Uncovering the selection criteria for the emergence of multi-building-block replicators from dynamic combinatorial libraries.

A family of self-replicating macrocycles was developed using dynamic combinatorial chemistry. Replication is driven by self-assembly of the replicators into fibrils and relies critically on mechanically induced fibril fragmentation. Analysis of separate dynamic combinatorial libraries made from one of six peptide-functionalized building blocks of different hydrophobicity revealed two selection criteria that govern the emergence of replicators from these systems. First, the replicators need to have a critical macrocycle size that endows them with sufficient multivalency to enable their self-assembly into fibrils. Second, efficient replication occurs only for library members that are of low abundance in the absence of a replication pathway. This work has led to spontaneous emergence of replicators with unrivalled structural complexity, being built from up to eight identical subunits and reaching a MW of up to 5.6 kDa. The insights obtained in this work provide valuable guidance that should facilitate future discovery of new complex self-replicating molecules. They may also assist in the development of new self-synthesizing materials, where self-assembly drives the synthesis of the very molecules that self-assemble. To illustrate the potential of this concept, the present system enables access to self-assembling materials made from self-synthesizing macrocycles with tunable ring size ranging from trimers to octamers.

Outcome of infection of C57BL/6 IL-10(-/-) mice with Campylobacter jejuni strains is correlated with genome content of open reading frames up- and down-regulated in vivo.

Human Campylobacter jejuni infection can result in an asymptomatic carrier state, watery or bloody diarrhea, bacteremia, meningitis, or autoimmune neurological sequelae. Infection outcomes of C57BL/6 IL-10(-/-) mice orally infected with twenty-two phylogenetically diverse C. jejuni strains were evaluated to correlate colonization and disease phenotypes with genetic composition of the strains. Variation between strains was observed in colonization, timing of development of clinical signs, and occurrence of enteric lesions. Five pathotypes of C. jejuni in C57BL/6 IL-10(-/-) mice were delineated: little or no colonization, colonization without disease, colonization with enteritis, colonization with hemorrhagic enteritis, and colonization with neurological signs with or without enteritis. Virulence gene content of ten sequenced strains was compared in silico; virulence gene content of twelve additional strains was compared using a C. jejuni pan-genome microarray. Neither total nor virulence gene content predicted pathotype; nor was pathotype correlated with multilocus sequence type. Each strain was unique with regard to absences of known virulence-related loci and/or possession of point mutations and indels, including phase variation, in virulence-related genes. An experiment in C. jejuni 11168-infected germ-free mice showed that expression levels of ninety open reading frames (ORFs) were significantly up- or down-regulated in the mouse cecum at least two-fold compared to in vitro growth. Genomic content of these ninety C. jejuni 11168 ORFs was significantly correlated with the capacity to colonize and cause enteritis in C57BL/6 IL-10(-/-) mice. Differences in gene expression levels and patterns are thus an important determinant of pathotype in C. jejuni strains in this mouse model.

Mechanosensitive self-replication driven by self-organization.

Self-replicating molecules are likely to have played an important role in the origin of life, and a small number of fully synthetic self-replicators have already been described. Yet it remains an open question which factors most effectively bias the replication toward the far-from-equilibrium distributions characterizing even simple organisms. We report here two self-replicating peptide-derived macrocycles that emerge from a small dynamic combinatorial library and compete for a common feedstock. Replication is driven by nanostructure formation, resulting from the assembly of the peptides into fibers held together by beta sheets. Which of the two replicators becomes dominant is influenced by whether the sample is shaken or stirred. These results establish that mechanical forces can act as a selection pressure in the competition between replicators and can determine the outcome of a covalent synthesis.

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates.

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

Directed evolution of hybrid enzymes: Evolving enantioselectivity of an achiral Rh-complex anchored to a protein.

The concept of utilizing the methods of directed evolution for tuning the enantioselectivity of synthetic achiral metal-ligand centers anchored to proteins has been implemented experimentally for the first time.