2'-Azido RNA, a versatile tool for chemical biology: synthesis, X-ray structure, siRNA applications, click labeling

Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2'-azido cytidine and 2'-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2'-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2'-azido uridine and 2'-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2'-azido group supports the C3'-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2'-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2'-azido modifications are compatible with 2'-fluoro and/or 2'-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2'-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2'-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification.     

Acetabular cup geometry and bone-implant interference have more influence on initial periprosthetic joint space than joint loading and surgical cup insertion

Environmental variations in patient-dependent and surgical factors were modeled using robust optimization with a finite element acetabular cup-pelvis model. A previously developed statistical optimization scheme was used to: (1) determine the cup geometry and the optimal cup-bone interference that maximized bone-implant contact areas and minimized changes in the gap volume between the implant and bone surface during gait loading and unloading; and (2) determine the relative contributions of design, patient-dependent, and surgical factors to variations in bone-implant contact areas and a change in gap volume. The statistical analyses indicated that the design variables, namely the equatorial diameter and eccentricity, explained most of the variations in the performance measures. Further, the hemispherical designs performed better than the nonhemispherical designs. The 58 mm hemispherical cup, with 2 mm diametral interferences, minimized the change in gap volume and attained 82% and 81% of the maximum predicted total and rim contact areas, respectively. The equatorial diameter and eccentricity, not the patient-dependent and surgical factors, explained most of the variations in the performance measures. Perfect surface apposition was not attained with any of the cup designs.     

The WAG1 and WAG2 protein kinases negatively regulate root waving in Arabidopsis

The WAG1 and WAG2 genes of Arabidopsis thaliana encode protein-serine/threonine kinases that are closely related to PINOID. In order to determine what roles WAG1 and WAG2 play in seedling development, we used a reverse genetics approach to study the wag1, wag2 and wag1/wag2 mutant phenotypes for clues. Although the wag mutants do not contain detectable amounts of the corresponding mRNA, they are wild type in most respects. However, wag1/wag2 double mutants exhibit a pronounced wavy root phenotype when grown vertically on agar plates, a phenotype observed in wild-type plants only on plates inclined to angles less than 90 degrees. The wag1 and wag2 mutants also demonstrate enhanced root waving, but to a lesser extent. Moreover, the double mutant roots are more resistant to the effects of N-1-naphthylphthalamic acid on the inhibition of root curling, raising the possibility that transport of auxin is affected in the wag mutants. Promoter fusions to the gusA reporter gene demonstrate that the WAG promoters are most active in root tips, consistent with the observed phenotypes in the wag mutants.