dz13) could be improved with judicious use of sequence and chemical modifications. Catalytic activity was measured to establish correlations between catalytic activity and biological potency.

Proteomic and computational analysis of secreted proteins with type I signal peptides from the Antarctic archaeon Methanococcoides burtonii.

LC-MS/MS was used to identify secreted proteins in the Antarctic archaeon Methanococcoides burtonii. Seven proteins possessing a classical class 1 signal peptide were identified in the supernatant from cultures grown at 4 and 23 degrees C. The proteins included a putative S-layer cell surface protein, cell surface protein involved with cell adhesion, and trypsin-like serine protease.

Cold adaptation of the Antarctic archaeon, Methanococcoides burtonii assessed by proteomics using ICAT.

Using isotope coded affinity tag (ICAT) chromatography and liquid chromatography-mass spectrometry, 163 proteins were identified from the cold-adapted archaeon, Methanococcoides burtonii. 14 proteins were differentially expressed during growth at 4 degrees C and 23 degrees C. Knowledge of protein abundance, protein identity and gene arrangement was used to determine mechanisms of cold adaptation.

Predicted roles for hypothetical proteins in the low-temperature expressed proteome of the Antarctic archaeon Methanococcoides burtonii.

Using liquid chromatography-mass spectrometry, 528 proteins were identified that are expressed during growth at 4 degrees C in the cold adapted archaeon, Methanococcoides burtonii. Of those, 135 were annotated previously as unique or conserved hypothetical proteins. We have performed a comprehensive, integrated analysis of the latter proteins using threading, InterProScan, predicted subcellular localization and visualization of conserved gene context across multiple prokaryotic genomes.

Biology of the cold adapted archaeon, Methanococcoides burtonii determined by proteomics using liquid chromatography-tandem mass spectrometry.

Genome sequence data of the cold-adapted archaeon, Methanococcoides burtonii, was linked to liquid chromatography-mass spectrometry analysis of the expressed-proteome to define the key biological processes functioning at 4 degrees C. 528 proteins ranging in pI from 3.5 to 13.

Cold adaptation in the Antarctic Archaeon Methanococcoides burtonii involves membrane lipid unsaturation.

Direct analysis of membrane lipids by liquid chromatography-electrospray mass spectrometry was used to demonstrate the role of unsaturation in ether lipids in the adaptation of Methanococcoides burtonii to low temperature. A proteomics approach using two-dimensional liquid chromatography-mass spectrometry was used to identify enzymes involved in lipid biosynthesis, and a pathway for lipid biosynthesis was reconstructed from the M. burtonii draft genome sequence.

A proteomic determination of cold adaptation in the Antarctic archaeon, Methanococcoides burtonii.

A global view of the biology of the cold-adapted archaeon Methanococcoides burtonii was achieved using proteomics. Proteins specific to growth at 4 degrees C versus T(opt) (23 degrees C) were identified by mass spectrometry using the draft genome sequence of M. burtonii.