June 2024
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2 Citations
Israel Journal of Chemistry
Proteins are synthesized within ribosomes through the polymerization of amino acids (AAs). This process requires prior activation of AAs through aminoacylation that attaches them to their corresponding transfer RNAs (tRNAs). Within cells, this attachment is facilitated by aminoacyl‐tRNA synthetase, resulting in a tRNA:AA conjugate. A set of ribozymes developed to acylate tRNA with non‐canonical substrates enables this process outside the confines of living cells, thereby facilitating the synthesis of novel bio‐based products. In modern biotechnology, aminoacylating ribozymes contribute to the production of innovative bio‐based materials bearing functional non‐canonical chemical substrates (NCSs) and fill the gaps in synthesizing unique polymeric backbones, extending the scope beyond traditional peptide bonds. This review summarizes current understanding of flexizymes at the molecular level and their application in generating exceptional polymeric backbones through ribosome‐mediated synthesis in vitro .
![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 Ser60,61 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.](https://www.researchgate.net/publication/364679508/figure/fig2/AS:11431281091938676@1666667499084/Design-of-g-keto-and-hydrazino-esters-and-ribosome-mediated-synthesis-of-pyridazinone_Q320.jpg)
![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 tRNAPro1E2(GGU) and tRNAGluE2(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.](https://www.researchgate.net/publication/364679508/figure/fig4/AS:11431281091929601@1666667499167/Ribosomal-synthesis-of-alternating-copolymers-with-a-pyridazinone-backbone-A-We-designed_Q320.jpg)