Systematic screening of LNA/2′-O-methyl chimeric derivatives of a TAR RNA aptamer

AbstractWe synthesized and evaluated by surface plasmon resonance 64 LNA/2′-O-methyl sequences corresponding to all possible combinations of such residues in a kissing aptamer loop complementary to the 6-nt loop of the TAR element of HIV-1. Three combinations of LNA/2′-O-methyl nucleoside analogues where one or two LNA units are located on the 3′ side of the aptamer loop display an affinity for TAR below 1nM, i.e. one order of magnitude higher than the parent RNA aptamer. One of these combinations inhibits the TAR-dependent luciferase expression in a cell assay

A Boost for the Emerging Field of RNA Nanotechnology: Report on the First International Conference on RNA Nanotechnology

This Nano Focus article highlights recent advances in RNA nanotechnology as presented at the First International Conference of RNA Nanotechnology and Therapeutics, which took place in Cleveland, OH, USA (October 23-25, 2010) (http;//www.eng.uc.edu/nanomedidne/RNA2010/), chaired by Peixuan Guo and co-chaired by David Rueda and Scott Tenenbaum. The conference was the first of its kind to bring together more than 30 invited speakers in the frontier of RNA nanotechnology from France, Sweden, South Korea, China, and throughout the United States to discuss RNA nanotechnology and Its applications. It provided a platform for researchers from academia, government, and the pharmaceutical industry to share existing knowledge, vision, technology, and challenges in the field and promoted collaborations among researchers interested in advancing this emerging scientific discipline. The meeting covered a range of topics, including biophysical and single-molecule approaches for characterization of RNA nanostructures; structure studies on RNA nanoparticles by chemical or biochemical approaches, computation, prediction, and modeling of RNA nanoparticle structures; methods for the assembly of RNA nanoparticles; chemistry for RNA synthesis, conjugation, and labeling; and application of RNA nanoparticles in therapeutics. A special invited talk on the well-established principles of DNA nanotechnology was arranged to provide models for RNA nanotechnology. An Administrator from National Institutes of Health (NIH) National Cancer Institute (NCI) Alliance for Nanotechnology in Cancer discussed the current nanocancer research directions and future funding opportunities at NCl. As indicated by the feedback received from the invited speakers and the meeting participants, this meeting was extremely successful, exciting, and informative, covering many groundbreaking findings, pioneering ideas, and novel discoveries

Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex

Eukaryotic RNase H2 is a heterotrimeric enzyme. Here, we show that the biochemical composition and stoichiometry of the human RNase H2 complex is consistent with the properties previously deduced from genetic studies. The catalytic subunit of eukaryotic RNase H2, RNASEH2A, is well conserved and similar to the monomeric prokaryotic RNase HII. In contrast, the RNASEH2B and RNASEH2C subunits from human and Saccharomyces cerevisiae share very little homology, although they both form soluble B/C complexes that may serve as a nucleation site for the addition of RNASEH2A to form an active RNase H2, or for interactions with other proteins to support different functions. The RNASEH2B subunit has a PIP-box and confers PCNA binding to human RNase H2. Unlike Escherichia coli RNase HII, eukaryotic RNase H2 acts processively and hydrolyzes a variety of RNA/DNA hybrids with similar efficiencies, suggesting multiple cellular substrates. Moreover, of five analyzed mutations in human RNASEH2B and RNASEH2C linked to Aicardi-Goutières Syndrome (AGS), only one, R69W in the RNASEH2C protein, exhibits a significant reduction in specific activity, revealing a role for the C subunit in enzymatic activity. Near-normal activity of four AGS-related mutant enzymes was unexpected in light of their predicted impairment causing the AGS phenotype

P2X4 receptors in activated C8-B4 cells of cerebellar microglial origin

We investigated the properties and regulation of P2X receptors in immortalized C8-B4 cells of cerebellar microglial origin. Resting C8-B4 cells expressed virtually no functional P2X receptors, but largely increased functional expression of P2X4 receptors within 2–6 h of entering the activated state. Using real-time polymerase chain reaction, we found that P2X4 transcripts were increased during the activated state by 2.4-fold, but this increase was not reflected by a parallel increase in total P2X4 proteins. In resting C8-B4 cells, P2X4 subunits were mainly localized within intracellular compartments, including lysosomes. We found that cell surface P2X4 receptor levels increased by ∼3.5-fold during the activated state. This change was accompanied by a decrease in the lysosomal pool of P2X4 proteins. We next exploited our findings with C8-B4 cells to investigate the mechanism by which antidepressants reduce P2X4 responses. We found little evidence to suggest that several antidepressants were antagonists of P2X4 receptors in C8-B4 cells. However, we found that moderate concentrations of the same antidepressants reduced P2X4 responses in activated microglia by affecting lysosomal function, which indirectly reduced cell surface P2X4 levels. In summary, our data suggest that activated C8-B4 cells express P2X4 receptors when the membrane insertion of these proteins by lysosomal secretion exceeds their removal, and that antidepressants indirectly reduce P2X4 responses by interfering with lysosomal trafficking

The binding of an antisense oligonucleotide to a hairpin structure via triplex formation inhibits chemical and biological reactions.

We have investigated the binding of a 26-mer antisense oligodeoxynucleotide to a 69-mer DNA hairpin with a 13 base pair stem, bearing an Rsa1 restriction site. The 5' part of the 26-mer annealed to a stretch of six purines at the bottom of the hairpin. The 3' part was designed to fold back to form a triplex with both the stem of the hairpin and with the sequence paired to its own 5' region. Using non-denaturing polyacrylamide gel electrophoresis, melting curves (Tm) and chemical footprinting, we were able to show the formation of a 'double-hairpin' complex between the 69-mer and the 26-mer antisense oligopyrimidines. The association was both sequence and pH-dependent. The formation of a double hairpin complex was shown to prevent the alkylation of the 69-mer DNA target by an oligonucleotide-nitrogen mustard conjugate and to selectively inhibit the action of Rsa1

The binding of T4 gene 32 protein to MS2 virus RNA and transfer RNA.

Fluorescence titrations, absorption spectroscopy and stopped-flow techniques were used to study the interaction of T4 coded 32-protein (P 32) with MS2 RNA and total tRNA from E. coli under different ionic conditions. It is shown that the amount of MS2 RNA and tRNA secondary structure melted by P 32 varies markedly and reversibly within a range of ionic conditions under which the binding constant of P 32 to single-stranded nucleic acids unable to form stable hairpins remains higher than 10(8) M-1. Kinetic experiments suggest that P 32 dissociates from the MS2 RNA rewinding strand with a similar rate constant as calculated for the dissociation from single-stranded regions. Possible in vivo consequences of these findings are discussed