Mirror-image trypsin digestion and sequencing of D-proteins.

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.

Mirror-image T7 transcription of chirally inverted ribosomal and functional RNAs.

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 5, 16, and 23 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.

Directed evolution and selection of biostable L-DNA aptamers with a mirror-image DNA polymerase.

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.

Bioorthogonal information storage in L-DNA with a high-fidelity mirror-image Pfu DNA polymerase.

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.

Longitudinal survey of microbiome associated with particulate matter in a megacity.

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 PM and PM 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 PM and PM, although considerable temporal variations existed.

Mirror-Image 5S Ribonucleoprotein Complexes.

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 in vitro despite limited efficiency and assemble with enzymatically transcribed mirror-image 5S ribosomal RNA into ribonucleoprotein complexes.

Ligation of Soluble but Unreactive Peptide Segments in the Chemical Synthesis of Haemophilus Influenzae DNA Ligase.

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.

Sequencing Mirror-Image DNA Chemically.

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.

Metagenomic analysis of basal ice from an Alaskan glacier.

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.

Mirror-image polymerase chain reaction.

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 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.

Microbial Diversity in a Hypersaline Sulfate Lake: A Terrestrial Analog of Ancient Mars.

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.

Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction.

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 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.

The essential mycobacterial amidotransferase GatCAB is a modulator of specific translational fidelity.

Although regulation of translation fidelity is an essential process, diverse organisms and organelles have differing requirements of translational accuracy, and errors in gene translation serve an adaptive function under certain conditions. 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 GatCAB, but it had not been appreciated that GatCAB may play a role in modulating mistranslation rates.

A synthetic molecular system capable of mirror-image genetic replication and transcription.

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.

Targeted isolation and cloning of 100-kb microbial genomic sequences by Cas9-assisted targeting of chromosome segments.

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.

Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters.

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.

Optimized DNA extraction and metagenomic sequencing of airborne microbial communities.

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.

Inhalable microorganisms in Beijing's PM2.5 and PM10 pollutants during a severe smog event.

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.

Preparation of fatty acid micelles.

Here we describe a method for preparing fatty acid micelles. The method for adding micelles to a buffered solution containing fatty acid or phospholipid vesicles is also discussed.

Vesicle extrusion through polycarbonate track-etched membranes using a hand-held mini-extruder.

The vesicle extrusion method is used for preparing small (e.g., 100 nm diameter) monodisperse (uniform-sized) vesicles.

Preparation of fatty acid or phospholipid vesicles by thin-film rehydration.

Prepare polydisperse, multilamellar vesicles by rehydrating a thin film of fatty acids or phospholipids.

Photochemically driven redox chemistry induces protocell membrane pearling and division.

Prior to the evolution of complex biochemical machinery, the growth and division of simple primitive cells (protocells) must have been driven by environmental factors. We have previously demonstrated two pathways for fatty acid vesicle growth in which initially spherical vesicles grow into long filamentous vesicles; division is then mediated by fluid shear forces. Here we describe a different pathway for division that is independent of external mechanical forces.

The origins of cellular life.

Understanding the origin of cellular life on Earth requires the discovery of plausible pathways for the transition from complex prebiotic chemistry to simple biology, defined as the emergence of chemical assemblies capable of Darwinian evolution. We have proposed that a simple primitive cell, or protocell, would consist of two key components: a protocell membrane that defines a spatially localized compartment, and an informational polymer that allows for the replication and inheritance of functional information. Recent studies of vesicles composed of fatty-acid membranes have shed considerable light on pathways for protocell growth and division, as well as means by which protocells could take up nutrients from their environment.

Preparation of large monodisperse vesicles.

Preparation of monodisperse vesicles is important both for research purposes and for practical applications. While the extrusion of vesicles through small pores (approximately 100 nm in diameter) results in relatively uniform populations of vesicles, extrusion to larger sizes results in very heterogeneous populations of vesicles. Here we report a simple method for preparing large monodisperse multilamellar vesicles through a combination of extrusion and large-pore dialysis.