RESUMO
The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for inâ vitro protein synthesis. Using phi29â DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 10(4) â clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription-translation reactions performed inâ vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis inâ vitro.
Assuntos
DNA/química , Nanopartículas , Proteínas/síntese química , Fluorescência , Dispositivos Lab-On-A-Chip , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de TransmissãoRESUMO
New enzyme catalysts are usually engineered by repurposing the active sites of natural proteins. Here we show that design and directed evolution can be used to transform a non-natural, functionally naive zinc-binding protein into a highly active catalyst for an abiological hetero-Diels-Alder reaction. The artificial metalloenzyme achieves >104 turnovers per active site, exerts absolute control over reaction pathway and product stereochemistry, and displays a catalytic proficiency (1/KTS = 2.9 × 1010 M-1) that exceeds all previously characterized Diels-Alderases. These properties capitalize on effective Lewis acid catalysis, a chemical strategy for accelerating Diels-Alder reactions common in the laboratory but so far unknown in nature. Extension of this approach to other metal ions and other de novo scaffolds may propel the design field in exciting new directions.
Assuntos
Ácidos de Lewis/química , Metaloproteínas/metabolismo , Catálise , Domínio Catalítico , Reação de Cicloadição , Teoria da Densidade Funcional , Evolução Molecular Direcionada , Ligação de Hidrogênio , Cinética , Metaloproteínas/química , Simulação de Acoplamento Molecular , Especificidade por SubstratoRESUMO
Severe congenital neutropenia (SCN) is a life-threatening disorder most often caused by dominant mutations of ELANE that interfere with neutrophil maturation. We conducted a pooled CRISPR screen in human hematopoietic stem and progenitor cells (HSPCs) that correlated ELANE mutations with neutrophil maturation potential. Highly efficient gene editing of early exons elicited nonsense-mediated decay (NMD), overcame neutrophil maturation arrest in HSPCs from ELANE-mutant SCN patients, and produced normal hematopoietic engraftment function. Conversely, terminal exon frameshift alleles that mimic SCN-associated mutations escaped NMD, recapitulated neutrophil maturation arrest, and established an animal model of ELANE-mutant SCN. Surprisingly, only -1 frame insertions or deletions (indels) impeded neutrophil maturation, whereas -2 frame late exon indels repressed translation and supported neutrophil maturation. Gene editing of primary HSPCs allowed faithful identification of variant pathogenicity to clarify molecular mechanisms of disease and encourage a universal therapeutic approach to ELANE-mutant neutropenia, returning normal neutrophil production and preserving HSPC function.
Assuntos
Elastase de Leucócito , Neutropenia , Animais , Síndrome Congênita de Insuficiência da Medula Óssea , Edição de Genes , Humanos , Elastase de Leucócito/genética , Mutação/genética , Neutropenia/genética , VirulênciaRESUMO
Primordial sequence signatures in modern proteins imply ancestral origins tracing back to simple peptides. Although short peptides seldom adopt unique folds, metal ions might have templated their assembly into higher-order structures in early evolution and imparted useful chemical reactivity. Recapitulating such a biogenetic scenario, we have combined design and laboratory evolution to transform a zinc-binding peptide into a globular enzyme capable of accelerating ester cleavage with exacting enantiospecificity and high catalytic efficiency (k cat/K M ~ 106 M-1 s-1). The simultaneous optimization of structure and function in a naïve peptide scaffold not only illustrates a plausible enzyme evolutionary pathway from the distant past to the present but also proffers exciting future opportunities for enzyme design and engineering.