A Robust Growth-Based Selection Platform to Evolve an Enzyme via Dependency on Noncanonical Tyrosine Analogues.

Growth-based selections evaluate the fitness of individual organisms at a population level. In enzyme engineering, such growth selections allow for the rapid and straightforward identification of highly efficient biocatalysts from extensive libraries. However, selection-based improvement of (synthetically useful) biocatalysts is challenging, as they require highly dependable strategies that artificially link their activities to host survival. Here, we showcase a robust and scalable growth-based selection platform centered around the complementation of noncanonical amino acid-dependent bacteria. Specifically, we demonstrate how serial passaging of populations featuring millions of carbamoylase variants autonomously selects biocatalysts with up to 90,000-fold higher initial rates. Notably, selection of replicate populations enriched diverse biocatalysts, which feature distinct amino acid motifs that drastically boost carbamoylase activity. As beneficial substitutions also originated from unintended copying errors during library preparation or cell division, we anticipate that our growth-based selection platform will be applicable to the continuous, autonomous evolution of diverse biocatalysts in the future.

Tethered Ribosomes: Toward the Synthesis of Nonproteinogenic Polymers in Bacteria.

The ribosome is the core element of the translational apparatus and displays unrivaled fidelity and efficiency in the synthesis of long polymers with defined sequences and diverse compositions. Repurposing ribosomes for the assembly of nonproteinogenic (bio)polymers is an enticing prospect with implications for fundamental science, bioengineering and synthetic biology alike. Here, we review tethered ribosomes, which feature inseparable large and small subunits that can be evolved for novel function without interfering with native translation. Following a tutorial summary of ribosome structure, function, and biogenesis, we introduce design and optimization strategies for the creation of orthogonal and tethered ribosomes. We also highlight studies, in which (rational) engineering efforts of these designer ribosomes enabled the evolution of new functions. Lastly, we discuss future prospects and challenges that remain for the ribosomal synthesis of tailor-made (bio)polymers.

Selecting Better Biocatalysts by Complementing Recoded Bacteria.

In vivo selections are powerful tools for the directed evolution of enzymes. However, the need to link enzymatic activity to cellular survival makes selections for enzymes that do not fulfill a metabolic function challenging. Here, we present an in vivo selection strategy that leverages recoded organisms addicted to non-canonical amino acids (ncAAs) to evolve biocatalysts that can provide these building blocks from synthetic precursors. We exemplify our platform by engineering carbamoylases that display catalytic efficiencies more than five orders of magnitude higher than those observed for the wild-type enzyme for ncAA-precursors. As growth rates of bacteria under selective conditions correlate with enzymatic activities, we were able to elicit improved variants from populations by performing serial passaging. By requiring minimal human intervention and no specialized equipment, we surmise that our strategy will become a versatile tool for the in vivo directed evolution of diverse biocatalysts.

A Strategy to Select Macrocyclic Peptides Featuring Asymmetric Molecular Scaffolds as Cyclization Units by Phage Display.

Macrocyclic peptides (MPs) have positioned themselves as a privileged class of compounds for the discovery of therapeutics and development of chemical probes. Aided by the development of powerful selection strategies, high-affinity binders against biomolecular targets can readily be elicited from massive, genetically encoded libraries by affinity selection. For example, in phage display, MPs are accessed on the surface of whole bacteriophages via disulfide formation, the use of (symmetric) crosslinkers, or the incorporation of non-canonical amino acids. To facilitate a straightforward cyclization of linear precursors with asymmetric molecular scaffolds, which are often found at the core of naturally occurring MPs, we report an efficient two-step strategy to access MPs via the programmed modification of a unique cysteine residue and an N-terminal amine. We demonstrate that this approach yields MPs featuring asymmetric cyclization units from both synthetic peptides and when linear precursors are appended onto a phage-coat protein. Finally, we showcase that our cyclization strategy is compatible with traditional phage-display protocols and enables the selection of MP binders against a model target protein from naïve libraries. By enabling the incorporation of non-peptidic moieties that (1) can serve as cyclization units, (2) provide interactions for binding, and/or (3) tailor pharmacological properties, our head-to-side-chain cyclization strategy provides access to a currently under-explored chemical space for the development of chemical probes and therapeutics.

Catalytic and structural properties of ATP-dependent caprolactamase from Pseudomonas jessenii.

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases. The caprolactamase was produced in Escherichia coli, isolated by His-tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6-aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG0 '= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αß)2 tetramer and revealed an ATP-binding site in CapA and a Zn-coordinating site in CapB. Mutations in the ATP-binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild-type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP-dependent lactam hydrolysis.

Addicting Escherichia coli to New-to-Nature Reactions.

Biocontainment is an essential feature when deploying genetically modified organisms (GMOs) in open system applications, as variants escaping their intended operating environments could negatively impact ecosystems and human health. To avoid breaches resulting from metabolic cross-feeding, horizontal gene transfer, and/or genetic mutations, synthetic auxotrophs have been engineered to become dependent on exogenously supplied xenobiotics, such as noncanonical amino acids (ncAAs). The incorporation of these abiological building blocks into essential proteins constitutes a first step toward constructing xenobiological barriers between GMOs and their environments. To transition synthetic auxotrophs further away from familiar biology, we demonstrate how bacterial growth can be confined by transition-metal complexes that catalyze the formation of an essential ncAA through new-to-nature reactions. Specifically, using a homogeneous ruthenium complex enabled us to localize bacterial growth on solid media, while heterogeneous palladium nanoparticles could be recycled and deployed up to five consecutive times to ensure the survival of synthetic auxotrophs in liquid cultures.

Genetic-Code-Expansion Strategies for Vaccine Development.

By providing long-term protection against infectious diseases, vaccinations have significantly reduced death and morbidity worldwide. In the 21st century, (bio)technological advances have paved the way for developing prophylactic vaccines that are safer and more effective as well as enabling the use of vaccines as therapeutics to treat human diseases. Here, we provide a focused review of the utility of genetic code expansion as an emerging tool for the development of vaccines. Specifically, we discuss how the incorporation of immunogenic noncanonical amino acids can aid in eliciting immune responses against adverse self-proteins and highlight the potential of an expanded genetic code for the construction of replication-incompetent viruses. We close the review by discussing the future prospects and remaining challenges for the application of these approaches in the development of both prophylactic and therapeutic vaccines in the near future.

Affinity-Selected Bicyclic Peptide G-Quadruplex Ligands Mimic a Protein-like Binding Mechanism.

The study of G-quadruplexes (G4s) in a cellular context has demonstrated links between these nucleic acid secondary structures, gene expression, and DNA replication. Ligands that bind to the G4 structure therefore present an excellent opportunity for influencing gene expression through the targeting of a nucleic acid structure rather than sequence. Here, we explore cyclic peptides as an alternative class of G4 ligands. Specifically, we describe the development of de novo G4-binding bicyclic peptides selected by phage display. Selected bicyclic peptides display submicromolar affinity to G4 structures and high selectivity over double helix DNA. Molecular simulations of the bicyclic peptide-G4 complexes corroborate the experimental binding strengths and reveal molecular insights into G4 recognition by bicyclic peptides via the precise positioning of amino acid side chains, a binding mechanism reminiscent of endogenous G4-binding proteins. Overall, our results demonstrate that selection of (bi)cyclic peptides unlocks a valuable chemical space for targeting nucleic acid structures.

A Screening Platform to Identify and Tailor Biocompatible Small-Molecule Catalysts.

Interfacing biocompatible, small-molecule catalysis with cellular metabolism promises a straightforward introduction of new function into organisms without the need for genetic manipulation. However, identifying and optimizing synthetic catalysts that perform new-to-nature transformations under conditions that support life is a cumbersome task. To enable the rapid discovery and fine-tuning of biocompatible catalysts, we describe a 96-well screening platform that couples the activity of synthetic catalysts to yield non-canonical amino acids from appropriate precursors with the subsequent incorporation of these nonstandard building blocks into GFP (quantifiable readout). Critically, this strategy does not only provide a common readout (fluorescence) for different reaction/catalyst combinations, but also informs on the organism's fitness, as stop codon suppression relies on all steps of the central dogma of molecular biology. To showcase our approach, we have applied it to the evaluation and optimization of transition-metal-catalyzed deprotection reactions.

Correction to: Molecular and pharmacological modulators of the tumor immune contexture revealed by deconvolution of RNA-seq data.

It was highlighted that the original article [1] contained a typesetting mistake in the name of Noel Filipe da Cunha Carvalho de Miranda. This was incorrectly captured as Noel Filipe da Cunha Carvahlo de Miranda. It was also highlighted that in Fig. 3C the left panels Y-axis were cropped and in Fig. 5C, CD8 bar was cropped. This Correction article shows the correct Figs. 3 and 5. The original article has been updated.

Molecular and pharmacological modulators of the tumor immune contexture revealed by deconvolution of RNA-seq data.

We introduce quanTIseq, a method to quantify the fractions of ten immune cell types from bulk RNA-sequencing data. quanTIseq was extensively validated in blood and tumor samples using simulated, flow cytometry, and immunohistochemistry data.quanTIseq analysis of 8000 tumor samples revealed that cytotoxic T cell infiltration is more strongly associated with the activation of the CXCR3/CXCL9 axis than with mutational load and that deconvolution-based cell scores have prognostic value in several solid cancers. Finally, we used quanTIseq to show how kinase inhibitors modulate the immune contexture and to reveal immune-cell types that underlie differential patients' responses to checkpoint blockers.Availability: quanTIseq is available at http://icbi.at/quantiseq .

Selection, Addiction and Catalysis: Emerging Trends for the Incorporation of Noncanonical Amino Acids into Peptides and Proteins in Vivo.

Expanding the genetic code of organisms by incorporating noncanonical amino acids (ncAAs) into target proteins through the suppression of stop codons in vivo has profoundly impacted how we perform protein modification or detect proteins and their interaction partners in their native environment. Yet, with genetic code expansion strategies maturing over the past 15 years, new applications that make use-or indeed repurpose-these techniques are beginning to emerge. This Concept article highlights three of these developments: 1) The incorporation of ncAAs for the biosynthesis and selection of bioactive macrocyclic peptides with novel ring architectures, 2) synthetic biocontainment strategies based on the addiction of microorganisms to ncAAs, and 3) enzyme design strategies, in which ncAAs with unique functionalities enable the catalysis of new-to-nature reactions. Key advances in all three areas are presented and potential future applications discussed.

Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid.

The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding pocket. Such fine-tuning is also possible for catalytic unnatural amino acids. Specifically, the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction, is reported. Consecutive rounds of directed evolution identified several mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency (kcat ) of the designer enzyme by almost 100-fold. This results from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by more than 200-fold.

Analysis of NRAS RNA G-quadruplex binding proteins reveals DDX3X as a novel interactor of cellular G-quadruplex containing transcripts.

RNA G-quadruplexes (rG4s) are secondary structures in mRNAs known to influence RNA post-transcriptional mechanisms thereby impacting neurodegenerative disease and cancer. A detailed knowledge of rG4-protein interactions is vital to understand rG4 function. Herein, we describe a systematic affinity proteomics approach that identified 80 high-confidence interactors that assemble on the rG4 located in the 5'-untranslated region (UTR) of the NRAS oncogene. Novel rG4 interactors included DDX3X, DDX5, DDX17, GRSF1 and NSUN5. The majority of identified proteins contained a glycine-arginine (GAR) domain and notably GAR-domain mutation in DDX3X and DDX17 abrogated rG4 binding. Identification of DDX3X targets by transcriptome-wide individual-nucleotide resolution UV-crosslinking and affinity enrichment (iCLAE) revealed a striking association with 5'-UTR rG4-containing transcripts which was reduced upon GAR-domain mutation. Our work highlights hitherto unrecognized features of rG4 structure-protein interactions that highlight new roles of rG4 structures in mRNA post-transcriptional control.

A designer enzyme for hydrazone and oxime formation featuring an unnatural catalytic aniline residue.

Creating designer enzymes with the ability to catalyse abiological transformations is a formidable challenge. Efforts toward this goal typically consider only canonical amino acids in the initial design process. However, incorporating unnatural amino acids that feature uniquely reactive side chains could significantly expand the catalytic repertoire of designer enzymes. To explore the potential of such artificial building blocks for enzyme design, here we selected p-aminophenylalanine as a potentially novel catalytic residue. We demonstrate that the catalytic activity of the aniline side chain for hydrazone and oxime formation reactions is increased by embedding p-aminophenylalanine into the hydrophobic pore of the multidrug transcriptional regulator from Lactococcus lactis. Both the recruitment of reactants by the promiscuous binding pocket and a judiciously placed aniline that functions as a catalytic residue contribute to the success of the identified artificial enzyme. We anticipate that our design strategy will prove rewarding to significantly expand the catalytic repertoire of designer enzymes in the future.

TIminer: NGS data mining pipeline for cancer immunology and immunotherapy.

SUMMARY: Recently, a number of powerful computational tools for dissecting tumor-immune cell interactions from next-generation sequencing data have been developed. However, the assembly of analytical pipelines and execution of multi-step workflows are laborious and involve a large number of intermediate steps with many dependencies and parameter settings. Here we present TIminer, an easy-to-use computational pipeline for mining tumor-immune cell interactions from next-generation sequencing data. TIminer enables integrative immunogenomic analyses, including: human leukocyte antigens typing, neoantigen prediction, characterization of immune infiltrates and quantification of tumor immunogenicity. AVAILABILITY AND IMPLEMENTATION: TIminer is freely available at http://icbi.i-med.ac.at/software/timiner/timiner.shtml. CONTACT: zlatko.trajanoski@i-med.ac.at. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pan-cancer Immunogenomic Analyses Reveal Genotype-Immunophenotype Relationships and Predictors of Response to Checkpoint Blockade.

The Cancer Genome Atlas revealed the genomic landscapes of human cancers. In parallel, immunotherapy is transforming the treatment of advanced cancers. Unfortunately, the majority of patients do not respond to immunotherapy, making the identification of predictive markers and the mechanisms of resistance an area of intense research. To increase our understanding of tumor-immune cell interactions, we characterized the intratumoral immune landscapes and the cancer antigenomes from 20 solid cancers and created The Cancer Immunome Atlas (https://tcia.at/). Cellular characterization of the immune infiltrates showed that tumor genotypes determine immunophenotypes and tumor escape mechanisms. Using machine learning, we identified determinants of tumor immunogenicity and developed a scoring scheme for the quantification termed immunophenoscore. The immunophenoscore was a superior predictor of response to anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) and anti-programmed cell death protein 1 (anti-PD-1) antibodies in two independent validation cohorts. Our findings and this resource may help inform cancer immunotherapy and facilitate the development of precision immuno-oncology.

G-quadruplex structures within the 3' UTR of LINE-1 elements stimulate retrotransposition.

Long interspersed nuclear elements (LINEs) are ubiquitous transposable elements in higher eukaryotes that have a significant role in shaping genomes, owing to their abundance. Here we report that guanine-rich sequences in the 3' untranslated regions (UTRs) of hominoid-specific LINE-1 elements are coupled with retrotransposon speciation and contribute to retrotransposition through the formation of G-quadruplex (G4) structures. We demonstrate that stabilization of the G4 motif of a human-specific LINE-1 element by small-molecule ligands stimulates retrotransposition.

An Epigenetics-Inspired DNA-Based Data Storage System.

Biopolymers are an attractive alternative to store and circulate information. DNA, for example, combines remarkable longevity with high data storage densities and has been demonstrated as a means for preserving digital information. Inspired by the dynamic, biological regulation of (epi)genetic information, we herein present how binary data can undergo controlled changes when encoded in synthetic DNA strands. By exploiting differential kinetics of hydrolytic deamination reactions of cytosine and its naturally occurring derivatives, we demonstrate how multiple layers of information can be stored in a single DNA template. Moreover, we show that controlled redox reactions allow for interconversion of these DNA-encoded layers of information. Overall, such interlacing of multiple messages on synthetic DNA libraries showcases the potential of chemical reactions to manipulate digital information on (bio)polymers.

Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase.

By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.