RESUMEN
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.
Asunto(s)
Técnicas Químicas Combinatorias , Cromatografía Líquida de Alta Presión , Microscopía Electrónica de Transmisión , Modelos Moleculares , Péptidos/químicaRESUMEN
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.
Asunto(s)
Compuestos Macrocíclicos/química , Péptidos/química , Fenómenos Químicos , Dicroismo Circular , Técnicas Químicas Combinatorias , Microscopía por Crioelectrón , Evolución Química , Concentración de Iones de Hidrógeno , Cinética , Leucina/química , Lisina/química , Fenómenos Mecánicos , Modelos Químicos , Conformación Molecular , Origen de la Vida , Biblioteca de Péptidos , Análisis Espectral , Estrés Mecánico , Compuestos de Sulfhidrilo/química , TermodinámicaRESUMEN
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.
Asunto(s)
Evolución Molecular Dirigida/métodos , Biblioteca de Genes , Mutagénesis , Reacción en Cadena de la Polimerasa/métodos , Aspergillus niger/enzimología , Aspergillus niger/genética , Bacillus megaterium/enzimología , Bacillus megaterium/genética , Proteínas Bacterianas/genética , Candida/enzimología , Candida/genética , Sistema Enzimático del Citocromo P-450/genética , Epóxido Hidrolasas/genética , Lipasa/genética , NADPH-Ferrihemoproteína Reductasa/genética , Plásmidos , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genéticaRESUMEN
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.