In RNA interference (RNAi), short double-stranded RNA (known as siRNA) inhibits expression from homologous genes. Clinical or pre-clinical use of siRNAs is likely to require stabilizing modifications because of the prevalence of intracellular and extracellular nucleases. In order to examine the effect of modification on siRNA efficacy and stability, we developed a new method for synthesizing stereoregular boranophosphate siRNAs. This work demonstrates that boranophosphate siRNAs are consistently more effective than siRNAs with the widely used phosphorothioate modification. Furthermore, boranophosphate siRNAs are frequently more active than native siRNA if the center of the antisense strand is not modified. Boranophosphate modification also increases siRNA potency. The finding that boranophosphate siRNAs are at least ten times more nuclease resistant than unmodified siRNAs may explain some of the positive effects of boranophosphate modification. The biochemical properties of boranophosphate siRNAs make them promising candidates for an RNAi-based therapeutic.

The human papillomavirus oncoproteins E6 and E7 promote cell proliferation and contribute to carcinogenesis by interfering with the activities of cellular tumor suppressors. We used a small interfering RNA molecule targeting the E7 region of the bicistronic E6 and E7 mRNA to induce RNA interference, thereby reducing expression of E6 and E7 in HeLa cells. RNA interference of E6 and E7 also inhibited cellular DNA synthesis and induced morphological and biochemical changes characteristic of cellular senescence. These results demonstrate that reducing E6 and E7 expression is sufficient to cause HeLa cells to become senescent.Human papillomavirus (HPV) infection contributes to the development of over 90% of anogenital cancers (2). HPVs are oncogenic because they lack the DNA and RNA polymerases required for the viral life cycle and therefore must induce production of host cell replication proteins by driving the host cell into a proliferative state. The HPV oncoproteins E6 and E7 direct the progression of the cell cycle and play a major role in HPV-induced carcinogenesis by interfering with host cell regulatory proteins (14). The E7 protein inhibits the function of the retinoblastoma (Rb) family proteins, leading to cell cycle progression (13). In response to abnormal E7-driven proliferation, the host cell triggers apoptosis or senescence by activating the p53 tumor suppressor. To defeat this antiproliferative host cell response, HPVs produce the E6 protein, which targets p53 for degradation (12).While E6 and E7 mask the activities of p53 and Rb proteins, HPV-induced malignancies generally maintain wild-type copies of the p53 and Rb genes. This suggests that reducing E6 and E7 expression in cells transformed with HPV would restore the activity of endogenous tumor suppressors and, thus, prevent proliferation of cells transformed with E6 and E7. Several laboratories have shown that the introduction of the HPV transcriptional regulator E2 into cells transformed with HPV induces either apoptosis or senescence, at least in part by inhibiting E6 and E7 expression (4,7,8,9,17). These studies raise the question of whether E2 expression is necessary to inhibit growth or whether a reduction in E6 and E7 expression alone would be sufficient. In order to determine the effects of reducing E6 and E7 levels in cells without the introduction of additional proteins, we used RNA interference (RNAi).RNAi is a process in which double-stranded RNA (dsRNA) homologous to a specific mRNA dramatically inhibits the expression of the protein made from that mRNA (3, 16). It has been shown that small interfering dsRNA of approximately 21 bases (siRNA) potently induces RNAi in mammalian cells without provoking a nonspecific interferon response (6). Using RNAi to specifically reduce expression of E6 and E7, we found that inhibiting E6 and E7 expression alone is sufficient to induce senescence in HeLa cells.siRNA targeting E7 reduces expression of the E6 and E7 viral oncogenes in HeLa cells. To inhibit E6 and E7 expression in HeLa cells (a cell lin...

BackgroundWe present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development.ResultsThe genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements.ConclusionsAnalyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution.

Elucidation of maternal immune correlates of protection against congenital cytomegalovirus (CMV) is necessary to inform future vaccine design. Here, we present a novel rhesus macaque model of placental rhesus CMV (rhCMV) transmission and use it to dissect determinants of protection against congenital transmission following primary maternal rhCMV infection. In this model, asymptomatic intrauterine infection was observed following i.v. rhCMV inoculation during the early second trimester in two of three rhCMV-seronegative pregnant females. In contrast, fetal loss or infant CMV-associated sequelae occurred in four rhCMV-seronegative pregnant macaques that were CD4 + T-cell depleted at the time of inoculation. Animals that received the CD4 +