Proximity extension of circular DNA aptamers with real-time protein detection

Multivalent circular aptamers or ‘captamers’ have recently been introduced through the merger of aptameric recognition functions with the basic principles of DNA nanotechnology. Aptamers have strong utility as protein-binding motifs for diagnostic applications, where their ease of discovery, thermal stability and low cost make them ideal components for incorporation into targeted protein assays. Here we report upon a property specific to circular DNA aptamers: their intrinsic compatibility with a highly sensitive protein detection method termed the ‘proximity extension’ assay. The circular DNA architecture facilitates the integration of multiple functional elements into a single molecule: aptameric target recognition, nucleic acid hybridization specificity and rolling circle amplification. Successful exploitation of these properties is demonstrated for the molecular analysis of thrombin, with the assay delivering a detection limit nearly three orders of magnitude below the dissociation constants of the two contributing aptamer–thrombin interactions. Real-time signal amplification and detection under isothermal conditions points towards potential clinical applications, with both fluorescent and bioelectronic methods of detection achieved. This application elaborates the pleiotropic properties of circular DNA aptamers beyond the stability, potency and multitargeting characteristics described earlier

A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB

BACKGROUND: Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation. CONCLUSIONS/SIGNIFICANCE: In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein

Rhodium- and iridium-catalyzed double hydroalkoxylation of alkynes, an efficient method for the synthesis of O,O-acetals: catalytic and mechanistic studies

An efficient method for the synthesis of O,O-acetals via metal-catalyzed double hydroalkoxylation of alkynes was developed using the Ir(I) and Rh(I) complexes [Ir(PyP)(CO)2]BPh4 (1) and [Rh(bim)(CO) 2]BPh4 (2), where PyP = 1-[2-(diphenylphosphino)ethyl] pyrazole and bim = bis(N-methylimidazol-2-yl)-methane, as catalysts for the consecutive addition of two alcohol functional groups to terminal and nonterminal alkynes to form O,O-acetals. When the catalyzed cyclization of alkynols was performed in the presence of an excess amount of methanol as a cosolvent, a molecule of methanol was incorporated into the acetal product. The catalyzed cyclization of alkynols in the absence of an alcoholic solvent led to cyclization with incorporation of a second molecule of substrate in the final acetal product. Complexes 1 and 2 were also effective as catalysts for the cyclization of alkyne diols to form bicyclic O,O-acetals. The iridium complex 1 was more efficient than the rhodium complex 2 in promoting the reactions of aliphatic alkyne diols. On the other hand, the rhodium complex 2 was more effective for promoting the reactions of aromatic substrates. Mechanistic investigation using low-temperature NMR spectroscopy showed that the catalytic cycle proceeded via π coordination of the alkyne of the substrate to the metal center followed by the sequential addition of two hydroxyl groups to form O,O-acetals. Deuteration studies and analysis of reaction intermediates supported the proposed mechanism

Relayed coherence transfer from a heternucleus through an extended spin system

Heteronuclear relayed coherence transfer X → H → H → …, involving several proton-proton coherence-transfer steps, is demonstrated. All (or selected parts) of the proton spectrum of a molecule containing a heteronucleus can be obtained by inducing coherence in the X spin system and transferring it, stepwise, throughout the coupled-proton spin system. The method permits the proton spectrum of a molecule which contains an NMR-active label to be obtained selectively in the presence of overwhelming amounts of other protonated material. The influence of nuclear relaxation on “extended” coherence-transfer sequences is discussed.8 page(s

Heteronuclear relayed coherence transfer. The selective observation of protons in spin systems containing heteronuclei

A pulse sequence for heteronuclear relayed coherence transfer (RCT) X → H → H is described and used to selectively observe the protons of a spin system which contains a heteronucleus. A two-dimensional extension of the sequence permits the protons of spin systems containing heteronuclei to be separated by virtue of the chemical shift of the heteronucleus. Performing the relay from the heteronucleus to the proton spin system is preferable to the conventional heteronuclear RCT sequence (H → H → X) since the ultimate detection of proton rather than heteronuclear magnetization affords an increase in the sensitivity of the experiment.8 page(s

Rh(I) and Ir(I) catalysed intermolecular hydroamination with substituted hydrazines

The catalysed intermolecular hydroamination of a series of terminal alkynes with substituted hydrazines was achieved using Rh(I) and Ir(I) complexes.4 page(s

Synthesis of spiroketals by iridium-catalyzed double hydroalkoxylation

A highly efficient approach to the synthesis of spiroketals involves the double cyclization of alkynyl diols using transition-metal catalysts. The iridium complex [Ir(PyP)(CO)₂]BPh₄ where PyP = 1-[(2-diphenylphosphino)ethyl]pyrazole is an effective catalyst for promoting the formation of spiroketals via this double hydroalkoxylation reaction. The complex promotes the formation of a series of spiroketal products from alkynyl diol starting materials such as 3-ethynylpentane-1,5-diol and 2-(4-hydroxybut-1-ynyl)benzyl alcohol. Stereoselective cyclization occurs for 3-ethynylpentane-1,5-diol, 3-ethynylhexane-1,6-diol. The cycloadditions occur in all but one case with quantitative conversion in under 24h at 120 °C.6 page(s

Unusual reactivity of the bis(pyrazolyl)borate chelate : B-H for B-X (X = F, Cl, OH) substitution in complexes of ruthenium

Reaction of the complex [RuTp(Bp)PPh₃] (6, where Tp = tris(pyrazolyl)borate and Bp = bis(pyrazolyl)borate) with AgBF₄, AgOTf,and SOCl₂ was observed to yield the highly unusual B-F- and B-OH-containing compounds [RuTp(Bpᶠ²)PPh₃] (7), [RuTp(Bpᴼᴴ)PPh₃]OTf (9), and [RuTp-(Bpᴼᴴ) PPh₃]Cl (10), respectively, via substitution of the Bp boron-hydride groups. The complexes 7 and 9 were structurally characterized by X-ray crystallography. Formation of the B-F product 7 represents a rare example of BF 4 anion reactivity under relatively mild conditions, whereas the B-OH product 9 contains the first example of a stable trivalent pyrazolyl-borane moiety. These substitution processes are thought to be assisted by the interaction of the boron-hydride moieties of 6 with the Lewis acidic Ag(I) and S(IV) centers of the reactive agents, which polarize the B-H bond and activate the boron center toward electrophilic attack of available halogen- and hydroxy-containing species.7 page(s

1H-detected multinuclear NMR experiments for the measurement of small heteronuclear coupling constants in transition metal complexes

The scalar coupling constants between protons, nitrogens, and phosphorus in the metal complex chloro(triphenylphosphine)bis[bis(1-pyrazolyl)methane]ruthenium(II) chloride, [RuCl(PPh3)(BPM)2]+Cl- (1), were measured with a set of specially adapted NMR experiments. The absolute sign of the coupling constants was determined by relating the signs of the measured couplings to that of a one-bond proton−carbon coupling constant. A complete set of coupling constants >|0.4| Hz was obtained with use of a single sample with 99% 15N-labeled bis-pyrazolyl ligands. The data show that the two-bond 15N−15N and 31P−15N couplings across the metal center are significantly larger, if the two metal-ligating atoms are trans rather than cis with respect to one another. Furthermore, all trans couplings 2JPN and 2JNN are positive, while the corresponding cis couplings are negative or too small to be measured. The conformation dependence of the scalar coupling constants supports the rapid structural characterization of catalytically active organometallic complexes by NMR spectroscopy. The proposed set of NMR experiments includes HSQC experiments with small flip angles, a quantitative long-range 15N−15N correlation experiment, and DQ/ZQ experiments for the determination of the sign and size of JNN and JPN coupling constants in linear spin systems