Selection-based discovery of druglike macrocyclic peptides.

Macrocyclic peptides are an emerging class of therapeutics that can modulate protein-protein interactions. In contrast to the heavily automated high-throughput screening systems traditionally used for the identification of chemically synthesized small-molecule drugs, peptide-based macrocycles can be synthesized by ribosomal translation and identified using in vitro selection techniques, allowing for extremely rapid (hours to days) screening of compound libraries comprising more than 10(13) different species. Furthermore, chemical modification of translated peptides and engineering of the genetic code have greatly expanded the structural diversity of the available peptide libraries. In this review, we discuss the use of these technologies for the identification of bioactive macrocyclic peptides, emphasizing recent developments.

Ribosomal Incorporation of Aromatic Oligoamides as Peptide Sidechain Appendages.

Derivatives of 4-aminomethyl-l-phenylalanine with aromatic oligoamide foldamers as sidechain appendages were successfully charged on tRNA by means of flexizymes. Their subsequent incorporation both at the C-terminus of, and within, peptide sequences by the ribosome, was demonstrated. These results expand the registry of chemical structures tolerated by the ribosome to sidechains significantly larger and more structurally defined than previously demonstrated.

Reprogramming the genetic code in vitro.

The site-specific introduction of non-canonical amino acids into polypeptides through genetic code reprogramming has become a powerful tool for biochemical studies and bioorganic synthesis. Although a variety of such techniques have been developed, all are based on the 'mis-acylation' of tRNA molecules with non-canonical amino acids. Multiple strategies have been devised to synthesize such non-canonical aminoacyl-tRNAs; for example, those based on protein or ribozyme aminoacyl-tRNA synthetase enzymes are particularly useful. Such techniques have enabled the incorporation of hundreds of different non-canonical amino acids into polypeptides in vitro. This review discusses the development and application of in vitro genetic code reprogramming techniques, especially enzymatic mis-acylation, and examines recent efforts to engineer the translational machinery to increase the range of translatable non-canonical amino acids.

tRNA engineering for manipulating genetic code.

In ribosomal translation, only 20 kinds of proteinogenic amino acids (pAAs), namely 19 l-amino acids and glycine, are exclusively incorporated into polypeptide chain. To overcome this limitation, various methods to introduce non-proteinogenic amino acids (npAAs) other than the 20 pAAs have been developed to date. However, the repertoire of amino acids that can be simultaneously introduced is still limited. Moreover, the efficiency of npAA incorporation is not always sufficient depending on their structures. Fidelity of translation is sometimes low due to misincorporation of competing pAAs and/or undesired translation termination. Here, we provide an overview of efforts to solve these issues, focusing on the engineering of tRNAs.

tRid, an enabling method to isolate previously inaccessible small RNA fractions.

Detection of rare small RNA species whose sizes are overlapping with tRNAs often suffers from insufficient sensitivity due to the overwhelming abundance of tRNAs. We here report a method, named tRid (tRNA rid), for removing abundant tRNAs from small RNA fractions regardless of tRNA sequence species. By means of tRid, we are able to selectively enrich small RNAs which have been previously difficult to access due to mass existence of tRNAs in such fractions. A flexible tRNA-acylation ribozyme, known as flexizyme, is a key tool where the total tRNAs are aminoacylated with N-biotinylated phenylalanine regardless of tRNA sequences, and therefore the biotin-tagged tRNAs could be readily removed from the small RNA fractions by the use of streptavidin-immobilized magnetic beads. Next generation sequencing of the isolated small RNA fraction revealed that small RNAs with less than 200nt were effectively enriched, allowing us to identify previously unknown small RNAs in HeLa and E. coli.

Dual Genetic Encoding of Acetyl-lysine and Non-deacetylatable Thioacetyl-lysine Mediated by Flexizyme.

Acetylation of lysine residues is an important post-translational protein modification. Lysine acetylation in histones and its crosstalk with other post-translational modifications in histone and non-histone proteins are crucial to DNA replication, DNA repair, and transcriptional regulation. We incorporated acetyl-lysine (AcK) and the non-hydrolyzable thioacetyl-lysine (ThioAcK) into full-length proteins in vitro, mediated by flexizyme. ThioAcK and AcK were site-specifically incorporated at different lysine positions into human histone H3, either individually or in pairs. We demonstrate that the thioacetyl group in histone H3 could not be removed by the histone deacetylase sirtuin type 1. This method provides a powerful tool to study protein acetylation and its role in crosstalk between post-translational modifications.

Misacylation of tRNA with Ser-Pro Dipeptide for In Vitro Transcription-Translation.

Serine-proline (Ser-Pro) backbone-modified dipeptide analogues are powerful tools to investigate the role of cis-trans isomerization in the regulation of the cell cycle and transcription. These studies have previously been limited to synthetic peptides, whose synthesis is a challenge for larger peptides due to the compounding yield loss incurred in each step. We now introduce a method for the aminoacylation of tRNA with dipeptides and dipeptide analogs to permit the installation of cis- and trans-locked Ser-Pro analogues into full-length proteins. To that end, we synthesized the 3,5-dinitrobenzyl (DNB)-activated esters of a native Ser-Pro dipeptide and its cis- and trans-locked alkene analogs. Murakami et al. created the DNB flexizyme (dFx), a ribozyme that acylates tRNA with DNB esters of amino acids to permit unnatural amino acids to be incorporated into proteins. A tRNA from yeast that recognizes the amber stop codon, along with the dFx flexizyme, were generated by in vitro transcription with T7 RNA polymerase. dFx was used to successfully catalyze the chemical misacylation of truncated amber tRNA with the Ser-Pro-DNB activated dipeptide. This method allows the introduction of non-native Ser-Pro dipeptide mimics into full-length proteins by in vitro transcription-translation. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 3,5-dinitrobenzyl activated esters of Ser-Pro Basic Protocol 2: Preparation of truncated amber tRNA Basic Protocol 3: Acylation of amber-tRNA by the dFx flexizyme Basic Protocol 4: PAGE electrophoresis of tRNASerPro.

Ribosomal Synthesis of Peptides Bearing Noncanonical Backbone Structures via Chemical Posttranslational Modifications.

Noncanonical peptide backbone structures, such as heterocycles and non-α-amino acids, are characteristic building blocks present in peptidic natural products. To achieve ribosomal synthesis of designer peptides bearing such noncanonical backbone structures, we have devised translation-compatible precursor residues and their chemical posttranslational modification processes. In this chapter, we describe the detailed procedures for the in vitro translation of peptides containing the precursor residues by means of genetic code reprogramming technology and posttranslational generation of objective noncanonical backbone structures.

Chemical insights into flexizyme-mediated tRNA acylation.

A critical step in repurposing the cellular translation machinery for the synthesis of polymeric products is the acylation of transfer RNA (tRNA) with unnatural monomers. Toward this goal, flexizymes, ribozymes capable of aminoacylation, have emerged as a uniquely adept tool for charging tRNA with ever increasingly diverse substrates. In this review, we present a library of monomer substrates that have been tested for tRNA acylation with the flexizyme system. From this mile-high view, we provide insights for understanding the chemical factors that influence flexizyme-mediated tRNA acylation. We conclude that flexizymes are primitive esterification catalysts that display a modest binding affinity to the monomer's aromatic recognition element. Together, these robust, yet flexible, flexizyme systems provide researchers with unprecedented access for preparing unnatural acyl-tRNA and the opportunity to repurpose the translation machinery for the synthesis of novel biologically derived structures beyond native proteins and peptides.

In Vitro Selection of Thioether-Closed Macrocyclic Peptide Ligands by Means of the RaPID System.

The Random nonstandard Peptides Integrated Discovery (RaPID) system enables efficient screening of macrocyclic peptides with high affinities against target molecules. Random peptide libraries are prepared by in vitro translation using the Flexible In vitro Translation (FIT) system, which allows for incorporation of diverse nonproteinogenic amino acids into peptides by genetic code reprogramming. By introducing an N-chloroacetyl amino acid at the N-terminus and a Cys at the downstream, macrocyclic peptide libraries can be readily generated via posttranslational thioether formation. Here, we describe how to prepare a thioether-closed macrocyclic peptide library, and its application to the RaPID screening.

Mechanistic studies of non-canonical amino acid mutagenesis.

Over the past decade, harnessing the cellular protein synthesis machinery to incorporate non-canonical amino acids (ncAAs) into tailor-made peptides has significantly advanced many aspects of molecular science. More recently, groundbreaking progress in our ability to engineer this machinery for improved ncAA incorporation has led to significant enhancements of this powerful tool for biology and chemistry. By revealing the molecular basis for the poor or improved incorporation of ncAAs, mechanistic studies of ncAA incorporation by the protein synthesis machinery have tremendous potential for informing and directing such engineering efforts. In this chapter, we describe a set of complementary biochemical and single-molecule fluorescence assays that we have adapted for mechanistic studies of ncAA incorporation. Collectively, these assays provide data that can guide engineering of the protein synthesis machinery to expand the range of ncAAs that can be incorporated into peptides and increase the efficiency with which they can be incorporated, thereby enabling the full potential of ncAA mutagenesis technology to be realized.

Multiple Site-Specific One-Pot Synthesis of Two Proteins by the Bio-Orthogonal Flexizyme System.

Lysine acetylation is a reversible post-translational modification (PTM) vastly employed in many biological events, including regulating gene expression and dynamic transitions in chromatin remodeling. We have developed the first one-pot bio-orthogonal flexizyme system in which both acetyl-lysine (AcK) and non-hydrolysable thioacetyl-lysine (ThioAcK) were site-specifically incorporated into human histone H3 and H4 at different lysine positions in vitro, either individually or in pairs. In addition, the high accuracy of this system moving toward one-pot synthesis of desired histone variants is also reported.

Discovery of Functional Macrocyclic Peptides by Means of the RaPID System.

Flexizymes, highly flexible tRNA aminoacylation ribozymes, have enabled charging of virtually any amino acid (including non-proteogenic ones) onto tRNA molecules. Coupling to a custom-made in vitro translation system, namely the flexible in vitro translation (FIT) system, has unveiled the remarkable tolerance of the ribosome toward molecules, remote from what nature has selected to carry out its elaborate functions. Among the very diverse molecules and chemistries that have been ribosomally incorporated, a plethora of entities capable of mediating intramolecular cyclization have revolutionized the design and discovery of macrocyclic peptides. These macrocyclization reactions (which can be spontaneous, chemical, or enzymatic) have all served as tools for the discovery of peptides with natural-like structures and properties. Coupling of the FIT system and mRNA display techniques, known as the random non-standard peptide integrated discovery (RaPID) system, has in turn allowed for the simultaneous screening of trillions of macrocyclic peptides against challenging biological targets. The macrocyclization methodologies are chosen depending on the structural and functional characteristics of the desired molecule. Thus, they can emanate from the peptide's N-terminus or its side chains, attributing flexibility or rigidity, or even result in the installation of fluorescent probes.

Artificial Division of Codon Boxes for Expansion of the Amino Acid Repertoire of Ribosomal Polypeptide Synthesis.

In ribosomal polypeptide synthesis, the 61 sense codons redundantly code for the 20 proteinogenic amino acids. The genetic code contains eight family codon boxes consisting of synonymous codons that redundantly code for the same amino acid. Here, we describe the protocol of a recently published method to artificially divide such family codon boxes and encode multiple nonproteinogenic amino acids in addition to the 20 proteinogenic ones in a reprogrammed genetic code. To achieve this, an in vitro translation system reconstituted with 32 in vitro transcribed tRNASNN's (S = C or G; N = U, C, A or G) was first developed, where the 32 tRNA transcripts can be charged with 20 proteinogenic amino acids by aminoacyl-tRNA synthetases in situ and orthogonally decode the corresponding 31 NNS sense codons as well as the AUG initiation codon. When some redundant tRNAGNN's are replaced with tRNAGNN's precharged with nonproteinogenic amino acids by means of flexizymes, the nonproteinogenic and proteinogenic aminoacyl-tRNAs can decode the NNC and NNG codons in the same family codon box independently. In this protocol, we describe expression of model peptides, including a macrocyclic peptide containing three kinds of N-methyl-amino acids reassigned to the vacant codons generated by the method of artificial division of codon boxes.

A flexible, scalable method for preparation of homogeneous aminoacylated tRNAs.

Transfer RNAs (tRNAs) are cellular courier molecules that decipher the genetic code in messenger RNAs and enable the transfer of appropriate esterified amino acids to the growing peptide chain. The preparation of biophysical quantities of homogeneous aminoacylated tRNAs has remained a significant technical challenge. This is primarily due to the difficulty in removing contaminating nonaminoacylated tRNAs that are have very similar properties overall, as well as the hydrolytic instability of the aminoacyl linkage. We describe a flexible, scalable method to prepare homogeneous aminoacylated tRNAs that is also broadly compatible with mutant, misacylated, or otherwise aberrant tRNAs and other RNAs. This method combines ribozyme-mediated aminoacylation with reversible N-pentenoylation of the esterified amino acid, which not only protects against spontaneous deacylation but also provides a hydrophobic purification handle. This protocol makes it straightforward to produce biophysical quantities of natural and unnatural aminoacylated tRNAs and has proven essential for mechanistic investigations of the T-box riboswitches.