Explore the vast range of titles available.

A simple and general chemical structure change to a panel of cell-permeable small-molecule fluorophores increases their brightness and photostability, which will enable improved single-molecule studies and super-resolution imaging. Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N , N -dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

This Review discusses large-scale de novo DNA synthesis via oligos or arrays, describes gene assembly and error correction and considers applications for large-scale DNA synthesis. For over 60 years, the synthetic production of new DNA sequences has helped researchers understand and engineer biology. Here we summarize methods and caveats for the de novo synthesis of DNA, with particular emphasis on recent technologies that allow for large-scale and low-cost production. In addition, we discuss emerging applications enabled by large-scale de novo DNA constructs, as well as the challenges and opportunities that lie ahead.

Small molecules are developed that irreversibly bind to the common G12C mutant of K-Ras but not the wild-type protein; crystallographic studies reveal the formation of an allosteric pocket that is not apparent in previous Ras studies, and the small molecules shift the affinity of K-Ras to favour GDP over GTP. Drug-targeting strategy for Ras protein Mutations in the oncogenic small GTPase K-Ras are common in cancer making the enzyme an obvious drug target, but directly inhibiting K-Ras function with small molecules has proved difficult. Here, Shokat and colleagues report the development of small molecules that irreversibly bind to the common G12C mutant of K-Ras but not to the wild-type protein. Crystallographic studies reveal the formation of an allosteric pocket that is not apparent in previous structures of Ras, and the small molecules shift the affinity of K-Ras to favour GDP over GTP. These findings should provide a starting point for drug-discovery efforts targeting this mutant Ras protein. Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies 1 , 2 , 3 . Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP 4 and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis 5 , 6 . With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP 7 and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.

This protocol provides a detailed procedure for the chemical synthesis of proteins through native chemical ligation of peptide hydrazides. The two crucial stages of this protocol are (i) the solid-phase synthesis of peptide hydrazides via Fmoc chemistry and (ii) the native chemical ligation of peptide hydrazides through in situ NaNO 2 activation and thiolysis. This protocol may be of help in the synthesis of proteins that are not easily produced by recombinant technology and that include acid-sensitive modifications; it also does not involve the use of hazardous HF. The utility of the protocol is shown for the total synthesis of 140-aa-long α-synuclein, a protein that has an important role in the development of Parkinson's disease. The whole synthesis of the target protein α-synuclein in milligram scale takes ∼30 working days.

Histone post-translational modifications are important regulators of chromatin structure and gene expression. Lysine 2-hydroxisobutyrylation sites, discovered by MS and validated by chemical synthesis, are found in active chromatin and associated with male germ cell differentiation. We report the identification of a new type of histone mark, lysine 2-hydroxyisobutyrylation (K hib ), and identify the mark at 63 human and mouse histone K hib sites, including 27 unique lysine sites that are not known to be modified by lysine acetylation (K ac ) and lysine crotonylation (K cr ). This histone mark was initially identified by MS and then validated by chemical and biochemical methods. Histone K hib shows distinct genomic distributions from histone K ac or histone K cr during male germ cell differentiation. Using chromatin immunoprecipitation sequencing, gene expression analysis and immunodetection, we show that in male germ cells, H4K8 hib is associated with active gene transcription in meiotic and post-meiotic cells. In addition, H4K8 ac -associated genes are included in and constitute only a subfraction of H4K8 hib -labeled genes. The histone K hib mark is conserved and widely distributed, has high stoichiometry and induces a large structural change. These findings suggest its critical role on the regulation of chromatin functions.

Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α- L -threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson–Crick base pairing. Droplet-based optical polymerase sorting employs a fluorescent sensor to monitor polymerase activity inside the microenvironment of uniform water-in-oil emulsions. Here, the authors use this technique to select and isolate single cells for evolution of an unnatural nucleic acid polymerase.

Long-term noninvasive cell tracing by fluorescent probes is of great importance to life science and biomedical engineering. For example, understanding genesis, development, invasion and metastasis of cancerous cells and monitoring tissue regeneration after stem cell transplantation require continual tracing of the biological processes by cytocompatible fluorescent probes over a long period of time. In this work, we successfully developed organic far-red/near-infrared dots with aggregation-induced emission (AIE dots) and demonstrated their utilities as long-term cell trackers. The high emission efficiency, large absorptivity, excellent biocompatibility and strong photobleaching resistance of the AIE dots functionalized by cell penetrating peptides derived from transactivator of transcription proteins ensured outstanding long-term noninvasive in vitro and in vivo cell tracing. The organic AIE dots outperform their counterparts of inorganic quantum dots, opening a new avenue in the development of fluorescent probes for following biological processes such as carcinogenesis.

Leguminous plants establish a symbiosis with rhizobia to enable nitrogen fixation in root nodules under the control of the presumed root-to-shoot-to-root negative feedback called autoregulation of nodulation. In Lotus japonicus , autoregulation is mediated by CLE-RS genes that are specifically expressed in the root, and the receptor kinase HAR1 that functions in the shoot. However, the mature functional structures of CLE-RS gene products and the molecular nature of CLE-RS/HAR1 signalling governed by these spatially distant components remain elusive. Here we show that CLE-RS2 is a post-translationally arabinosylated glycopeptide derived from the CLE domain. Chemically synthesized CLE-RS glycopeptides cause significant suppression of nodulation and directly bind to HAR1 in an arabinose-chain and sequence-dependent manner. In addition, CLE-RS2 glycopeptide specifically produced in the root is found in xylem sap collected from the shoot. We propose that CLE-RS glycopeptides are the long sought mobile signals responsible for the initial step of autoregulation of nodulation. Symbiotic bacteria form nodules with plant roots and this is controlled by CLE-RS genes found in the plant. In this study, the CLE-RS2 gene product is shown to be a glycopeptide that can travel from the roots to the shoot of plants and binds to the receptor kinase HAR1.

A single trimethylated species is obtained in an on-resin N-methylation reaction of a cyclic hexapeptide. This regioselectivity is driven by conformation and the presence of intramolecular hydrogen bonds, and is correlated with membrane permeability of the peptides. Backbone N-methylation is common among peptide natural products and has a substantial impact on both the physical properties and the conformational states of cyclic peptides. However, the specific impact of N-methylation on passive membrane diffusion in cyclic peptides has not been investigated systematically. Here we report a method for the selective, on-resin N-methylation of cyclic peptides to generate compounds with drug-like membrane permeability and oral bioavailability. The selectivity and degree of N-methylation of the cyclic peptide was dependent on backbone stereochemistry, suggesting that conformation dictates the regiochemistry of the N-methylation reaction. The permeabilities of the N -methyl variants were corroborated by computational studies on a 1,024-member virtual library of N -methyl cyclic peptides. One of the most permeable compounds, a cyclic hexapeptide (molecular mass = 755 Da) with three N -methyl groups, showed an oral bioavailability of 28% in rat.

Graphene is in many ways an ideal sample support for cryo-electron microscopy, but its hydrophobicity prevents adsorption of protein from aqueous solution. Low-energy hydrogen-plasma treatment renders graphene hydrophilic and enables controlled adsorption of protein to its surface. Despite its many favorable properties as a sample support for biological electron microscopy, graphene is not widely used because its hydrophobicity precludes reliable protein deposition. We describe a method to modify graphene with a low-energy hydrogen plasma, which reduces hydrophobicity without degrading the graphene lattice. Use of plasma-treated graphene enables better control of protein distribution in ice for electron cryo-microscopy and improves image quality by reducing radiation-induced sample motion.