Improved synthesis of the unnatural base NaM, and evaluation of its orthogonality in transcription and translation.

Unnatural base pairs (UBP) promise to diversify cellular function through expansion of the genetic code. Some of the most successful UBPs are the hydrophobic base pairs 5SICS:NaM and TPT3:NaM developed by Romesberg. Much of the research on these UBPs has emphasized strategies to enable their efficient replication, transcription and translation in living organisms.

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.

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.

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).

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.

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.

Spontaneous, co-translational peptide macrocyclization using -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.

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.

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.

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.

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.

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.

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.

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 Pt 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.

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 β-amino acids.

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.

Genetic incorporation of 4-fluorohistidine into peptides enables selective affinity purification.

Due to the lowered pK of 4-fluorohistidine relative to histidine, peptides and proteins containing this amino acid are potentially endowed with novel properties. We report here the optimized synthesis of 4-fluorohistidine and show that it can efficiently replace histidine in in vitro translation reactions. Moreover, peptides containing 6×-fluorohistidine tags are able to be selectively captured and eluted from nickel resin in the presence of his-tagged protein mixtures.

Highly Constrained Bicyclic Scaffolds for the Discovery of Protease-Stable Peptides via mRNA Display.

Highly constrained peptides such as the knotted peptide natural products are promising medicinal agents because of their impressive biostability and potent activity. Yet, libraries of highly constrained peptides are challenging to prepare. Here, we present a method which utilizes two robust, orthogonal chemical steps to create highly constrained bicyclic peptide libraries.

Light induced drug release from a folic acid-drug conjugate.

A major area of cancer research focuses on improving the specificity of therapeutic agents by engineering drug-delivery vehicles that target overexpressed receptors on tumor cells. One of the most commonly used approaches involves targeting of folate receptors using folic acid conjugated to a drug-containing macromolecular cargo. Once internalized via endocytosis, the drugs must be released from these constructs in order to avoid being trapped in the endosomes.

Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair.

DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol at the first (Tg1), second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3' terminus, was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break.