RNA interference is a mechanism by which double-stranded RNA triggers the loss of RNA of homologous sequence (1). Long double strand RNAs are processed by the double strand endonuclease Dicer into short RNA duplexes (siRNA) 2 ranging from 21 to 23 nucleotides in length (2). The double strand RNA-binding proteins Dicer and human immunodeficiency virus, type 1, trans-activating response RNA-binding protein (TRBP) transfer the siRNAs to the RNA-induced silencing complex (RISC) (3). The antisense strand of the siRNA binds to the RISC endonuclease Argonaute 2 (Ago2), which then cleaves the target mRNA at a single phosphodiester bond bridging the ribonucleotides opposing the 10th and 11th nucleotide from the 5Ј terminus of the antisense strand (4 -11).The structure-activity relationships of siRNAs in human cultured cells have been studied extensively, but these types of studies offer few insights into the underlying mechanisms contributing to the observed activities of the siRNA and, in particular, their interaction with the RISC endonuclease human Ago2. Surprisingly, the little that is known about the interaction between human Ago2 and the substrate comes from a single report describing the preliminary characterization of recombinant human Ago2 (11). Specifically, human Ago2 cleavage activity was magnesium-dependent, and the antisense RNA containing a phosphate at the 5Ј terminus exhibited greater cleavage activity compared with the antisense RNA with a 5Ј-hydroxyl. The enzyme was unable to cleave a DNA target or use a DNA antisense strand to trigger the cleavage of a complementary RNA (11). In addition, UV cross-linking experiments showed that single strand but not double strand RNA was able to cross-link with the recombinant enzyme. Finally, unlike RISC activity from cellular extracts, which has been shown to catalyze multiple rounds of cleavage, recombinant Ago2 exhibited single-turnover kinetics (11,12).The architecture of the human Ago2 protein consists of a PIWI domain at the amino terminus, a centrally located Mid domain and a PAZ domain at the carboxyl terminus (13-17). The PIWI domain constitutes the catalytic domain of the enzyme and exhibits a three-dimensional structure similar to RNase H, sharing the same aspartic acid-aspartic acid-glutamic acid (DDE) catalytic triad and metal cofactor requirements (10,16,17). Recently, the structures of argonaute from Thermus thermophilus and Archaeoglobus fulgidus bound to the antisense strand have been solved (15,18). The structures show that the PAZ, Mid, and PIWI domains form an extended nucleic acid binding surface for the antisense strand. In addition, a basic binding pocket positioned within the Mid domain and a basic cleft in the PIWI domain were shown to bind, respectively, the 5Ј-terminal phosphate and the backbone at the 5Ј-pole of the antisense strand (15,18). Aside from the two 3Ј-terminal nucleotides of the antisense strand, which were shown to bind a hydrophobic pocket within the PAZ domain, no interactions were observed between the enzyme and the 3Ј-pol...

Administration of small interfering RNAs (siRNAs) leads to degradation of specific mRNAs utilizing the cellular RNA interference (RNAi) machinery. It has been demonstrated that co-administration of siRNAs may lead to attenuation of activity of one of the siRNAs. Utilizing antisense and siRNA-mediated RNA-induced silencing complex (RISC) gene reduction we show that siRNA competition is correlated with differences in the cellular expression levels of Ago2, while levels of other RISC proteins have no effect on competition. We also show that under certain conditions siRNA competition rather than reduction of cellular RISC levels may be responsible for apparent reduction in siRNA activity. Furthermore, exploiting siRNA competition, we show that the RISC pathway loads and results in detectable cleavage of the target RNA in ∼2 h after transfection. The RISC pathway is also capable of being reloaded even in the absence of new protein synthesis. RISC reloading and subsequent induction of detectable cleavage of a new target RNA, requires about 9–12 h following the initial transfection.

Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.

Human RNase H1 cleaves RNA exclusively in an RNA/ DNA duplex; neither double-strand DNA nor doublestrand RNA is a viable substrate. Previous studies suggest that the helical geometry and sugar conformation of the DNA and RNA may play a role in the selective recognition of the heteroduplex substrate by the enzyme. We systematically evaluated the influence of sugar conformation, minor groove bulk, and conformational flexibility of the heteroduplex on enzyme efficiency. Modified nucleotides were introduced into the oligodeoxyribonucleotide at the catalytic site of the heteroduplex and consisted of southern, northern, and eastern biased sugars with and without 2-substituents, non-hydrogen bonding base modifications, abasic deoxyribonucleotides, intranucleotide hydrocarbon linkers, and a ganciclovir-modified deoxyribonucleotide. Heteroduplexes containing modifications exhibiting strong northern or southern conformational biases with and without a bulky 2-substituent were cleaved at a significantly slower rate than the unmodified substrate. Modifications imparting the greatest degree of conformational flexibility were the poorest substrates, resulting in dramatically slower cleavage rates for the ribonucleotide opposing the modification and the surrounding ribonucleotides. Finally, modified heteroduplexes containing modifications predicted to mimic the sugar pucker and conformational flexibility of the deoxyribonucleotide exhibited cleavage rates comparable with those of the unmodified substrate. These data suggest that sugar conformation, minor groove width, and the relative positions of the intra-and internucleotide phosphates are the crucial determinants in the selective recognition of the heteroduplex substrate by human RNase H1 and offer immediate steps to improve the performance of DNA-like antisense oligonucleotides.RNase H hydrolyzes RNA in RNA-DNA hybrids (1). RNase H activity appears to be ubiquitous in eukaryotes and bacteria (2-7). Although RNases H constitute a family of proteins of varying molecular mass, the nucleolytic activity and substrate requirements appear to be similar for the various isotypes. For example, all RNases H studied to date function as endonucleases, exhibiting limited sequence specificity and requiring divalent cations (e.g. Mg 2ϩ and Mn 2ϩ ) to produce cleavage products with 5Ј-phosphate and 3Ј-hydroxyl termini (8). Recently, two human RNase H genes have been cloned and expressed (9 -11). RNase H1 is a 286-amino acid protein and is expressed ubiquitously in human cells and tissues (9). The amino acid sequence of human RNase H1 displays strong homology to RNase H1 from yeast, chicken, Escherichia coli, and mouse (9). The human RNase H2 enzyme is a 299-amino acid protein with a calculated mass of 33.4 kDa and has also been shown to be ubiquitously expressed in human cells and tissues (10).1 Human RNase H2 shares strong amino acid sequence homology with RNase H2 from Caenorhabditis elegans, yeast, and E. coli (10). Although the biological roles for the human enzymes are not fully under...

To better understand the factors that influence the activity and specificity of antisense oligonucleotides (ASOs), we designed a minigene encoding superoxide dismutase 1 (SOD-1) and cloned the minigene into vectors for T7 transcription of pre-mRNA and splicing in a nuclear extract or for stable integration in cells. We designed a series of ASOs that covered the entire mRNA and determined the binding affinities and activities of the ASOs in a cell-free system and in cells. The mRNA bound known RNA-binding proteins on predicted binding sites in the mRNA. The higher order structure of the mRNA had a significantly greater effect than the RNA-binding proteins on ASO binding affinities as the ASO activities in cells and in the cell-free systems were consistent. We identified several ASOs that exhibited off-target hybridization to the SOD-1 minigene mRNA in the cell-free system. Off-target hybridization occurred only at highly accessible unstructured sites in the mRNA and these interactions were inhibited by both the higher order structure of the mRNA and by RNA-binding proteins. The same off-target hybridization interactions were identified in cells that overexpress E. coli RNase H1. No off-target activity was observed for cells expressing only endogenous human RNase H1. Neither were these off-target heteroduplexes substrates for recombinant human RNase H1 under multiple-turnover kinetics suggesting that the endogenous enzyme functions under similar kinetic parameters in cells and in the cell-free system. These results provide a blueprint for design of more potent and more specific ASOs.