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, 10 (1), 5097

Expanding the Limits of the Second Genetic Code With Ribozymes

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Expanding the Limits of the Second Genetic Code With Ribozymes

Joongoo Lee et al. Nat Commun.

Abstract

The site-specific incorporation of noncanonical monomers into polypeptides through genetic code reprogramming permits synthesis of bio-based products that extend beyond natural limits. To better enable such efforts, flexizymes (transfer RNA (tRNA) synthetase-like ribozymes that recognize synthetic leaving groups) have been used to expand the scope of chemical substrates for ribosome-directed polymerization. The development of design rules for flexizyme-catalyzed acylation should allow scalable and rational expansion of genetic code reprogramming. Here we report the systematic synthesis of 37 substrates based on 4 chemically diverse scaffolds (phenylalanine, benzoic acid, heteroaromatic, and aliphatic monomers) with different electronic and steric factors. Of these substrates, 32 were acylated onto tRNA and incorporated into peptides by in vitro translation. Based on the design rules derived from this expanded alphabet, we successfully predicted the acylation of 6 additional monomers that could uniquely be incorporated into peptides and direct N-terminal incorporation of an aldehyde group for orthogonal bioconjugation reactions.

Conflict of interest statement

M.C.J., J.S.M., J.Lee, K.E.S., and K.J.S. are co-inventors on the US provisional patent application 62/679,350 that incorporates discoveries described in this manuscript. All other authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Expanding the chemical substrate scope of flexizymes for genetic code reprogramming. a Flexizyme (Fx) recognizes the 3’-CCA sequence of tRNAs and catalyzes the acylation of tRNA using acid substrates. We seek to develop substrate design rules for flexizyme-mediated acylation reactions that expand the scope of chemical substrates used in ribosome-directed polymerization. b An Escherichia coli cell-free protein synthesis system reconstituted from the purified wild-type translational machinery (PURExpress) is used to produce peptide, containing noncanonical acid substrates. This approach for incorporating noncanonical monomers at the N-terminus of peptides is well established. Flexizyme-Leaving Group (FLG) alternatives include CME, DNBE, and ABT
Fig. 2
Fig. 2
Expanding the Fx substrate scope to analogs with various scaffolds. a Systematic design of noncanonical substrates for ribosome mediated polymerization. Phe (A) and structurally diversified noncanonical substrates (B–G). b Fx-catalyzed acylation under optimized conditions. Acid (pH 5.2) denaturing PAGE analysis under various conditions for Fx-catalyzed acylations of a microhelix tRNA (22 nt) with A–G. The acylation reactions were performed using eFx (45 nt) or aFx (47 nt) and monitored over 120 h at two different pHs (7.5 vs. 8.8). Reaction condition: 50 mM HEPES (pH 7.5) or bicine (pH 8.8), 60 mM MgCl2, 1 µM microhelix, 5 µM Fx, and 5 mM substrates in 20 % (v/v) DMSO solution. c The range of noncanonical substrates compatible with Fx was further extended on four different monomer structure (Phe analogs, benzoic acid derivatives, heteroaromatic, and aliphatic substrates). All acylation heat maps are shaded by percent conversion of microhelix. The blue and green color codes are used for the reaction with the CME leaving group:eFx pair and the ABT leaving group:aFx pair, respectively. See in Supplementary Fig. 3 for the numerical values of acylation
Fig. 3
Fig. 3
General substrate design rules for flexizyme tRNA-charging. Substrates with structural similarity to Phe, electron-deficient characteristics, and reduced steric hindrance around the carbonyl group show high compatibility with the flexizyme system
Fig. 4
Fig. 4
Simulated molecular interactions between selected substrates and the binding pocket of eFx. Tetrahedral intermediate models of the CME esters were optimized and subjected to Monte Carlo energy optimization via Rosetta. Dark yellow represents a Phe (A), b hydrocinnamic acid (B), c cinnamic acid (C), d benzoic acid (D), e phenylacetic acid (E). No strong interaction with the guanine residue (top red) is observed for f pyrrole-2-carboxylic acid (25) and g 2-thiophenecarboxylic acid (26); (green: substrate-charged tRNA)
Fig. 5
Fig. 5
Ribosomal synthesis of N-terminal functionalized peptides with noncanonical substrates. a Schematic overview of peptide synthesis and characterization. N-terminal functionalized peptides were prepared in the PURExpress system by using Fx-charged tRNAfMet, purified via the Strep tag, denatured with SDS, and characterized by MALDI mass spectrometry. b Mass spectrum of the peptide in the presence of all 20 natural amino acids and absence of Fx-charged tRNA. c Mass spectrum of the peptide in the absence of methionine and Fx-charged tRNA. di Mass spectra of peptides with N-terminally incorporated noncanonical substrates. *A minor amount of peptide containing phenylalanine at the N-terminus was unformylated. NH2-FWSHPQFEKST-OH; [M + Na]+ = 1415, A: phenylalanine, B: hydrocinnamic acid, C: cinnamic acid, D: benzoic acid, E: phenylacetic acid, G: propanoic acid
Fig. 6
Fig. 6
Putting flexizyme design rules into action for aldehyde and hydrazine bioconjugation. a We designed and synthesized 6 additional substrates (3336). Before Fx-mediated acylation, we estimated acylation of the substrates would give a low (<20 %, red), moderate (20–50%, blue), and high (>50%, green) yield. b 3335 and 37–38 were charged to mihx with the predicted acylation yields (upper panel). In contrast, 36 containing an electron-donating group was not charged to mihx at any pHs, reaction times, and flexizymes, whereas hydrocinnamic acid (B) was charged in 100% yield (lower panel), suggesting our design rules are in a good agreement with our prediction and useful as an efficient tool for predicting a substrate tRNA-charging yield. The obtained acylation yields (OAY, Fig. 6a) were determined by quantifying the relative band intensity on the gel using ImageJ software. c, d Mass spectra of the 35 peptides incubated with a hydrazide dye (Alex Fluor 488) at 37 °C for 1 and 14 h, respectively. The peaks correspond to the peptide with the dye chemically attached to 35. e The purified products obtained from the PURExpress reaction at the time point of 1 h and 14 h showed fluorescence after exposure of UV light filtered by 560/50 nm

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