Continuous Fluorescence Assay for In Vitro Translation Compatible with Noncanonical Amino Acids.

The tolerance of the translation apparatus toward noncanonical amino acids (ncAAs) has enabled the creation of diverse natural-product-like peptide libraries using mRNA display for use in drug discovery. Typical experiments testing for ribosomal ncAA incorporation involve radioactive end point assays to measure yield alongside mass spectrometry experiments to validate incorporation. These end point assays require significant postexperimental manipulation for analysis and prevent higher throughput analysis and optimization experiments. Continuous assays for in vitro translation involve the synthesis of fluorescent proteins which require the full complement of canonical AAs for function and are therefore of limited utility for testing of ncAAs. Here, we describe a new, continuous fluorescence assay for in vitro translation based on detection of a short peptide tag using an affinity clamp protein, which exhibits changes in its fluorescent properties upon binding. Using this assay in a 384-well format, we were able to validate the incorporation of a variety of ncAAs and also quickly test for the codon reading specificities of a variety of Escherichia coli tRNAs. This assay enables rapid assessment of ncAAs and optimization of translation components and is therefore expected to advance the engineering of the translation apparatus for drug discovery and synthetic biology.

Extensive breaking of genetic code degeneracy with non-canonical amino acids.

Genetic code expansion (GCE) offers many exciting opportunities for the creation of synthetic organisms and for drug discovery methods that utilize in vitro translation. One type of GCE, sense codon reassignment (SCR), focuses on breaking the degeneracy of the 61 sense codons which encode for only 20 amino acids. SCR has great potential for genetic code expansion, but extensive SCR is limited by the post-transcriptional modifications on tRNAs and wobble reading of these tRNAs by the ribosome. To better understand codon-tRNA pairing, here we develop an assay to evaluate the ability of aminoacyl-tRNAs to compete with each other for a given codon. We then show that hyperaccurate ribosome mutants demonstrate reduced wobble reading, and when paired with unmodified tRNAs lead to extensive and predictable SCR. Together, we encode seven distinct amino acids across nine codons spanning just two codon boxes, thereby demonstrating that the genetic code hosts far more re-assignable space than previously expected, opening the door to extensive genetic code engineering.

p-Chloropropynyl Phenylalanine, a Versatile Non-Canonical Amino Acid for Co-Translational Peptide Macrocyclization and Side Chain Diversification.

Macrocyclization has proven to be a beneficial strategy to improve upon some of the disadvantages of peptides as therapeutics. Nevertheless, many peptide cyclization strategies are not compatible with in vitro display technologies like mRNA display. Here we describe the novel amino acid p-chloropropynyl phenylalanine (pCPF). pCPF is a substrate for a mutant phenylalanyl-tRNA synthetase and its introduction into peptides via in vitro translation leads to spontaneous peptide macrocyclization in the presence of peptides containing cysteine. Macrocyclization occurs efficiently with a wide variety of ring sizes. Moreover, pCPF can be reacted with thiols after charging onto tRNA, enabling the testing of diverse ncAAs in translation. The versatility of pCPF should facilitate downstream studies of translation and enable the creation of novel macrocyclic peptide libraries.

Development of a Cyclic, Cell Penetrating Peptide Compatible with In Vitro Selection Strategies.

A key limitation for the development of peptides as therapeutics is their lack of cell permeability. Recent work has shown that short, arginine-rich macrocyclic peptides containing hydrophobic amino acids are able to penetrate cells and reach the cytosol. Here, we have developed a new strategy for developing cyclic cell penetrating peptides (CPPs) that shifts some of the hydrophobic character to the peptide cyclization linker, allowing us to do a linker screen to find cyclic CPPs with improved cellular uptake. We demonstrate that both hydrophobicity and position of the alkylation points on the linker affect uptake of macrocyclic cell penetrating peptides (CPPs). Our best peptide, 4i, is on par with or better than prototypical CPPs Arg9 (R9) and CPP12 under assays measuring total cellular uptake and cytosolic delivery. 4i was also able to carry a peptide previously discovered from an in vitro selection, 8.6, and a cytotoxic peptide into the cytosol. A bicyclic variant of 4i showed even better cytosolic entry than 4i, highlighting the plasticity of this class of peptides toward modifications. Since our CPPs are cyclized via their side chains (as opposed to head-to-tail cyclization), they are compatible with powerful technologies for peptide ligand discovery including phage display and mRNA display. Access to diverse libraries with inherent cell permeability will afford the ability to find cell permeable hits to many challenging intracellular targets.

Expansion of the genetic code through reassignment of redundant sense codons using fully modified tRNA.

Breaking codon degeneracy for the introduction of non-canonical amino acids offers many opportunities in synthetic biology. Yet, despite the existence of 64 codons, the code has only been expanded to 25 amino acids in vitro. A limiting factor could be the over-reliance on synthetic tRNAs which lack the post-transcriptional modifications that improve translational fidelity. To determine whether modified, wild-type tRNA could improve sense codon reassignment, we developed a new fluorous method for tRNA capture and applied it to the isolation of roughly half of the Escherichia coli tRNA isoacceptors. We then performed codon competition experiments between the five captured wild-type leucyl-tRNAs and their synthetic counterparts, revealing a strong preference for wild-type tRNA in an in vitro translation system. Finally, we compared the ability of wild-type and synthetic leucyl-tRNA to break the degeneracy of the leucine codon box, showing that only captured wild-type tRNAs are discriminated with enough fidelity to accurately split the leucine codon box for the encoding of three separate amino acids. Wild-type tRNAs are therefore enabling reagents for maximizing the reassignment potential of the genetic code.

Hyperaccurate Ribosomes for Improved Genetic Code Reprogramming.

The reprogramming of the genetic code through the introduction of noncanonical amino acids (ncAAs) has enabled exciting advances in synthetic biology and peptide drug discovery. Ribosomes that function with high efficiency and fidelity are necessary for all of these efforts, but for challenging ncAAs, the competing processes of near-cognate readthrough and peptidyl-tRNA dropoff can be issues. Here we uncover the surprising extent of these competing pathways in the PURE translation system using mRNAs encoding peptides with affinity tags at the N- and C-termini. We also show that hyperaccurate or error restrictive ribosomes with mutations in ribosomal protein S12 lead to significant improvements in yield and fidelity in the context of both canonical AAs and a challenging α,α-disubstituted ncAA. Hyperaccurate ribosomes also improve yields for quadruplet codon readthrough for a tRNA containing an expanded anticodon stem-loop, although they are not able to eliminate triplet codon reading by this tRNA. The impressive improvements in fidelity and the simplicity of introducing this mutation alongside other efforts to engineer the translation apparatus make hyperaccurate ribosomes an important advance for synthetic biology.

Spontaneous, co-translational peptide macrocyclization using p-cyanoacetylene-phenylalanine.

Peptide macrocycles (PMCs) are increasingly popular for the development of inhibitors of protein-protein interactions (PPIs). Large libraries of PMCs are accessible using display technologies like mRNA display and phage display. These technologies require macrocyclization chemistries to be compatible with biological milieu, severely limiting the types of technologies available for cyclization. Here, we introduce the novel non-canonical amino acid (ncAA) p-cyanoacetylene-L-Phe (pCAF), which facilitates spontaneous, co-translational cyclization through Michael addition with cysteine under physiological conditions. This new, robust chemistry creates stable macrocycles of a wide variety of ring sizes including bicyclic structures.

Non-canonical Amino Acid Substrates of E. coli Aminoacyl-tRNA Synthetases.

In this comprehensive review, I focus on the twenty E. coli aminoacyl-tRNA synthetases and their ability to charge non-canonical amino acids (ncAAs) onto tRNAs. The promiscuity of these enzymes has been harnessed for diverse applications including understanding and engineering of protein function, creation of organisms with an expanded genetic code, and the synthesis of diverse peptide libraries for drug discovery. The review catalogues the structures of all known ncAA substrates for each of the 20 E. coli aminoacyl-tRNA synthetases, including ncAA substrates for engineered versions of these enzymes. Drawing from the structures in the list, I highlight trends and novel opportunities for further exploitation of these ncAAs in the engineering of protein function, synthetic biology, and in drug discovery.

Silicon phthalocyanines: synthesis and resurgent applications.

The intense far-red absorption and emission features have made silicon phthalocyanines (SiPcs) distinct from the structurally related porphyrin analogues. Unlike most other phthalocyanines, SiPcs possess two additional axial bonds which reduce aggregation in solution and can be synthetically tailored, thereby creating further scope for modulation of optical, chemical and electronic properties. Multiple synthetic strategies have been employed for facile construction of symmetrical or unsymmetrical SiPc variants bearing desired substitutents at the axial and the aromatic ring positions. The overarching motive of this concise review article is to highlight and summarize the key synthetic routes and the fast-emerging applications of SiPcs in photouncaging techniques, photothermal and photoimmunotherapy, photovoltaics, optoelectronics and photocatalysis.

Direct, Competitive Comparison of Linear, Monocyclic, and Bicyclic Libraries Using mRNA Display.

Peptide macrocyclization is typically associated with the development of higher affinity and more protease stable protein ligands, and, as such, is an important tool in peptide drug discovery. Yet, within the context of a diverse library, does cyclization give inherent advantages over linear peptides? Here, we used mRNA display to create a peptide library of diverse ring sizes and topologies (monocyclic, bicyclic, and linear). Several rounds of in vitro selection against streptavidin were performed and the winning peptide sequences were analyzed for their binding affinities and overall topologies. The effect of adding a protease challenge on the enrichment of various peptides was also investigated. Taken together, the selection output yields insights about the relative abundance of binders of various topologies within a structurally diverse library.

Photocontrolled activation of small molecule cancer therapeutics.

Cancer remains one of the leading causes of death worldwide. Conventional treatment of the disease is comprised of chemotherapy, radiation and surgery among other treatment approaches. Chemotherapy is plagued by multiple side-effects caused due to non-specific drug action. Light-based therapies offer an alternative treatment approach that can be fine tuned to achieve the desired effect to treat the disease and address challenges posed by chemotherapeutic side-effects. Photodynamic therapy (PDT) is one of the light mediated treatment modalities that has been successfully applied to treat superficial malignancies with high-efficiency, although its dependence on normoxic conditions limits its efficiency to treat deep-seated tumors. On the other hand, light-sensitive drug-mimetics and drug-release platforms have been deemed efficient in preclinical settings to induce cancer cell death with minimal collateral damage. Drawing from about a decade's worth of examples, we highlight the application of photosensitive molecules as an alternative therapeutic option to PDT and describe their designs that influence the biology of the cancer cells, in turn affecting their viability with high spatio-temporal control.

A new strategy for the in vitro selection of stapled peptide inhibitors by mRNA display.

Hydrocarbon stapled peptides are promising therapeutics for inhibition of intracellular protein-protein interactions. Here we develop a new high-throughput strategy for hydrocarbon stapled peptide discovery based on mRNA display of peptides containing α-methyl cysteine and cyclized with m-dibromoxylene. We focus on development of a peptide binder to the HPV16 E2 protein.

Photo-controlled delivery of a potent analogue of doxorubicin.

Highly cytotoxic agents have found an important niche in targeted anticancer therapy. Here we develop a new light release strategy for the targeting of one of these agents, 2-pyrrolinodoxorubicin, showing dramatic enhancements in toxicity with light and single digit nM potency.

A Platinum(II) Complex of Heptamethine Cyanine for Photoenhanced Cytotoxicity and Cellular Imaging in Near-IR Light.

Controlled generation of cytotoxic agents with near-IR light is a current focus of photoactivated cancer therapy, including that involving cytotoxic platinum species. A heptamethine cyanine scaffolded PtII complex, IR797-Platin exhibits unprecedented Pt-O bond scission and enhancement in DNA platination in near-IR light. This complex also displayed significant singlet oxygen quantum yield thereby qualifying as a near-IR photodynamic therapeutic agent. The complex showed 30-60 fold enhancement of cytotoxicity in near-IR light in various cancer cell lines. The cellular imaging properties were also leveraged to observe its significant co-localization in cytoplasmic organelles. This is the first demonstration of a near-IR light-initiated therapy involving the cytotoxic effects of both active cisplatin and singlet oxygen.

Ribosomal incorporation of backbone modified amino acids via an editing-deficient aminoacyl-tRNA synthetase.

The ability to incorporate non-canonical amino acids (ncAA) using translation offers researchers the ability to extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P). Using this enzyme, we demonstrate ribosomal translation of 11 ncAAs including those with novel side chains, α,α-disubstitutions, and cyclic ß-amino acids.

Hyaluronic Acid Grafted Nanoparticles of a Platinum(II)-Silicon(IV) Phthalocyanine Conjugate for Tumor and Mitochondria-Targeted Photodynamic Therapy in Red Light.

Herein, we report novel hyaluronic acid formulated nanoparticles containing a platinum(II) conjugated silicon(IV) phthalocyanine (SiPc-Pt-HA) for tumor targeted red light photodynamic therapy and chemotherapy. The SiPc-Pt-HA conjugate showed specific uptake, photo-enhanced cytotoxicity (~1500 fold) and mitochondrial accumulation in breast cancer over normal cells.

Calcein represses human papillomavirus 16 E1-E2 mediated DNA replication via blocking their binding to the viral origin of replication.

Human papillomaviruses are causative agents in several human diseases ranging from genital warts to ano-genital and oropharyngeal cancers. Currently only symptoms of HPV induced disease are treated; there are no antivirals available that directly target the viral life cycle. Previously, we determined that the cellular protein TopBP1 interacts with the HPV16 replication/transcription factor E2. This E2-TopBP1 interaction is essential for optimal E1-E2 DNA replication and for the viral life cycle. The drug calcein disrupts the interaction of TopBP1 with itself and other host proteins to promote cell death. Here we demonstrate that calcein blocks HPV16 E1-E2 DNA replication via blocking the viral replication complex forming at the origin of replication. This occurs at non-toxic levels of calcein and demonstrates specificity as it does not block the ability of E2 to regulate transcription. We propose that calcein or derivatives could be developed as an anti-HPV therapeutic.