Chemists have developed a variety of synthetic oligomers “foldamers” that adopt folded conformations. Foldamers offer new opportunities for molecular design but are not amenable to the powerful directed evolution techniques that have been developed for biopolymers, which allow the rapid emergence of function in peptidic or nucleotidic aptamer backbones. This project intends to alleviate these limitations by combining foldamer chemical synthesis with in vitro translation technologies.
This interdisciplinary project aimed at developing a new molecular technology that combines the benefits of the high conformational stability and structural predictability of aromatic amide foldamers, and the powerful in vitro expression and selection of peptides using the Flexible In-vitro Translation (FIT) and Random nonstandard Peptide Integrated Discovery (RaPID) systems. The project demonstrated the ability of stable foldamer helices to template the folding of short peptides. Remarkably, it was found that the ribosome tolerates the presence of foldamer appendages on peptides during translation of mRNA into peptidic sequences. The benefit of the presence of the foldamers being that they carry folding information. This opens up the perspective of achieving directed evolution of peptide-foldamer hybrids with the idea that a hybrid can achieve more than a peptide alone or a foldamer alone. Eventually, ligands for difficult targets based on these hybrids may be identified for diagnostic or therapeutic purposes.
The joint work first required the chemical synthesis of various foldamers with a propensity to fold into a helical conformation, each linked to an amino acid. The ligation of this amino acid bearing a foldamer to a tRNA was then carried out, followed by the ribosomal expression of peptides integrating this amino acid. In many cases mass spectrometry allowed for the demonstration of the correct integration of the foldamer-functionalized amino acid into the peptide sequence. These hybrid structures were then separately synthesized on a larger scale and their structure was elucidated by various spectrocopic methods and by X-ray crystallography.
The project delivered a number of foldamer sequences of varying lengths and propensity to adopt helical conformations. It demonstrated their successful attachment to a tRNA by means of an amino acid. When this tRNA loaded with a foldamer was used as the initiation unit for in vitro peptide translation, peptides were produced that possessed the foldamer as an N-terminal attachment. Further studies showed that foldamers were also integrated during ribosomal peptide synthesis as an amino acid side chain attachment. The benefit of appending the foldamers to these short peptides comes from the folding information that the foldamer carries, which allows one to induce defined conformations in the peptide, as was demonstrated in macrocyclic foldamer-peptide hybrids. These results go far beyond what was expected at the start of the project and demonstrate a high tolerance of the ribosome for non-peptidic entities. Peptides are prototypical biologically active molecules but also have inherent limitations including a poor bioavailability and fast biodegradation. It is expected that foldamer-peptide hybrids offer opportunities for applications where peptide alone or foldamers alone would not work, and that ribosomal expression of these hybrids will accelerate the discovery process.
Approaches for expanding the range of chemical entities that can be produced by the ribosome may accelerate the discovery of molecules that can perform functions for which poorly folded, short peptidic sequences are ill suited. The discovery that the ribosome accepts objects far larger and distinct from peptides than those previously considered has broad implications. It invites to further exploration of the ribosome’s capabilities and bodes well for applications in e.g. therapeutics and diagnostics.
The scientific production at the term of the research contract includes an article entitled “Ribosomal synthesis and folding of peptide-helical aromatic foldamer hybrids“, involving both partners to equal levels, accepted for publication in Nature Chemist
This interdisciplinary project aims at developing a new molecular technology that combines the benefits of the high conformational stability and structural predictability of aromatic amide foldamers, and the powerful in vitro expression and selection of peptides using the Flexible In-vitro Translation (FIT) and Random nonstandard Peptide Integrated Discovery (RaPID) systems. Four specific objectives have been set: 1°) Proof-of-concept demonstration of the foldamer-templated folding of medium sized peptides. Directed evolution of peptides will deliver binders for given foldamer targets. The structure of the bound peptides will be determined; 2°) Challenging the ribosomal expression of mRNA into peptides with an appended foldamer that serves as a covalently bound structural template, and the directed evolution of such foldamer-peptide hybrids; 3°) Incorporation of monomeric units of foldamers into peptide sequences by direct ribosomal expression of mRNAs. In potentially ground-breaking experiments, we will explore the ability of in vitro ribosomal expression to incorporate non-natural monomers to enhance peptide folding. 4°) Implementation of function into hybrid foldamer-peptide architectures: new ligands for hydroxyapatite (HA) and collagen. The specific objectives above all aim at constructing peptide-foldamer hybrids which, because of their improved folding, are potent candidates to bind to a third, difficult, partner. A mineral (HA) and a protein (collagen) involved in bone and tissue repair have been selected.
Added value to the state of the art.
We will develop protocols to routinely combine foldamer chemical synthesis and in vitro peptide translation to produce functional hybrid folded molecular architectures. Earlier attempts towards such combinations are very scarce. The concept of using an entire aromatic amide foldamer to template peptide folding is unexplored and entirely new as of today. Progress towards this very challenging objective is anticipated to generate ground-breaking science in the field of folded molecular architecture design with potential applications in the field of bone and tissue repair and regeneration. In addition, our development of the technology for discovery of these hybrid molecules is anticipated to have to generate novel molecular architectures for a much wider range of industrial and academic applications.
Task 1: aromatic oligoamide foldamers will be produced via solid phase synthesis for the directed evolution via FIT and RaPID technologies of peptides demonstrating binding affinity to a given foldamer surface. Important binding motifs will be probed by NMR and X-ray crystallography and combined with peptides having an ability to bind HA or collagen. Task 2: short foldamer sequences will be incorporated into the FIT methodology in order to act as structural templates for the directed evolution of peptide-foldamer hybrids possessing stable conformations with the ability to bind to HA or collagen. Task 3: individual aromatic monomer units will be chemically synthesized to explore the ability of in vitro ribosomal expression to incorporate these foldamer building blocks into peptide sequences, again to be evolved to bind HA or collagen.
With each task comes an incremental increase in difficulty, but also an increase in impact and significance. Expected results include: (i) the identification and characterization of stable peptide-foldamer hybrid complexes with diverging functional groups; (ii) the demonstration that aromatic amide foldamers can serve as templates to induce the folding of medium sized peptides; (iii) in vitro translation and selection of peptides having an appended foldamer function; (iv) possibly, the ribosomal incorporation of delta-amino acid monomers; (v) peptide foldamer hybrids capable of binding HA or collagen which can be novel “molecular glues” for bone and tissue regeneration.
Madame Céline DOUAT (Chimie et Biologie des Membrances et Nano-Objets - CNRS-Univ. Bordeaux-INP)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
The University of Tokyo Bioorganic Chemistry Lab, Department of Chemistry, Graduate School of Science
CBMN-CNRS Chimie et Biologie des Membrances et Nano-Objets - CNRS-Univ. Bordeaux-INP
Help of the ANR 225,564 euros
Beginning and duration of the scientific project: November 2014 - 36 Months