RESUMEN
The development of mirror-image biology systems and related applications is hindered by the lack of effective methods to sequence mirror-image (D-) proteins. Although natural-chirality (L-) proteins can be sequenced by bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS), the sequencing of long D-peptides and D-proteins with the same strategy requires digestion by a site-specific D-protease before mass analysis. Here we apply solid-phase peptide synthesis and native chemical ligation to chemically synthesize a mirror-image version of trypsin, a widely used protease for site-specific protein digestion. Using mirror-image trypsin digestion and LC-MS/MS, we sequence a mirror-image large subunit ribosomal protein (L25) and a mirror-image Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and distinguish between different mutants of D-Dpo4. We also perform writing and reading of digital information in a long D-peptide of 50 amino acids. Thus, mirror-image trypsin digestion in conjunction with LC-MS/MS may facilitate practical applications of D-peptides and D-proteins as potential therapeutic and informational tools.
Asunto(s)
Proteínas , Espectrometría de Masas en Tándem , Cromatografía Liquida/métodos , Tripsina/química , Tripsina/metabolismo , Espectrometría de Masas en Tándem/métodos , Péptidos/química , DigestiónRESUMEN
To synthesize a chirally inverted ribosome with the goal of building mirror-image biology systems requires the preparation of kilobase-long mirror-image ribosomal RNAs that make up the structural and catalytic core and about two-thirds of the molecular mass of the mirror-image ribosome. Here, we chemically synthesized a 100-kilodalton mirror-image T7 RNA polymerase, which enabled efficient and faithful transcription of the full-length mirror-image 5S, 16S, and 23S ribosomal RNAs from enzymatically assembled long mirror-image genes. We further exploited the versatile mirror-image T7 transcription system for practical applications such as biostable mirror-image riboswitch sensor, long-term storage of unprotected kilobase-long l-RNA in water, and l-ribozyme-catalyzed l-RNA polymerization to serve as a model system for basic RNA research.
Asunto(s)
ADN Polimerasa Dirigida por ADN , ARN Catalítico , ARN Ribosómico 23S , ARN Ribosómico 5S , Ribosomas , Transcripción Genética , Proteínas Virales , Conformación de Ácido Nucleico , ARN Catalítico/genética , ARN Ribosómico 23S/biosíntesis , ARN Ribosómico 23S/química , ARN Ribosómico 23S/genética , ARN Ribosómico 5S/biosíntesis , ARN Ribosómico 5S/genética , ADN Polimerasa Dirigida por ADN/síntesis química , ADN Polimerasa Dirigida por ADN/química , Proteínas Virales/síntesis química , Proteínas Virales/química , ARN Ribosómico 16S/biosíntesis , ARN Ribosómico 16S/genéticaRESUMEN
Mirror-image aptamers made from chirally inverted nucleic acids are nuclease-resistant and exceptionally biostable, opening up opportunities for unique applications. However, the directed evolution and selection of mirror-image aptamers directly from large randomized L-DNA libraries has, to our knowledge, not been demonstrated previously. Here, we developed a 'mirror-image selection' scheme for the directed evolution and selection of biostable L-DNA aptamers with a mirror-image DNA polymerase. We performed iterative rounds of enrichment and mirror-image polymerase chain reaction (PCR) amplification of L-DNA sequences that bind native human thrombin, in conjunction with denaturing gradient gel electrophoresis (DGGE) to isolate individual aptamers and L-DNA sequencing-by-synthesis to determine their sequences. Based on the selected L-DNA aptamers, we designed biostable thrombin sensors and inhibitors, which remained functional in physiologically relevant nuclease-rich environments, even in the presence of human serum that rapidly degraded D-DNA aptamers. Mirror-image selection of biostable L-DNA aptamers directly from large randomized L-DNA libraries greatly expands the range of biomolecules that can be targeted, broadening their applications as biostable sensors, therapeutics and basic research tools.
Asunto(s)
Aptámeros de Nucleótidos , Humanos , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Trombina/genética , Biblioteca de Genes , Análisis de Secuencia de ADN , ADN Polimerasa Dirigida por ADN/genéticaRESUMEN
Natural DNA is exquisitely evolved to store genetic information. The chirally inverted L-DNA, possessing the same informational capacity but resistant to biodegradation, may serve as a robust, bioorthogonal information repository. Here we chemically synthesize a 90-kDa high-fidelity mirror-image Pfu DNA polymerase that enables accurate assembly of a kilobase-sized mirror-image gene. We use the polymerase to encode in L-DNA an 1860 paragraph by Louis Pasteur that first proposed a mirror-image world of biology. We realize chiral steganography by embedding a chimeric D-DNA/L-DNA key molecule in a D-DNA storage library, which conveys a false or secret message depending on the chirality of reading. Furthermore, we show that a trace amount of an L-DNA barcode preserved in water from a local pond remains amplifiable and sequenceable for 1 year, whereas a D-DNA barcode under the same conditions could not be amplified after 1 day. These next-generation mirror-image molecular tools may transform the development of advanced mirror-image biology systems and pave the way for the realization of the mirror-image central dogma and exploration of their applications.
Asunto(s)
ADN Polimerasa Dirigida por ADN , ADN , ADN/química , ADN/genética , Almacenamiento y Recuperación de la Información , EstereoisomerismoRESUMEN
BACKGROUND: While the physical and chemical properties of airborne particulate matter (PM) have been extensively studied, their associated microbiome remains largely unexplored. Here, we performed a longitudinal metagenomic survey of 106 samples of airborne PM2.5 and PM10 in Beijing over a period of 6 months in 2012 and 2013, including those from several historically severe smog events. RESULTS: We observed that the microbiome composition and functional potential were conserved between PM2.5 and PM10, although considerable temporal variations existed. Among the airborne microorganisms, Propionibacterium acnes, Escherichia coli, Acinetobacter lwoffii, Lactobacillus amylovorus, and Lactobacillus reuteri dominated, along with several viral species. We further identified an extensive repertoire of genes involved in antibiotic resistance and detoxification, including transporters, transpeptidases, and thioredoxins. Sample stratification based on Air Quality Index (AQI) demonstrated that many microbial species, including those associated with human, dog, and mouse feces, exhibit AQI-dependent incidence dynamics. The phylogenetic and functional diversity of air microbiome is comparable to those of soil and water environments, as its composition likely derives from a wide variety of sources. CONCLUSIONS: Airborne particulate matter accommodates rich and dynamic microbial communities, including a range of microbial elements that are associated with potential health consequences.
Asunto(s)
Contaminación del Aire , Metagenoma , Microbiota , Esmog , China , Ciudades , Monitoreo del Ambiente/métodos , Metagenómica/métodosRESUMEN
After realizing mirror-image genetic replication, transcription, and reverse transcription, the biggest challenge in establishing a mirror-image version of the central dogma is to build a mirror-image ribosome-based translation machine. Here, we chemically synthesized the natural and mirror-image versions of three ribosomal proteins (L5, L18, and L25) in the large subunit of the Escherichia coli ribosome with post-translational modifications. We show that the synthetic mirror-image proteins can fold inâ vitro despite limited efficiency and assemble with enzymatically transcribed mirror-image 5S ribosomal RNA into ribonucleoprotein complexes. In addition, the RNA-protein interactions are chiral-specific in that the mirror-image ribosomal proteins do not bind with natural 5S ribosomal RNA and vice versa. The synthesis and assembly of mirror-image 5S ribonucleoprotein complexes are important steps towards building a functional mirror-image ribosome.
Asunto(s)
ARN Ribosómico 5S/metabolismo , Proteínas Ribosómicas/metabolismo , Secuencia de Aminoácidos , Dicroismo Circular , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Unión Proteica , ARN Ribosómico 5S/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Ribosómicas/síntesis química , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , EstereoisomerismoRESUMEN
During the total chemical synthesis of the water-soluble globular Haemophilus Influenzae DNA ligase (Hin-Lig), we observed the surprising phenomenon of a soluble peptide segment that failed to undergo native chemical ligation. Based on dynamic light scattering and transmission electron microscopy experiments, we determined that the peptide formed soluble colloidal particles in a homogeneous solution containing 6 m guanidine hydrochloride. Conventional peptide performance-improving strategies, such as installation of a terminal/side-chain Arg tag or O-acyl isopeptide, failed to enable the reaction, presumably because of their inability to disrupt the formation of soluble colloidal particles. However, a removable backbone modification strategy recently developed for the synthesis of membrane proteins did disrupt the formation of the colloids, and the desired ligation of this soluble but unreactive system was eventually accomplished. This work demonstrates that an appropriate solution dispersion state, in addition to good peptide solubility, is a prerequisite for successful peptide ligation.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Ligasas/metabolismo , Haemophilus influenzae/enzimología , Péptidos/síntesis química , Técnicas de Síntesis en Fase Sólida/métodos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cromatografía Líquida de Alta Presión , Coloides/química , ADN Ligasas/química , ADN Ligasas/genética , Guanidina/química , Histidina/genética , Histidina/metabolismo , Oligopéptidos/genética , Oligopéptidos/metabolismo , Péptidos/análisis , Péptidos/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Glaciers cover ~ 10% of land but are among the least explored environments on Earth. The basal portion of glaciers often harbors unique aquatic microbial ecosystems in the absence of sunlight, and knowledge on the microbial community structures and their metabolic potential is very limited. Here, we provide insights into the microbial lifestyle present at the base of the Matanuska Glacier, Alaska. RESULTS: DNA and RNA were extracted from samples of the Matanuska Glacier basal ice. Using Illumina MiSeq and HiSeq sequencing, we investigated the microbial diversity with the metagenomic shotgun reads and 16S ribosomal RNA data. We further assembled 9 partial and draft bacterial genomes from the metagenomic assembly, and identified key metabolic pathways such as sulfur oxidation and nitrification. Collectively, our analyses suggest a prevalence of lithotrophic and heterotrophic metabolisms in the subglacial microbiome. CONCLUSION: Our results present the first metagenomic assembly and bacterial draft genomes for a subglacial environment. These results extend our understanding of the chemical and biological processes in subglacial environments critically influenced by global climate change.
Asunto(s)
Bacterias/clasificación , Bacterias/aislamiento & purificación , Genoma Bacteriano/genética , Sedimentos Geológicos/microbiología , Cubierta de Hielo/microbiología , Metagenómica , Microbiota/genética , Alaska , Bacterias/genética , Bacterias/metabolismo , Secuencia de Bases , Biodiversidad , Ecosistema , Nitrificación/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Azufre/metabolismoRESUMEN
The development of mirror-image biology systems faces a crucial barrier of lacking an L-DNA sequencing technique. Here, we developed a practical method for sequencing mirror-image DNA by adopting the Maxam-Gilbert sequencing approach, through which specific nucleobases in an end-labeled L-DNA are cleaved by achiral chemicals. This technique may facilitate the therapeutic application of nuclease-resistant L-aptamer drugs, and bring the vision of building an alternative, mirror-image self-replicating system closer to reality.
Asunto(s)
Aptámeros de Nucleótidos/química , ADN/química , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , EstereoisomerismoRESUMEN
Life can persist under severe osmotic stress and low water activity in hypersaline environments. On Mars, evidence for the past presence of saline bodies of water is prevalent and resulted in the widespread deposition of sulfate and chloride salts. Here we investigate Spotted Lake (British Columbia, Canada), a hypersaline lake with extreme (>3 M) levels of sulfate salts as an exemplar of the conditions thought to be associated with ancient Mars. We provide the first characterization of microbial structure in Spotted Lake sediments through metagenomic sequencing, and report a bacteria-dominated community with abundant Proteobacteria, Firmicutes, and Bacteroidetes, as well as diverse extremophiles. Microbial abundance and functional comparisons reveal similarities to Ace Lake, a meromictic Antarctic lake with anoxic and sulfidic bottom waters. Our analysis suggests that hypersaline-associated species occupy niches characterized foremost by differential abundance of Archaea, uncharacterized Bacteria, and Cyanobacteria. Potential biosignatures in this environment are discussed, specifically the likelihood of a strong sulfur isotopic fractionation record within the sediments due to the presence of sulfate reducing bacteria. With its high sulfate levels and seasonal freeze-thaw cycles, Spotted Lake is an analog for ancient paleolakes on Mars in which sulfate salt deposits may have offered periodically habitable environments, and could have concentrated and preserved organic materials or their biomarkers over geologic time.
RESUMEN
The construction of mirror-image biological systems may open the next frontier for biomedical technology development and discovery. Here we have designed and chemically synthesized a mutant version of the thermostable Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) consisting of d-amino acids. With a total peptide length of 358 amino acid residues, it is the largest chemically synthesized d-amino acid protein reported to date. We show that the d-polymerase is able to amplify a 120-bp l-DNA sequence coding for the Escherichia coli 5S ribosomal RNA gene rrfB by mirror-image polymerase chain reaction, and that both the natural and mirror-image systems operate with strict chiral specificity. The development of efficient miPCR systems may lead to many practical applications, such as mirror-image systematic evolution of ligands by exponential enrichment for the selection of therapeutically promising nuclease-resistant l-nucleic acid aptamers.
RESUMEN
Polymerase chain reaction (PCR) has been a defining tool in modern biology. Towards realizing mirror-image PCR, we have designed and chemically synthesized a mutant version of the 352-residue thermostable Sulfolobus solfataricus P2 DNA polymerase IV with l-amino acids and tested its PCR activity biochemically. To the best of our knowledge, this enzyme is the largest chemically synthesized protein reported to date. We show that with optimization of PCR conditions, the fully synthetic polymerase is capable of amplifying template sequences of up to 1.5 kb. The establishment of this synthetic route for chemically synthesizing DNA polymerase IV is a stepping stone towards building a d-enzyme system for mirror-image PCR, which may open up an avenue for the creation of many mirror-image molecular tools such as mirror-image systematic evolution of ligands by exponential enrichment.
RESUMEN
Although regulation of translation fidelity is an essential process1-7, diverse organisms and organelles have differing requirements of translational accuracy8-15, and errors in gene translation serve an adaptive function under certain conditions16-20. Therefore, optimal levels of fidelity may vary according to context. Most bacteria utilize a two-step pathway for the specific synthesis of aminoacylated glutamine and/or asparagine tRNAs, involving the glutamine amidotransferase GatCAB21-25, but it had not been appreciated that GatCAB may play a role in modulating mistranslation rates. Here, by using a forward genetic screen, we show that the mycobacterial GatCAB enzyme complex mediates the translational fidelity of glutamine and asparagine codons. We identify mutations in gatA that cause partial loss of function in the holoenzyme, with a consequent increase in rates of mistranslation. By monitoring single-cell transcription dynamics, we demonstrate that reduced gatCAB expression leads to increased mistranslation rates, which result in enhanced rifampicin-specific phenotypic resistance. Consistent with this, strains with mutations in gatA from clinical isolates of Mycobacterium tuberculosis show increased mistranslation, with associated antibiotic tolerance, suggesting a role for mistranslation as an adaptive strategy in tuberculosis. Together, our findings demonstrate a potential role for the indirect tRNA aminoacylation pathway in regulating translational fidelity and adaptive mistranslation.
Asunto(s)
Mycobacterium smegmatis/enzimología , Transferasas de Grupos Nitrogenados/genética , Biosíntesis de Proteínas , Aminoacilación de ARN de Transferencia , Asparagina/metabolismo , Codón , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Glutamina/metabolismo , Mutación , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Transferasas de Grupos Nitrogenados/metabolismo , Rifampin/farmacologíaRESUMEN
The overwhelmingly homochiral nature of life has left a puzzle as to whether mirror-image biological systems based on a chirally inverted version of molecular machinery could also have existed. Here we report that two key steps in the central dogma of molecular biology, the template-directed polymerization of DNA and transcription into RNA, can be catalysed by a chemically synthesized D-amino acid polymerase on an L-DNA template. We also show that two chirally mirrored versions of the 174-residue African swine fever virus polymerase X could operate in a racemic mixture without significant enantiomeric cross-inhibition to the activity of each other. Furthermore, we demonstrate that a functionally active L-DNAzyme could be enzymatically produced using the D-amino acid polymerase. The establishment of such molecular systems with an opposite handedness highlights the potential to exploit enzymatically produced mirror-image biomolecules as research and therapeutic tools.
Asunto(s)
Aminoácidos/síntesis química , ADN/química , Estereoisomerismo , Aminoácidos/química , Replicación del ADN/genética , Replicación del ADN/fisiología , Conformación de Ácido Nucleico , Polimerizacion , ARN/química , Transcripción Genética/genéticaRESUMEN
Cloning of long microbial genomic sequences is an essential tool in synthetic biology and genome engineering. Such long sequences are often difficult to obtain directly by traditional PCR or restriction enzyme digestion, and therefore the cloning of these sequences has remained a technical obstacle in molecular biology. Based on the in vitro application of RNA-guided Cas9 nuclease, the method of Cas9-assisted targeting of chromosome segments (CATCH) cleaves target DNA in vitro from intact bacterial chromosomes embedded in agarose plugs, which can be subsequently ligated with cloning vector through Gibson assembly. Here we describe an optimized protocol of CATCH cloning for the targeted cloning of long genomic sequences of up to 100 kb from microorganisms. The protocol uses standard laboratory equipment and takes â¼8 h of bench time over several days, and it may potentially simplify and accelerate efforts to isolate and clone large gene clusters from microorganisms.
Asunto(s)
Cromosomas Bacterianos , Clonación Molecular/métodos , Genoma Bacteriano/genética , Proteínas Bacterianas/genética , Proteína 9 Asociada a CRISPR , Endonucleasas/genética , Escherichia coli/genética , Vectores Genéticos , Genómica/métodos , ARN Guía de Kinetoplastida , Biología Sintética/métodos , Flujo de TrabajoRESUMEN
The cloning of long DNA segments, especially those containing large gene clusters, is of particular importance to synthetic and chemical biology efforts for engineering organisms. While cloning has been a defining tool in molecular biology, the cloning of long genome segments has been challenging. Here we describe a technique that allows the targeted cloning of near-arbitrary, long bacterial genomic sequences of up to 100 kb to be accomplished in a single step. The target genome segment is excised from bacterial chromosomes in vitro by the RNA-guided Cas9 nuclease at two designated loci, and ligated to the cloning vector by Gibson assembly. This technique can be an effective molecular tool for the targeted cloning of large gene clusters that are often expensive to synthesize by gene synthesis or difficult to obtain directly by traditional PCR and restriction-enzyme-based methods.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/genética , Clonación Molecular/métodos , ADN Bacteriano/genética , Endonucleasas/metabolismo , Familia de Multigenes/genética , Bacillus subtilis/genética , Proteína 9 Asociada a CRISPR , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Vectores Genéticos , Genoma Bacteriano/genética , Streptomyces/genética , Streptomyces aureofaciens/genética , Proteínas Virales/metabolismoRESUMEN
Metagenomic sequencing has been widely used for the study of microbial communities from various environments such as soil, ocean, sediment and fresh water. Nonetheless, metagenomic sequencing of microbial communities in the air remains technically challenging, partly owing to the limited mass of collectable atmospheric particulate matter and the low biological content it contains. Here we present an optimized protocol for extracting up to tens of nanograms of airborne microbial genomic DNA from collected particulate matter. With an improved sequencing library preparation protocol, this quantity is sufficient for downstream applications, such as metagenomic sequencing for sampling various genes from the airborne microbial community. The described protocol takes â¼12 h of bench time over 2-3 d, and it can be performed with standard molecular biology equipment in the laboratory. A modified version of this protocol may also be used for genomic DNA extraction from other environmental samples of limited mass or low biological content.
Asunto(s)
Microbiología del Aire , Bioquímica/métodos , ADN/aislamiento & purificación , Monitoreo del Ambiente/métodos , Consorcios Microbianos/genética , Bioquímica/instrumentación , China , Monitoreo del Ambiente/instrumentación , Diseño de Equipo , Metagenoma , Material Particulado , Control de Calidad , Análisis de Secuencia de ADN , Flujo de TrabajoRESUMEN
Particulate matter (PM) air pollution poses a formidable public health threat to the city of Beijing. Among the various hazards of PM pollutants, microorganisms in PM2.5 and PM10 are thought to be responsible for various allergies and for the spread of respiratory diseases. While the physical and chemical properties of PM pollutants have been extensively studied, much less is known about the inhalable microorganisms. Most existing data on airborne microbial communities using 16S or 18S rRNA gene sequencing to categorize bacteria or fungi into the family or genus levels do not provide information on their allergenic and pathogenic potentials. Here we employed metagenomic methods to analyze the microbial composition of Beijing's PM pollutants during a severe January smog event. We show that with sufficient sequencing depth, airborne microbes including bacteria, archaea, fungi, and dsDNA viruses can be identified at the species level. Our results suggested that the majority of the inhalable microorganisms were soil-associated and nonpathogenic to human. Nevertheless, the sequences of several respiratory microbial allergens and pathogens were identified and their relative abundance appeared to have increased with increased concentrations of PM pollution. Our findings may serve as an important reference for environmental scientists, health workers, and city planners.
Asunto(s)
Microbiología del Aire , Contaminantes Atmosféricos/análisis , Bacterias/clasificación , Monitoreo del Ambiente/métodos , Hongos/clasificación , Esmog/análisis , Microbiología del Aire/normas , Contaminantes Atmosféricos/química , Bacterias/aislamiento & purificación , China , Ciudades , Hongos/aislamiento & purificación , Humanos , Exposición por Inhalación , Tamaño de la Partícula , Filogenia , Salud PúblicaRESUMEN
Prepare polydisperse, multilamellar vesicles by rehydrating a thin film of fatty acids or phospholipids.