Ribosome-mediated biosynthesis of pyridazinone oligomers in vitro

Abstract

The ribosome is a macromolecular machine that catalyzes the sequence-defined polymerization of L-α-amino acids into polypeptides. The catalysis of peptide bond formation between amino acid substrates is based on entropy trapping, wherein the adjacency of transfer RNA (tRNA)-coupled acyl bonds in the P-site and the α-amino groups in the A-site aligns the substrates for coupling. The plasticity of this catalytic mechanism has been observed in both remnants of the evolution of the genetic code and modern efforts to reprogram the genetic code (e.g., ribosomal incorporation of non-canonical amino acids, ribosomal ester formation). However, the limits of ribosome-mediated polymerization are underexplored. Here, rather than peptide bonds, we demonstrate ribosome-mediated polymerization of pyridazinone bonds via a cyclocondensation reaction between activated γ-keto and α-hydrazino ester monomers. In addition, we demonstrate the ribosome-catalyzed synthesis of peptide-hybrid oligomers composed of multiple sequence-defined alternating pyridazinone linkages. Our results highlight the plasticity of the ribosome's ancient bond-formation mechanism, expand the range of non-canonical polymeric backbones that can be synthesized by the ribosome, and open the door to new applications in synthetic biology.

Fig. 1. Ribosome-catalyzed formation of pyridazinone bonds in vitro.

Genetic code reprogramming using the flexizyme system enables the acylation of non-canonical substrates with tRNA. Upon flexizyme-mediated tRNA acylation of keto (orange) and hydrazino (green) activated esters, the programmed keto-tRNA (orange) and hydrazinyl-tRNA (green) were added to an in vitro transcription and translation platform using purified components and allowed to decode two consecutive codons programmed on an mRNA strand. The translation mixture produced a pyridazinone bond (pink). For comparison, the typical peptide bond (red) is shown on the right. Fx flexizyme, AG activating group, CME cyanomethyl ester, DNB dinitrobenzylester, and ABT amino-derivatized benzylthio ester.

Fig. 2. Design of γ-keto and hydrazino esters and ribosome-mediated synthesis of pyridazinone bonds.

A Four γ-keto (orange) and B two hydrazino (green) esters were synthesized with an activated leaving group (CME, DNB, and ABT). DNB or ABT were used for the substrates that do not contain an aromatic moiety and the ABT-activated substances were only synthesized when the DNB substrates were found to be water-insoluble (Supplementary Information; 1-CME, 2-CME, 3-DNB, 3-ABT, 4-DNB; 5-CME, 6-DNB, 6-ABT). The substrates were charged to tRNA by the appropriate Fx and introduced to an in vitro translation reaction containing wild-type ribosomes. C In vitro translation reactions were carried out with pairs of γ-keto ester substrates (A) and hydrazino ester substrates (B). Ribosome-catalyzed synthesis of eight different pyridazinone rings was observed. The relative percent yield of the target oligomer of all species was determined by the peak area corresponding to the theoretical mass/the sum of areas of the whole peaks shown in the mass spectrum, as shown in matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectra (Supplementary Information). Percent yield is based on n = 3 reactions. D, E MALDI-TOF mass spectra of oligomers polymerized by the ribosome in vitro with a pyridazinone bond formed between 1 and 5, and 1 and 6, respectively. The calculated masses of the products in D are [M + H]⁺ = 1362, [M + Na]⁺ = 1384 and in E are [M + H]⁺ = 1286, [M + Na]⁺ = 1308. See SI for MALDI-TOF mass spectra of the other pyridazinone bonds represented in C. The non-target products at masses 1058 and 1080 (#) and 1305 and 1327 (*) are a reporter strep-tag alone (TrpSerHisProGlnPheGluLys) and the peptide containing a misincorporated Ser^, at the Thr (ACC) codon (1(Ser)TrpSerHisProGlnPheGluLys, see Supplementary Fig. 2 for details). Spectra in D and E are representative of n = 3 independent experiments.

Fig. 3. The ribosome is required for pyridazinone formation under in vitro translation conditions.

A The in vitro polymerization reaction was conducted using the same conditions that produced an N-terminal pyridazinone bond in an oligomer, but without the presence of ribosomes. In the deconvoluted mass spectra, the compounds having a molar mass of 179.0701 and 181.1017 were observed as a single peak at 4.4 and 3.6 min, which corresponds to 4-oxo-4-phenylbutanoic acid (orange in B) aminophenylalanine (green in C) hydrolyzed from tRNA^fMet(CUA) and tRNA^Pro1E2(GGU), respectively. D No species corresponding to the theoretical mass of OPDP (322.1317) were observed in the reaction mixture. The extracted ion chromatograms were obtained based on theoretical average masses (Supplementary Information). Spectra are representative of n = 3 independent experiments.

Fig. 4. Ribosomal synthesis of alternating copolymers with a pyridazinone backbone.

A We designed an additional amino acid, γKPheA (7), bearing a ketone on its γ-carbon of the sidechain, for sequential polymerization of pyridazinones bonds on a biopolymer chain. Compounds 7 and 6 were charged to tRNA^Pro1E2(GGU) and tRNA^GluE2(GAU) by flexizyme, respectively, and added to an in vitro transcription and translation reaction. The genetic template was designed to consecutively incorporate the monomers in an alternating fashion (ABAB- or ABABAB-type). The resulting peptides-pyridazinone hybrids were purified via the streptavidin tag (WSHPQFEK) and characterized by MALDI-TOF mass spectrometry. B MALDI mass spectrum of the StrepII-7676 peptide (relative peak area: 14.8%) and its molecular structure, calculated mass: [M + H]⁺ = 1791; [M + Na]⁺ = 1813 (C) MALDI mass spectrum of the StrepII-767676 (relative peak area: 16.9%) peptide and its molecular structure, calculated mass: [M + H]⁺ = 2034; [M + Na]⁺ = 2056. Spectra are representative of n = 3 independent experiments.