Selective Nucleic Acid Capture with Shielded Covalent Probes

Nucleic acid probes are used for diverse applications in vitro, in situ, and in vivo. In any setting, their power is limited by imperfect selectivity (binding of undesired targets) and incomplete affinity (binding is reversible, and not all desired targets bound). These difficulties are fundamental, stemming from reliance on base pairing to provide both selectivity and affinity. Shielded covalent (SC) probes eliminate the longstanding trade-off between selectivity and durable target capture, achieving selectivity via programmable base pairing and molecular conformation change, and durable target capture via activatable covalent cross-linking. In pure and mixed samples, SC probes covalently capture complementary DNA or RNA oligo targets and reject two-nucleotide mismatched targets with near-quantitative yields at room temperature, achieving discrimination ratios of 2–3 orders of magnitude. Semiquantitative studies with full-length mRNA targets demonstrate selective covalent capture comparable to that for RNA oligo targets. Single-nucleotide DNA or RNA mismatches, including nearly isoenergetic RNA wobble pairs, can be efficiently rejected with discrimination ratios of 1–2 orders of magnitude. Covalent capture yields appear consistent with the thermodynamics of probe/target hybridization, facilitating rational probe design. If desired, cross-links can be reversed to release the target after capture. In contrast to existing probe chemistries, SC probes achieve the high sequence selectivity of a structured probe, yet durably retain their targets even under denaturing conditions. This previously incompatible combination of properties suggests diverse applications based on selective and stable binding of nucleic acid targets under conditions where base-pairing is disrupted (e.g., by stringent washes in vitro or in situ, or by enzymes in vivo)

Glycosaminoglycan Binding Facilitates Entry of a Bacterial Pathogen into Central Nervous Systems

Certain microbes invade brain microvascular endothelial cells (BMECs) to breach the blood-brain barrier (BBB) and establish central nervous system (CNS) infection. Here we use the leading meningitis pathogen group B Streptococcus (GBS) together with insect and mammalian infection models to probe a potential role of glycosaminoglycan (GAG) interactions in the pathogenesis of CNS entry. Site-directed mutagenesis of a GAG-binding domain of the surface GBS alpha C protein impeded GBS penetration of the Drosophila BBB in vivo and diminished GBS adherence to and invasion of human BMECs in vitro. Conversely, genetic impairment of GAG expression in flies or mice reduced GBS dissemination into the brain. These complementary approaches identify a role for bacterial-GAG interactions in the pathogenesis of CNS infection. Our results also highlight how the simpler yet genetically conserved Drosophila GAG pathways can provide a model organism to screen candidate molecules that can interrupt pathogen-GAG interactions for future therapeutic applications

Mechanism and Enantioselectivity in Palladium-Catalyzed Conjugate Addition of Arylboronic Acids to β‑Substituted Cyclic Enones: Insights from Computation and Experiment

Enantioselective conjugate additions of arylboronic acids to β-substituted cyclic enones have been previously reported from our laboratories. Air- and moisture-tolerant conditions were achieved with a catalyst derived in situ from palladium(II) trifluoroacetate and the chiral ligand (S)-t-BuPyOx. We now report a combined experimental and computational investigation on the mechanism, the nature of the active catalyst, the origins of the enantioselectivity, and the stereoelectronic effects of the ligand and the substrates of this transformation. Enantioselectivity is controlled primarily by steric repulsions between the t-Bu group of the chiral ligand and the α-methylene hydrogens of the enone substrate in the enantiodetermining carbopalladation step. Computations indicate that the reaction occurs via formation of a cationic arylpalladium(II) species, and subsequent carbopalladation of the enone olefin forms the key carbon–carbon bond. Studies of nonlinear effects and stoichiometric and catalytic reactions of isolated (PyOx)Pd(Ph)I complexes show that a monomeric arylpalladium–ligand complex is the active species in the selectivity-determining step. The addition of water and ammonium hexafluorophosphate synergistically increases the rate of the reaction, corroborating the hypothesis that a cationic palladium species is involved in the reaction pathway. These additives also allow the reaction to be performed at 40 °C and facilitate an expanded substrate scope

Sensitive and selective nucleic acid capture with shielded covalent probes

Nucleic acid probes are used for diverse applications in vitro, in situ, and in vivo. In any setting, their power is limited by imperfect selectivity (binding of undesired targets) and incomplete affinity (binding is reversible and not all desired targets are bound). These limitations stem from reliance on base pairing to both reject off-targets and retain desired targets. To address this selectivity/affinity tradeoff, shielded covalent probes achieve selectivity via conformation change and durable capture via covalent crosslinking of a photoactive nucleoside analog. In vitro assays show that mismatches are efficiently rejected and desired targets are durably captured. For probes designed to reject two-nucleotide mismatches, desired targets are captured nearly quant. Single-nucleotide mismatches are discriminated near the thermodn. limit. The probes operate isothermally and crosslinking activation is rapid with low-cost light sources. If desired, crosslinks can be reversed to release the target after capture. We envision a wide array of applications

Novel male trait prolongs survival in suicidal mating

Male redback spiders (Latrodectus hasselti) maximize paternity if they copulate twice with their cannibalistic mate. Facilitating cannibalistic attack during their first copulation yields paternity benefits. However, females have paired sperm-storage organs inseminated during two separate copulations, so males that succumb to partial cannibalism during the first copulation lose at least 50% of their paternity to rivals. In this paper, we describe a novel male trait—an abdominal constriction that appears during courtship—that allows males to survive and mate with females for a second time, despite the substantial cannibalistic damage inflicted during the first copulation. Constricted males that were wounded to simulate early cannibalism had higher endurance, greater survivorship, longer subsequent courtship and higher mating success than wounded males that were not constricted. Constriction was not found in a non-sacrificial congener that rarely survived simulated cannibalism, and the protective effect of constriction in redbacks was specific to the type of damage inflicted by females during the first copulation. Thus, the abdominal constriction allows males to overcome the potential fitness limit imposed by their own suicidal strategy—paradoxically, by prolonging survival across two cannibalistic copulations

Research Overview on Multi-Species Downstream Migration Measures at the Fithydro Test Case HPP Bannwil

Providing over 56% of the national electricity supply, hydropower plays the most important role in Swiss energy production. Its importance is justified both by the sheer amount of energy that is produced and by the ecological and economic advantages it presents. Not only is hydropower renewable, it can also supply base load energy through run-of-river power plants as well as peak load and power storage through pumped storage plants. The introduction of the revised Water Protection Act in January 2011 in Switzerland provided the fundament to further improve on the ecologic strengths hydropower offers by supporting operators to enhance the connectivity at hydro facilities. One important aspect of water body restoration is enabling or improving the up- and downstream migration of fish, which can be hindered by obstacles like sills, weirs, dams or hydro plants. The BKW Group (BKW) owns or operates within 14 catchment basins, which are in need of upstream migration improvements, 13 where downstream migration has to be addressed, 6 due for sediment management modifications and another 3 at which the effects of hydropeaking will have to be reduced. Since no generally accepted state-of-the-art measures for the implementation of safe downstream fish migration at large hydropower plants (HPP) have been developed yet, the group has been tasked to participate in the interdisciplinary research project “Fishfriendly Innovative Technologies for Hydropower” (FIThydro) to find new and innovative solutions on this subject. The project aims at finding cost-efficient solutions and strategies to mitigate unfavourable ecological effects of hydropower and to support the development of self-sustainable fish populations. The project is funded by the Horizon2020 framework programme of the European Union. The FIThydro consortium consists of 26 partners (13 research and 13 industrial) in 10 European countries. This article provides an overview on the BKW test case HPP Bannwil at the Aare River in Switzerland and describes the ongoing and planned research efforts