A sequential strand-displacement strategy enables efficient six-step DNA-templated synthesis.
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ABSTRACT:
SUBMITTER: He Y
PROVIDER: S-EPMC3125949 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Quenched autoligation probes have been employed previously in a target-templated nonenzymatic ligation strategy for detecting nucleic acids in cells by fluorescence. A common source of background signal in such probes is the undesired reaction with water and other cellular nucleophiles. Here, we describe a new class of self-ligating probes, double displacement (DD) probes, that rely on two displacement reactions to fully unquench a nearby fluorophore. Three potential double displacement architectures, all possessing two fluorescence quencher/leaving groups (dabsylate groups), were synthesized and evaluated for templated reaction with nucleophile (phosphorothioate) probes both in vitro and in intact bacterial cells. All three DD probe designs provided substantially better initial quenching than a single-Dabsyl control. In isothermal templated reactions in vitro, double displacement probes yielded considerably lower background signal than previous single displacement probes; investigation into the mechanism revealed that one dabsylate acts as a sacrificial leaving group, reacting nonspecifically with water, but yielding little signal because another quencher group remains. Templated reaction with the specific nucleophile probe is required to activate a signal. The double displacement probes provided a ca. 80-fold turn-on signal and yielded a 2-4-fold improvement in signal/background over single Dabsyl probes. The best-performing probe architecture was demonstrated in a two-color, FRET-based two-allele discrimination system in vitro and was shown to be capable of discriminating between two closely related species of bacteria differing by a single nucleotide at an rRNA target site.
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Project description:DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3'-5' exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3'-5' exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5' end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair.
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Project description:BackgroundThe general approach to the synthesis of metal complexes begins with ligand synthesis, followed by ligand reaction with metal salts to afford organometallic complexes. Our research group first reported a one-pot multicomponent synthesis of chiral oxazolinyl-zinc complexes, in the presence of a large amount of ZnCl2 (0.4-2.6 equiv.), with the yields of some products reaching 90%.ResultsOur prior strategy was extended to use copper, cobalt, nickel, manganese, palladium or platinum salts as the third component. The one-step method used 1.0 equivalent of a metal salt, such as M(OAc)2·nH2O or MCl2·nH2O (M: Cu, Co, Ni, Pd or Pt, n = 1, 2 or 4), as a reagent to generate chiral salicyloxazoline complexes 1-8 in the reaction of 2-cyanophenol with different d- and l-amino alcohols.ConclusionComplexes 1-8 were obtained using a one-pot method with a sequential strategy. The reaction outcome was demonstrated for three-component reactions between metal salts, amino alcohols and 2-hydroxybenzonitrile to afford organometallic complexes in good yields (65-95%).
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Project description:The cost-efficient large-scale production of novel carbon nanostructure with high performance is still a challenge, restricting their applications in catalysis. Herein, we present a low-cost one-pot and one-step approach for the synthesis of both N-doped graphene (NG) and N-doped carbon nanotubes (NCNTs) from self-templated organic nanoplates. By varying the FeCl3 concentration in the precursor, we can control the formation of graphene or CNTs. To the best of our knowledge, this is the first example for the controllable synthesis of graphene or CNTs by varying the precursors' compositions. This provides a simple and cost-effective route for the large-scale production of both NG and NCNTs for applications in catalysis. By example, we show how these unique structured nanocarbons can be applied in electrocatalysis for oxygen reduction reaction (ORR). The obtained NG and NCNTs show excellent ORR activities with long-term stability under alkaline conditions. The unique porous nanostructure, abundant defects, homogeneous N-doping and high N-content in the NG and NCNTs can provide abundant active sites, leading to the excellent ORR performance. This research not only displayed a simple and cost-effective approach for the large-scale production of novel carbon nanoarchitectures, but also revealed the exceptional application potential of these nanocarbons for electrocatalysis.
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