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Sci Rep ; 11(1): 11669, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083677


In vivo selection systems are powerful tools for directed evolution of enzymes. The selection pressure of the systems can be tuned by regulating the expression levels of the catalysts. In this work, we engineered a selection system for laboratory evolution of highly active enzymes by incorporating a translationally suppressing cis repressor as well as an inducible promoter to impart stringent and tunable selection pressure. We demonstrated the utility of our selection system by performing directed evolution experiments using TEM ß-lactamase as the model enzyme. Five evolutionary rounds afforded a highly active variant exhibiting 440-fold improvement in catalytic efficiency. We also showed that, without the cis repressor, the selection system cannot provide sufficient selection pressure required for evolving highly efficient TEM ß-lactamase. The selection system should be applicable for the exploration of catalytic perfection of a wide range of enzymes.

Evolução Molecular Direcionada , Regulação Enzimológica da Expressão Gênica , Seleção Genética , Sequência de Bases , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Conformação Proteica , Engenharia de Proteínas , RNA Bacteriano , Relação Estrutura-Atividade
J Am Chem Soc ; 139(8): 2892-2895, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28218835


RNA or single-stranded DNA aptamers with 2'-F pyrimidines have been pursued to increase resistance to nucleases, and while it seems likely that these and other modifications, including the modification of purines, could be used to optimize additional properties, this has been much less explored because such aptamers are challenging to discover. Using a thermostable DNA polymerase, SFM4-3, which was previously evolved to accept nucleotides with 2'-modifications, we now report the selection of 2'-F purine aptamers that bind human neutrophil elastase (HNE). Two aptamers were identified, 2fHNE-1 and 2fHNE-2, that bind HNE with reasonable affinity. Interestingly, the 2'-F substituents facilitate the selection of specific interactions with HNE and overcome nonspecific electrostatic interactions that can otherwise dominate. The data demonstrate that inclusion of only a few 2'-F substituents can optimize properties far beyond simple nuclease resistance and that SFM4-3 should prove valuable for the further exploration and production of aptamers with properties optimized for various applications.

Curr Opin Chem Biol ; 34: 80-87, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27565457


DNA and RNA are remarkable because they can both encode information and possess desired properties, including the ability to bind specific targets or catalyze specific reactions. Nucleotide modifications that do not interfere with enzymatic synthesis are now being used to bestow DNA or RNA with properties that further increase their utility, including phosphate and sugar modifications that increase nuclease resistance, nucleobase modifications that increase the range of activities possible, and even whole nucleobase replacement that results in selective pairing and the creation of unnatural base pairs that increase the information content. These modifications are increasingly being applied both in vitro and in vivo, including in efforts to create semi-synthetic organisms with altered or expanded genetic alphabets.

DNA/metabolismo , Proteínas/metabolismo , RNA/metabolismo , Pareamento de Bases , Catálise , Interações Hidrofóbicas e Hidrofílicas , Técnica de Seleção de Aptâmeros
Nat Chem ; 8(6): 556-62, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27219699


The PCR amplification of oligonucleotides enables the evolution of sequences called aptamers that bind specific targets with antibody-like affinity. However, in many applications the use of these aptamers is limited by nuclease-mediated degradation. In contrast, oligonucleotides that are modified at their sugar C2' positions with methoxy or fluorine substituents are stable to nucleases, but they cannot be synthesized by natural polymerases. Here we report the development of a polymerase-evolution system and its use to evolve thermostable polymerases that efficiently interconvert C2'-OMe-modified oligonucleotides and their DNA counterparts via 'transcription' and 'reverse transcription' or, more importantly, that PCR-amplify partially C2'-OMe- or C2'-F-modified oligonucleotides. A mechanistic analysis demonstrates that the ability to amplify the modified oligonucleotides evolved by optimizing interdomain interactions that stabilize the catalytically competent closed conformation of the polymerase. The evolved polymerases should find practical applications and the developed evolution system should be a powerful tool for tailoring polymerases to have other types of novel function.

DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , DNA/química , Aptâmeros de Nucleotídeos/química , Replicação do DNA/fisiologia , Evolução Molecular , Temperatura Alta , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos/síntese química
PLoS Genet ; 9(1): e1003187, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23300488


The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function.

Aminoácidos , Evolução Molecular Direcionada , Código Genético , Proteínas/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Corismato Mutase/química , Corismato Mutase/genética , Methanococcales/genética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação Puntual , Conformação Proteica , Estabilidade Proteica , Relação Estrutura-Atividade