Dicer processes long double-stranded RNA (dsRNA) and pre-microRNAs to generate the functional intermediates (short interfering RNAs and microRNAs) of the RNA interference pathway. Here we identify features of RNA structure that affect Dicer specificity and efficiency. The data presented show that various attributes of the 3 end structure, including overhang length and sequence composition, play a primary role in determining the position of Dicer cleavage in both dsRNA and unimolecular, short hairpin RNA (shRNA). We also demonstrate that siRNA end structure affects overall silencing functionality. Awareness of these new features of Dicer cleavage specificity as it is related to siRNA functionality provides a more detailed understanding of the RNAi mechanism and can shape the development of hairpins with enhanced functionality.

Long (27-29-bp dsRNA) Dicer-dependent substrates have been identified as potent mediators of RNAi-induced gene knockdown in HEK293 and HeLa cells. As the lengths of these molecules are reported to be below the threshold generally regarded as necessary for induction of the mammalian interferon (IFN) response, these long siRNA are being considered as RNAi substrates in both research and therapeutic settings. In this report, we demonstrate that >23-bp dsRNA can influence cell viability and induce a potent IFN response (highlighted by a strong up-regulation of the dsRNA receptor, Toll-like receptor 3) in a cell typespecific manner. This finding suggests that the length threshold for siRNA induction of the IFN response is not fixed but instead varies significantly among different cell types. Given the diversity of cell types that comprise whole organisms, these findings suggest great care should be taken when considering length variations of dsRNA molecules for RNAi experimentation, especially in therapeutic applications.

The local structure of nucleic acids can be determined from traditional solution NMR techniques, but it is usually not possible to uniquely define the global conformation of DNA or RNA double helices. This results from the short-range nature of the NOE-distance and torsion angle constraints used in generating the solution structures. However, new alignment techniques make it possible to readily measure residual dipolar couplings, which provide information on the relative orientation of individual bond vectors in the molecule. To determine the effects of incorporating dipolar couplings in the structure determinations of nucleic acids, molecular dynamics calculations were performed with simulated constraints derived from two DNA duplex target molecules. Refinements that included NOE, torsion angle, and dipolar coupling constraints were compared to refinements without dipolar couplings. These results show that dipolar couplings significantly improved the local structure while also dramatically improving the global structure of DNA duplexes. The model simulations also illustrate that molecular dynamics calculations induce changes in the local structure before the global structure, which can have important implications for refinements with dipolar coupling constraints. Results are presented that show that the inclusion of dipolar coupling constraints makes it possible to accurately and precisely reproduce the overall helical bend in a DNA duplex. The implications of including dipolar coupling constraints in defining DNA global structure and DNA bending in solution will be discussed.

Although the functional parameters of microRNAs (miRNAs) have been explored in some depth, the roles of these molecules in viral infections remain elusive. Here we report a general method for global analysis of miRNA function that compares the significance of both overexpressing and inhibiting each mouse miRNA on the growth properties of different viruses. Our comparative analysis of representatives of all three herpesvirus subfamilies identified host miRNAs with broad anti-and proviral properties which extend to a singlestranded RNA virus. Specifically, we demonstrate the broad antiviral capacity of miR-199a-3p and illustrate that this individual hostencoded miRNA regulates multiple pathways required and/or activated by viruses, including PI3K/AKT and ERK/MAPK signaling, oxidative stress signaling, and prostaglandin synthesis. Global miRNA expression analysis further demonstrated that the miR-199a/miR-214 cluster is down-regulated in both murine and human cytomegalovirus infection and manifests similar antiviral properties in mouse and human cells. Overall, we report a general strategy for examining the contributions of individual host miRNAs in viral infection and provide evidence that these molecules confer broad inhibitory potential against multiple viruses.RNAi | herpesvirus | RNA virus | RNA processing | phosphatidylinositol-3-kinase-Akt signalling S ince the discovery of the first microRNA (miRNA) in Caenorhabditis elegans, research in diverse organisms has illuminated the role of this class of small RNA in a wide range of cellular processes (reviewed in ref.

The discovery that the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) acquired immune system can be utilized to create double-strand breaks (DSBs) in eukaryotic genomes has resulted in the ability to create genomic changes more easily than with other genome engineering techniques. While there is significant potential for the CRISPR-Cas9 system to advance basic and applied research, several unknowns remain, including the specificity of the RNA-directed DNA cleavage by the small targeting RNA, the CRISPR RNA (crRNA). Here we describe a novel synthetic RNA approach that allows for high-throughput gene editing experiments. This was used with a functional assay for protein disruption to perform high-throughput analysis of crRNA activity and specificity. We performed a comprehensive test of target cleavage using crRNAs that contain one and two nucleotide mismatches to the DNA target in the 20mer targeting region of the crRNA, allowing for the evaluation of hundreds of potential mismatched target sites without the requirement for the off-target sequences and their adjacent PAMs to be present in the genome. Our results demonstrate that while many crRNAs are functional, less than 5% of crRNAs with two mismatches to their target are effective in gene editing; this suggests an overall high level of functionality but low level of off-targeting.