Abstract: Aspects of the present invention include the production and use of chemically modified RNAi agents (e.g., shRNAs) in gene silencing applications. The chemically modified RNAi agents disclosed herein have reduced immunostimulatory activity, increased serum stability, or both, as compared to a corresponding RNAi agent not having the chemical modification. Compositions containing chemically modified RNAi agents according to aspects of the present invention (including pharmaceutical compositions) and kits containing the same are also provided.

Abstract: Methods, compositions, and kits that include small hairpin RNA (shRNA) useful for inhibition of gene expression, such as viral-mediated gene expression, are described.

Abstract: We describe new hybridization probes and methods for their use in detection, identification, and quantitation of polynucleotides such as RNA and DNA. Ordinary short oligonucleotide probes usually provide higher sequence-specificity but lower efficacy of hybridization than longer ordinary polynucleotide probes where both are fully complementary to the target polynucleotide. Our new polynucleotide probes combine the hybridization efficacy of long probes with the sequence-specificity of short probes. The polynucleotide probes contain a target binding domain and a binding enhancer domain, where the binding enhancer domain does not for stable structures under hybridizing conditions with the target binding domain or its corresponding target. These binding enhancer domains are able to improve the hybridization features of the target binding domain as well as the signal-to-noise ratio for target detection.

Abstract: The invention provides allosterically regulatable polynucleotides capable of target-dependent circularization and topological linkage to a target nucleic acid molecule. Polynucleotides of the invention include a target binding sequence and a regulatory element which prevents circularization in the absence of the target binding. Polynucleotides may include a catalytic domain, allowing circularization to proceed via catalysis when the target binding sequence of the polynucleotide is bound to the target. Topologically linked polynucleotides may be used for detection of target molecules or to inhibit transcription or translation of the target.

Abstract: 5.5 Angstrom resolution x-ray crystallographic structures of 70S ribosome complexes containing messenger RNA and tranfer RNA (tRNA), or tRNA analogs, are provided. The resolution has been enhanced by fitting atomic resolution structures of 30S and 50S subunits onto the 5.5 anstrong electron density map. The enhanced structure reveals regions of structural differences between the 70S complex and the structures of the individual 30S and 50S components. Pharmacophore design to discover novel inhibitors or activators may be carried out using the enhanced 5.5 Angstrom 70S structure.

Abstract: Methods of preparing gene-specific oligonucleotide libraries are disclosed. In one embodiment a double-stranded RNA corresponding to both sense and antisense strands of mRNA is digested by ribonuclease to produce short RNA fragments. In subsequent ligation steps, flanking oligoribonucleotides of defined sequences may be attached to the 3- and 5-ends of each fragment by RNA ligase (such as T4 RNA ligase). The products of ligation can be reverse transcribed and PCR amplified (RT-PCR) using the oligonucleotides attached to the gene-derived sequences as primer-binding sites. Various methods for incorporating libraries into expression vectors allowing expression of either siRNAs or shRNAs are also disclosed.

Abstract: Structures of 70S ribosome complexes containing messenger RNA and transfer RNA (tRNA), or tRNA analogs, have been solved by x-ray crystallography at up to 5.5 Angstrom resolution. Many details of the interactions between tRNA and the ribosome, and of the packing arrangement of ribosomal RNA (rRNA) helices in and between the ribosomal subunits can be seen. Numerous contacts are made between the 30S subunit and the P-tRNA anticodon stem-loop; in contrast, the anticodon region of A-tRNA is much more exposed. A complex network of molecular interactions suggestive of a functional relay is centered around the long penultimate stem of 16S rRNA at the subunit interface, including interactions involving the “switch” helix and decoding site of 16S rRNA and RNA bridges from the 50S subunit. We have enhanced the resolution our 5.5 Angstrom resolution map by fitting atomic resolution structures of 30S and 50S subunits onto our 5.5 Angstrom electron density map.