Synthesis and Evaluation of Novel Ring-Strained Noncanonical Amino Acids for Residue-Specific Bioorthogonal Reactions in Living Cells

Bioorthogonal reactions are ideally suited to selectively modify proteins in complex environments, even in vivo. Kinetics and product stability of these reactions are crucial parameters to evaluate their usefulness for specific applications. Strain promoted inverse electron demand Diels–Alder cycloadditions (SPIEDAC) between tetrazines and strained alkenes or alkynes are particularly popular, as they allow ultrafast labeling inside cells. In combination with genetic code expansion (GCE)-a method that allows to incorporate noncanonical amino acids (ncAAs) site-specifically into proteins in vivo. These reactions enable residue-specific fluorophore attachment to proteins in living mammalian cells. Several SPIEDAC capable ncAAs have been presented and studied under diverse conditions, revealing different instabilities ranging from educt decomposition to product loss due to β-elimination. To identify which compounds yield the best labeling inside living mammalian cells has frequently been difficult. In this study we present a) the synthesis of four new SPIEDAC reactive ncAAs that cannot undergo β-elimination and b) a fluorescence flow cytometry based FRET-assay to measure reaction kinetics inside living cells. Our results, which at first sight can be seen conflicting with some other studies, capture GCE-specific experimental conditions, such as long-term exposure of the ring-strained ncAA to living cells, that are not taken into account in other assays.     

Debugging Eukaryotic Genetic Code Expansion for Site‐Specific Click‐PAINT Super‐Resolution Microscopy

Super‐resolution microscopy (SRM) greatly benefits from the ability to install small photostable fluorescent labels into proteins. Genetic code expansion (GCE) technology addresses this demand, allowing the introduction of small labeling sites, in the form of uniquely reactive noncanonical amino acids (ncAAs), at any residue in a target protein. However, low incorporation efficiency of ncAAs and high background fluorescence limit its current SRM applications. Redirecting the subcellular localization of the pyrrolysine‐based GCE system for click chemistry, combined with DNA‐PAINT microscopy, enables the visualization of even low‐abundance proteins inside mammalian cells. This approach links a versatile, biocompatible, and potentially unbleachable labeling method with residue‐specific precision. Moreover, our reengineered GCE system eliminates untargeted background fluorescence and substantially boosts the expression yield, which is of general interest for enhanced protein engineering in eukaryotes using GCE.     

Efficient Phage Display with Multiple Distinct Non‐Canonical Amino Acids Using Orthogonal Ribosome‐Mediated Genetic Code Expansion

Phage display is a powerful approach for evolving proteins and peptides with new functions, but the properties of the molecules that can be evolved are limited by the chemical diversity encoded. Herein, we report a system for incorporating non‐canonical amino acids (ncAAs) into proteins displayed on phage using the pyrrolysyl‐tRNA synthetase/tRNA pair. We improve the efficiency of ncAA incorporation using an evolved orthogonal ribosome (riboQ1), and encode a cyclopropene‐containing ncAA (CypK) at diverse sites on a displayed single‐chain antibody variable fragment (ScFv), in response to amber and quadruplet codons. CypK and an alkyne‐containing ncAA are incorporated at distinct sites, enabling the double labeling of ScFv with distinct probes, through mutually orthogonal reactions, in a one‐pot procedure. These advances expand the number of functionalities that can be encoded on phage‐displayed proteins and provide a foundation to further expand the scope of phage display applications.     

Initiation of Protein Synthesis with Non-Canonical Amino Acids In Vivo

By transplanting identity elements into E. coli tRNA^fMet, we have engineered an orthogonal initiator tRNA (itRNA^Ty2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA^Ty2 can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA^fMet from the genome afforded an E. coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.     

Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo

By transplanting identity elements into E. coli tRNA^fMet, we have engineered an orthogonal initiator tRNA (itRNA^Ty2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA^Ty2 can initiate translation in vivo with aromatic non‐canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA^fMet from the genome afforded an E. coli strain in which the efficiency of non‐canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.     

Structural Basis for Expansion of the Genetic Alphabet with an Artificial Nucleobase Pair

Hydrophobic artificial nucleobase pairs without the ability to pair through hydrogen bonds are promising candidates to expand the genetic alphabet. The most successful nucleobase surrogates show little similarity to each other and their natural counterparts. It is thus puzzling how these unnatural molecules are processed by DNA polymerases that have evolved to efficiently work with the natural building blocks. Here, we report structural insight into the insertion of one of the most promising hydrophobic unnatural base pairs, the dDs–dPx pair, into a DNA strand by a DNA polymerase. We solved a crystal structure of KlenTaq DNA polymerase with a modified template/primer duplex bound to the unnatural triphosphate. The ternary complex shows that the artificial pair adopts a planar structure just like a natural nucleobase pair, and identifies features that might hint at the mechanisms accounting for the lower incorporation efficiency observed when processing the unnatural substrates.     

Designing logical codon reassignment – Expanding the chemistry in biology

Over the last decade, the ability to genetically encode unnatural amino acids (UAAs) has evolved rapidly. The programmed incorporation of UAAs into recombinant proteins relies on the reassignment or suppression of canonical codons with an amino-acyl tRNA synthetase/tRNA (aaRS/tRNA) pair, selective for the UAA of choice. In order to achieve selective incorporation, the aaRS should be selective for the designed tRNA and UAA over the endogenous amino acids and tRNAs. Enhanced selectivity has been achieved by transferring an aaRS/tRNA pair from another kingdom to the organism of interest, and subsequent aaRS evolution to acquire enhanced selectivity for the desired UAA. Today, over 150 non-canonical amino acids have been incorporated using such methods. This enables the introduction of a large variety of structures into proteins, in organisms ranging from prokaryote, yeast and mammalian cells lines to whole animals, enabling the study of protein function at a level that could not previously be achieved. While most research to date has focused on the suppression of ‘non-sense’ codons, recent developments are beginning to open up the possibility of quadruplet codon decoding and the more selective reassignment of sense codons, offering a potentially powerful tool for incorporating multiple amino acids. Here, we aim to provide a focused review of methods for UAA incorporation with an emphasis in particular on the different tRNA synthetase/tRNA pairs exploited or developed, focusing upon the different UAA structures that have been incorporated and the logic behind the design and future creation of such systems. Our hope is that this will help rationalize the design of systems for incorporation of unexplored unnatural amino acids, as well as novel applications for those already known.     

Highly Stable trans‐Cyclooctene Amino Acids for Live‐Cell Labeling

trans‐Cyclooctene groups incorporated into proteins via non‐canonical amino acids (ncAAs) are emerging as specific handles for bioorthogonal chemistry. Here, we present a highly improved synthetic access to the axially and the equatorially linked trans‐cyclooct‐2‐ene isomers ( 1 a , b ). We further show that the axially connected isomer has a half‐life about 10 times higher than the equatorial isomer and reacts with tetrazines much faster, as determined by stopped‐flow experiments. The improved properties resulted in different labeling performance of the insulin receptor on the surface of intact cells.     

A general approach for the generation of orthogonal tRNAs

BACKGROUND: The addition of new amino acids to the genetic code of Escherichia coli requires an orthogonal suppressor tRNA that is uniquely acylated with a desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. A tRNA(Tyr)(CUA)-tyrosyl-tRNA synthetase pair imported from Methanococcus jannaschii can be used to generate such a pair. In vivo selections have been developed for selecting mutant suppressor tRNAs with enhanced orthogonality, which can be used to site-specifically incorporate unnatural amino acids into proteins in E. coli. RESULTS: A library of amber suppressor tRNAs derived from M. jannaschii tRNA(Tyr) was generated. tRNA(Tyr)(CUA)s that are substrates for endogenous E. coli aminoacyl-tRNA synthetases were deleted from the pool by a negative selection based on suppression of amber nonsense mutations in the barnase gene. The remaining tRNA(Tyr)(CUA)s were then selected for their ability to suppress amber nonsense codons in the beta-lactamase gene in the presence of the cognate M. jannaschii tyrosyl-tRNA synthetase (TyrRS). Four mutant suppressor tRNAs were selected that are poorer substrates for E. coli synthetases than M. jannaschii tRNA(Tyr)(CUA), but still can be charged efficiently by M. jannaschii TyrRS. CONCLUSIONS: The mutant suppressor tRNA(Tyr)(CUA) together with the M. jannaschii TyrRS is an excellent orthogonal tRNA-synthetase pair for the in vivo incorporation of unnatural amino acids into proteins. This general approach may be expanded to generate additional orthogonal tRNA-synthetase pairs as well as probe the interactions between tRNAs and their cognate synthetases.     

A biosynthetic route to photoclick chemistry on proteins

Light-induced chemical reactions exist in nature, regulating many important cellular and organismal functions, e.g., photosensing in prokaryotes and vision formation in mammals. Here, we report the genetic incorporation of a photoreactive unnatural amino acid, p-(2-tetrazole)phenylalanine (p-Tpa), into myoglobin site-specifically in E. coli by evolving an orthogonal tRNA/aminoacyl-tRNA synthetase pair and the use of p-Tpa as a bioorthogonal chemical "handle" for fluorescent labeling of p-Tpa-encoded myoglobin via the photoclick reaction. Moreover, we elucidated the structural basis for the biosynthetic incorporation of p-Tpa into proteins by solving the X-ray structure of p-Tpa-specific aminoacyl-tRNA synthetase in complex with p-Tpa. The genetic encoding of this photoreactive amino acid should make it possible in the future to photoregulate protein function in living systems.     

Monitoring the site-specific incorporation of dual fluorophore-quencher base analogues for target DNA detection by an unnatural base pair system

We developed intramolecular dual fluorophore-quencher base analogues for site-specific incorporation into DNA by an unnatural base pair replication system. An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) exhibits high fidelity in PCR amplification, and the 2-nitropyrrole moiety of Px acts as a quencher. Deoxyribonucleoside triphosphates of Px linked with a fluorophore (Cy3, Cy5 or FAM) were chemically synthesized, and the fluorescent properties and the enzymatic incorporation of the fluorophore-linked dPxTPs into DNA were examined in PCR amplification. The fluorophore-linked dPxTPs were site-specifically incorporated by PCR into DNA, opposite Ds in templates, with high selectivity. Furthermore, we found that the fluorescence of the triphosphates was partially quenched, but increased upon their incorporation into DNA. These dual fluorophore-quencher base analogues would be useful for site-specific DNA labeling and for monitoring the amplification products of target nucleic acid molecules with a specific sequence. We have demonstrated the utility of the fluorophore-linked Px substrates and the Ds-Px pairing in real-time quantitative PCR for target DNA molecule detection.     

Fragmentation of doubly-protonated peptide ion populations labeled by H/D exchange with CD(3)OD

Doubly-protonated bradykinin (RPPGFSPFR) and an angiotensin III analogue (RVYIFPF) were subjected to hydrogen/deuterium (H/D) exchange with CD(3)OD in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. A bimodal distribution of deuterium incorporation was present for bradykinin after H/D exchange for 90 s at a CD(3)OD pressure of 4 x 10(-7) Torr, indicating the existence of at least two distinct populations. Bradykinin ion populations corresponding to 0-2 and 5-11 deuteriums (i.e., D(0), D(1), D(2), D(5), D(6), D(7), D(8), D(9), D(10), and D(11)) were each monoisotopically selected and fragmented via sustained off-resonance irradiation (SORI) collision-induced dissociation (CID). The D(0)-D(2) ion populations, which correspond to the slower exchanging population, consistently require lower SORI amplitude to achieve a similar precursor ion survival yield as the faster-reacting (D(5)-D(11)) populations. These results demonstrate that conformation/protonation motif has an effect on fragmentation efficiency for bradykinin. Also, the partitioning of the deuterium atoms into fragment ions suggests that the C-terminal arginine residue exchanges more rapidly than the N-terminal arginine. Total deuterium incorporation in the b(1)/y(8) and b(2)/y(7) ion pairs matches very closely the theoretical values for all ion populations studied, indicating that the ions of a complementary pair are likely formed during the same fragmentation event, or that no scrambling occurs upon SORI. Deuterium incorporation into the y(1)/a(8) pseudo-ion pair does not closely match the expected theoretical values. The other peptide, doubly-protonated RVYIFPF, has a trimodal distribution of deuterium incorporation upon H/D exchange with CD(3)OD at a pressure of 1 x 10(-7) Torr for 600 s, indicating at least three distinct ion populations. After 90 s of H/D exchange where at least two distinct populations are detected, the D(0)-D(7) ion populations were monoisotopically selected and fragmented via SORI-CID over a range of SORI amplitudes. The precursor ion survival yield as a function of SORI amplitude falls into two distinct behaviors corresponding to slower- and faster-reacting ion populations. The slower-reacting population requires larger SORI amplitudes to achieve the same precursor ion survival yield as the faster exchanging population. Total deuterium incorporation into the y(2)/b(5) ion pairs matches closely the theoretical values over all ion populations and SORI amplitudes studied. This result indicates the y(2) and b(5) ions are likely formed by the same mechanism over the SORI amplitudes studied.     

Protein Crosslinking by Genetically Encoded Noncanonical Amino Acids with Reactive Aryl Carbamate Side Chains

The use of genetically encoded noncanonical amino acids (ncAAs) to construct crosslinks within or between proteins has emerged as a useful method to enhance protein stability, investigate protein–protein interactions, and improve the pharmacological properties of proteins. We report ncAAs with aryl carbamate side chains (PheK and FPheK) that can react with proximal nucleophilic residues to form intra‐ or intermolecular protein crosslinks. We evolved a pyrrolysyl‐tRNA synthetase that incorporates site‐specifically PheK and FPheK into proteins in both E. coli and mammalian cells. PheK and FPheK when incorporated into proteins showed good stability during protein expression and purification. FPheK reacted with adjacent Lys, Cys, and Tyr residues in thioredoxin in high yields. In addition, crosslinks could be formed between FPheK and Lys residue of two interacting proteins, including the heavy chain and light chain of an antibody Fab.     

A New Orthogonal Suppressor tRNA/Aminoacyl‐tRNA Synthetase Pair for Evolving an Organism with an Expanded Genetic Code

Several steps have been completed toward the development of a method for the site‐specific incorporation of unnatural amino acids into proteins in vivo. Our approach consists of the generation of amber suppressor tRNA/aminoacyl‐tRNA synthetase pairs that are orthogonal to all Escherichia coli endogenous tRNA/synthetase pairs, followed by directed evolution of the orthogonal aminoacyl‐tRNA synthetases to alter their amino‐acid specificities. A new orthogonal suppressor tRNA/aminoacyl‐tRNA synthetase pair in E. coli has been derived from the Saccharomyces cerevisiae tRNA^Asp and aspartyl‐tRNA synthetase, and the in vitro and in vivo characteristics of this pair were determined. Two different antibiotic resistance selections were compared using this novel pair in an effort to develop a tunable positive selection for a mutant synthetase capable of charging its cognate suppressor tRNA with an unnatural amino acid.     

An Evolutionary Strategy for Identification of Higher Order,Green Fluorescent Host–Guest Pairs Compatible with Living Systems

Engineered miniprotein host–small-molecule guest pairs could be utilized to design new processes within cells as well as investigate fundamental aspects of cell signaling mechanisms. However, the development of host–guest pairs capable of functioning in living systems has proven challenging. Moreover, few examples of host–guest pairs with stoichiometries other than 2:1 exist, significantly hindering the ability to study the influence of oligomerization state on signaling fidelity. Herein, we present an approach to identify host–guest systems for relatively small green fluorescent guests by incorporation into cyclic peptides. The optimal host–guest pair produced a 10-fold increase in green fluorescence signal upon binding. Biophysical characterization clearly demonstrated higher order supramolecular assembly, which could be visualized on the surface of living yeast cells using a turn-on fluorescence readout. This work further defines evolutionary design principles to afford host–guest pairs with stoichiometries other than 2:1 and enables the identification of spectrally orthogonal host–guest pairs.     

Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair

We report the discovery of a simple system through which variant pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pairs created in Escherichia coli can be used to expand the genetic code of Saccharomyces cerevisiae. In the process we have solved the key challenges of producing a functional tRNA(CUA Pyl) in yeast and discovered a pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pair that is orthogonal in yeast. Using our approach we have incorporated an alkyne-containing amino acid for click chemistry, an important post-translationally modified amino acid and one of its analogs, a photocaged amino acid and a photo-cross-linking amino acid into proteins in yeast. Extensions of our approach will allow the growing list of useful amino acids that have been incorporated in E. coli with variant pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pairs to be site-specifically incorporated into proteins in yeast.     

Site-specific incorporation of the mucin-type N-acetylgalactosamine-alpha-O-threonine into protein in Escherichia coli

Glycosylation is a prevalent posttranslational process capable of augmenting and modulating protein function. Efficient synthesis of high-purity, homogeneous glycoproteins is essential for the study of unique protein glycoforms and for the manufacture of therapeutically relevant forms. A promising new strategy for controlled in vivo synthesis of glycoproteins was recently established using suppressor tRNA technology. Using an evolved tRNA aminoacyl synthetase-tRNA pair from Methanococcus jannaschii, the glycosyl amino acid, N-acetylglucosamine-beta-O-serine (GlcNAc-beta-Ser), was site-specifically introduced into proteins cotranslationally in Escherichia coli. Herein, we report the evolution of a new tRNA aminoacyl synthetase-tRNA pair that has expanded the repertoire of glycoproteins that can be expressed in E. coli to contain the other major O-linked glycan, N-acetylgalactosamine-alpha-O-threonine (GalNAc-a-Thr).