Sequence-defined heteropolymer backbones

Abstract

Heteropolymer synthesis is achieved by incorporating a β2-backbone substrate using an orthogonal aminoacyl-tRNA synthetase to form a heteropolymer.

Description

Sequence-defined heteropolymer backbones

[0001] This invention was made with government support under grant 2002182 awarded by the National Science Foundation. The government has certain rights in the invention.

[0002] Cross-references to related application

[0003] This application claims priority to U.S. Provisional Application No.63 / 596,304; filed: Nov 05, 2023, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0004] Introduction

[0005] There is great interest in the synthesis and study of sequence-defined biomaterials whose monomer backbones diverge from canonical α-amino acids. Such hetero-oligomers are of interest as therapeutics1,2and bioplastics3and as tools to modulate cell function by providing proteins and polypeptides with proteolytic resistance,4–6improved membrane permeability,7,8and by stabilizing new and known secondary and tertiary structures.9–13As a class, sequence- defined biomaterials provide otherwise non-existent opportunitiesexpand and evolve protein and biomaterial structure and function and develop improved therapeutic agents.

[0006] One monomer family of particular interest for biomaterials consists of β-hydroxy acids (β-HAs) (Fig.1A). β-HAs embody both an expanded backbone and a non-proteinogenic nucleophile and assemble into biomaterials known as β-esters. Isolated β-esters are found in therapeutically relevant natural products (enterobactin, previoprolide),14biosurfactants with environmental applications (surfactin),15and FDA-approved therapeutics (romidepsin, rapamycin).16,17Polymeric β-esters are naturally found in polyhydroxyalkanoate (PHA) polyesters, which are currently in development as bioplastics18and drug encapsulation / delivery systems.19

[0007] Although there exists one report in which a single β2-ester has been introduced into a protein using the wild-type E. coli ribosome in vitro,20there are no examples in which any β2- ester has been introduced into a ribosomal product in vivo. Indeed, there remains only one example21in which a β-backbone of any type has been introduced into a protein in a cell, and that effort required the endogenous and non-orthogonal E. coli PheRS synthetase and an engineered ribosome with a structurally characterized assembly defect.21,22A primary challenge limiting the in vivo ribosomal synthesis of sequence-defined β-ester biomaterials is the absence of an orthogonal aminoacyl-tRNA synthetase (aaRS) / tRNA pair that accepts a β-HA as a substrate. 1 B24-055-2WO

[0008] Previous work has shown that the widely employed and orthogonal pyrrolysyl-tRNA synthetase (PylRS)23can accept several non-α-amino acid substrates, including those in which the α-NH2group is replaced with α-H, α-OH, α-SH, α-N-methyl, and α-N-formyl substituents.24–27 PylRS from Methanomethylophilus alvus (MaPylRS), as well as an engineered derivative MaFRSA,28also accept substrates with α-carboxy substituents in place of the α-amine.

[0009] Examination of the structure of MaFRSA bound to one such substrate, m-CF3-2- benzylmalonate, provided two insights into how PylRS enzymes might engage expanded backbone monomers such as a β2-HA. First, although the structure of PylRS bound to pyrrolysine shows the substrate α-amine coordinated to the homodimeric enzyme in a single conformation via a well-defined active site water molecule,29that water is absent in the structure of FRSA bound to m-CF3-2-benzylmalonate (Fig.1B). In the absence of this bound water, new amide backbone H-bonds from residues L121 and A122 engage the α-carboxy group instead of the bound water (Fig.1B). Second, in the structure of FRSA bound to m-CF3-2-benzylmalonate, the two subunits of the dimeric enzyme bind the prochiral m-CF3-2-benzylmalonate substrate in stereochemically distinct configurations. In one active site, the pro-S carboxylate of m-CF3-2- benzylmalonate engages the backbone amides of L121 and A122 (Fig.1B); in the other, the substrate rotates and the pro-R carboxylate is coordinated instead (Fig.1B).

[0010] Summary of the Invention

[0011] The invention provides methods for the synthesis of sequence-defined heteropolymer backbones and related compositions.

[0012] In an aspect the invention deploys an orthogonal synthetase that accepts a β2-backbone substrate to produce protein hetero-oligomers; heteropolymer synthesis is achieved by incorporating a β2-backbone substrate using an orthogonal aminoacyl-tRNA synthetase to form a heteropolymer.

[0013] In aspects and embodiments the invention provides:

[0014] 1. A method of heteropolymer synthesis comprising incorporating a β2-backbone substrate using an orthogonal aminoacyl-tRNA synthetase to form a β2-hydroxy acid (β2-HA) or β2-amino acid (β2-AA) / α-amino acid (α-AA) hybrid heteropolymer.

[0015] 2. A method herein, wherein the synthetase is a pyrrolysyl-tRNA synthetase (PylRS).

[0016] 3. A method herein, wherein the synthetase is a pyrrolysyl-tRNA synthetase (PylRS), that is Methanomethylophilus alvus (MaPylRS).

[0017] 4. A method herein, wherein the synthetase is a pyrrolysyl-tRNA synthetase (PylRS) mutant with an altered substrate spectrum. 2 B24-055-2WO

[0018] 5. A method herein, wherein the synthetase is a pyrrolysyl-tRNA synthetase (PylRS) mutant with an altered substrate spectrum, selected from: MaPylRS N346A / C348A, MaPylRS N346A / C348L, MaPylRS N346A / C348K, MaFRS1 (N166A / V168L), MaFRS2 (N166A / V168K), and MaFRSA (N166A / V168A).

[0019] 6. A method herein, wherein the aminoacyl-tRNA is MatRNAPylpair.

[0020] 7. A method herein, wherein the substrate is an (S)-β2-amino acid.

[0021] 8. A method herein, wherein the - hydroxy acid.

[0022] 9. A method herein, wherein the β2-hydroxy acid or (S)-β2-amino acid, with (R) or (S) stereochemistry at the carbon.

[0023] 10. A method herein, wherein β2-hydroxy acid (β2-HA) or β2- amino acid (β2-AA) substrate provides a heteropolymer with a functionality compared with a corresponding nonengineered protein, that is a functionality of improved utility in or as a biosurfactant, therapeutic, bioplastic, or and drug encapsulation / delivery system.

[0024] 11. A method herein, wherein the synthesis is in cellulo.

[0025] 12. A heteropolymer incorporating a β2-backbone substrate to form a β2-hydroxy acid (β2-HA) or β2-amino acid (β2-AA) / α-amino acid (α-AA) hybrid heteropolymer.

[0026] 13. A heteropolymer produced by a method herein.

[0027] 14. A method or heteropolymer substantially as described herein.

[0028] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[0029] Brief Description of the Drawings

[0030] Figs.1A-C. β-hydroxy acids in natural products and as substrates for pyrrolysyl- tRNA synthetase (PylRS) variants. Fig.1A. Structures of β2- and β3-hydroxy acids and natural products and biomaterials that contain β2- or β3-hydroxy esters. Fig.1B. Structure of MaFRSA bound to m-CF3-2-benzylmalonate (PDB 7UOR) illustrating the absence of a bound active site water and direct H-bonds between L121 and A122 (Ma numbering) and the (C) pro-S α- carboxyl or (D) pro-R α-carboxyl. Fig.1C. Structures of (S)- and (R)-β2-hydroxy acids.

[0031] Figs.2A-C. MaPylRS and MaFRSA accept β2-hydroxy acids as substrates in vitro. Fig. 2A. Workflow for and LC–HRMS analysis of in vitro tRNAPylacylation reactions containing 12.5 µM MaPylRS and 10 mM of the monomer shown in Fig.2B. Fig.2C. Monomers 5-8. 3 B24-055-2WO

[0032] Description of Particular Embodiments of the Invention

[0033] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and / or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[0034] Incorporation of multiple β2-backbones into proteins in vivo using an orthogonal aminoacyl-tRNA synthetase

[0035] Here we report that β2-hydroxy acids and β2-amino acids possessing both (R) and (S) absolute configurations (Fig.1C) are substrates for PylRS enzymes and report the cellular synthesis of model proteins containing β2-HA and β2-AA residues at internal positions.

[0036] These examples demonstrates an orthogonal synthetase that accepts a β2-backbone substrate and protein hetero-oligomers containing multiple expanded-backbone monomers produced in cellulo.

[0037] MaPylRS accept β2-hydroxy acids as substrates in vitro

[0038] To assess if PylRS-like enzymes would accept β2-hydroxy acids as substrates, we performed in vitro tRNA acylation reactions using purified enzymes and directly analyzed the products using intact tRNA LC-HRMS (Fig.2A).25,30We began with MaPylRS and compared the yields of acylated tRNAPylfrom reactions containing the known substrates (S)-α-NH2-Nε- Boc-Lysine ((S)-α-ΝΗ21) and (S)-α-OH-Nε-Boc-Lysine ((S)-α-OH 2) as well as the enantiopure β2-OH analogs 3 and 4. Reactions performed using 12.5 μM MaPylRS, 25 μΜ MatRNAPyl, and 10 mM 1 or 2 and incubated for 2 hours at 37oC generated the expected mono-acylated tRNA products 1-acyl-tRNAPyl(23188.4 Da) and 2-acyl-tRNAPyl(23189.6 Da) (Fig.2B, in yields of 56% and 59%, respectively. As observed previously,25MaPylRS-promoted acylation of tRNAPylwith (S)-α-OH 2 also generated a di-acylated tRNAPylproduct. Analogous reactions supplemented with (S)-β2-OH 3 or (R)-β2-OH 4 also generated the expected diastereomeric mono-acylated tRNA products 3-acyl-tRNAPyland 4-acyl-tRNAPyl(23203.6 Da) in yields of 27% and 79%, respectively (Fig.2B). Again, under these conditions both substrates also generated detectable levels of di-acylated tRNAPyl; 68% for (S)-β2-OH 3 and 11% for (R)-β2-OH 4. Thus, under these conditions, both β2-OH acids 3 and 4 are substrates for MaPylRS, as anticipated by the FRSA-bound structure of the pro-chiral substrate m-CF3-2-benzylmalonate.25

[0039] It has been estimated that the concentration of a single aaRS enzyme expressed from an endogenous promoter in E. coli falls in the low µM range.31Thus, these initial reactions, 4 B24-055-2WOperformed at high enzyme concentration (12.5 µM, 50 mol% of tRNAPyl), could mask reactivity differences that are relevant under conditions that better mimic the E. coli interior. To more carefully characterize the relative reactivity of monomers 1-4, we evaluated the yield of both mono- and di-acylated tRNAPylas the concentration of MaPylRS was reduced stepwise from 12.5 µM (50 mol%) to 625 nΜ (2.5 mol%). At low PylRS concentrations, the yield of tRNAPylmono-acylated with (S)-β2-OH 3 was comparable to that of (S)-α-OH 2 and higher than that of α-NH21; at higher the di-acylated product predominates. The yield of tRNAPylmono-enantiomer (R)-β2-OH 4 is highest at high enzyme concentrations. These results imply that (S)-β2-OH 3 is a better substrate for MaPylRS than its enantiomer (R)-β2-OH 4, especially under conditions where enzyme concentration is limiting.

[0040] MaFRSA accepts β2-hydroxy acids as substrates in vitro

[0041] The PylRS derivative FRSA contains two active site mutations (N166A and V168A) that favor substrates with substituted Phe side chains.28One of the best substrates for FRSA is the α-amino acid m-CF3-Phe 5 (Fig.2C). To determine if MaFRSA also accepts β2-HA substrates, we performed in vitro tRNA acylation reactions supplemented with m-CF3-Phe 5 alongside analogous reactions containing (S)-α-OH 6 and the enantiopure β2-OH analogs 7 and 8. Reactions performed with 12.5 μM MaFRSA, 25 μΜ MatRNAPyland 10 mM 5 or 6 cleanly generated the expected mono-acylated tRNA products 5-acyl-tRNAPyl(23175.6 Da) and 6-acyl- tRNAPyl(23176.8 Da) (Fig.2C). Quantification of the monoacyl-tRNA and unacylated tRNA pools indicates yields of 45% and 29% for 5-acyl-tRNAPyland 6-acyl-tRNAPylrespectively. Aminoacylation reactions supplemented with 10 mM (S)-β2-HA monomer 7 yielded only a single low signal peak in the TIC corresponding to the molecular weight of monoacyl-tRNA (23189.5 Da) with a yield of <1% 7-acyl-tRNAPyl. By contrast, supplementation of an analogous aminoacylation reaction with (R)-β2-HA 8 yielded two peaks in the TIC corresponding to monoacylated (23189.5 Da) and diacylated 8-acyl-tRNAPyl(23420.1 Da) (Fig. 2C) in 28% and 11% yield, respectively. Interestingly, while MaPylRS processes (S)-β2-OH 3 more efficiently than (R)-β2-OH 4, MaFRSA shows the opposite preference,2-β - HA 8 more efficiently than (S)-β2-HA 7.

[0042] MaPylRS supports in vivo synthesis of a protein containing a single β2-HA.

[0043] We next asked whether the in vitro tRNAPylacylation efficiencies observed with β2-OH substrates would support the incorporation of these monomers into proteins biosynthesized in E. coli. Experiments were performed using the recoded E. coli strain C321.ΔΑ.exp, which lacks all endogenous TAG codons and release factor 1 (RF1).32Cells were co-transformed with a pMega plasmid25encoding the MaPylRS / MatRNAPylpair (pMega-MaPylRS) as well as a pET22b plasmid encoding sfGFP with an in-frame TAG codon at position 3 (sfGFP-3TAG). MatRNAPyl5 B24-055-2WOnaturally decodes TAG codons,33so successful translation of full-length sfGFP is dependent on the concentration and activity of acyl-tRNAPyl. Test expressions were performed in the presence of 0.5 to 2 mM 1-4 and both OD600and 528 nm emission (F528) were monitored as a function of time as a proxy (albeit imperfect)34for sfGFP expression. Although the rate of increase in F528 / OD600was greater for monomers with a natural α-backbone ((S)-α-NH21 and (S)-α-OH 2), by 24 h all growth curves had reached saturation and hence this time point was used for comparisons of F528 / OD600.

[0044] Comparison of F528 / OD600values after 24 h revealed a clear concentration-dependent increase when cultures were supplemented with BocK 1 relative to those in which substrate was withheld (ΔAA) or supplemented with Lys, which is not a substrate for MaPylRS.23Although no concentration dependence of the F528 / OD600value was observed when cultures were supplemented with (S)-α-OH BocK 2, the F528 / OD600values observed after 24 h were comparable to those observed in the presence of 0.5 mM 1. Cultures supplemented with (S)-β2- OH 3 also showed an increase in F528 / OD600relative to those in which substrate was withheld (ΔAA) or supplemented with Lys. Interestingly, like growths supplemented with (S)-α-OH BocK 2, the F528 / OD600values observed after 24 h, although low, were independent of the concentration of (S)-β2-OH 3 over an 80-fold range of concentration. No increases in F528 / OD600 relative to background were observed when cultures were supplemented with (R)-β2-OH 4. Identical protein expression assays with C321.ΔΑ.exp cells harboring MaFRSA and supplemented with either enantiomer of β2-ΟΗ-m-CF3-Phe yielded no significant sfGFP expression over a ΔAA control.

[0045] Two experiments were performed in an attempt to increase the F528 / OD600values of cells expressing sfGFP in the presence of (S)-β2-OH 3. The growth experiments described above were repeated using either Top10 or BL21 (DE3) E. coli in place of C321.ΔΑ.exp, but neither improved the observed F528 / OD600values of growths supplemented with (S)-β2-OH 3. We also examined whether the F528 / OD600values of growths supplemented with (S)-β2-OH 3 could be improved via mutation of MatRNAPyl. Previous work has emphasized the effect of tRNA identity on the efficiency of non-canonical α-amino acid incorporation into proteins35and designer tRNAs such as Pro1E236are often essential to support incorporation of non-α-amino acid monomers during in vitro translation reactions. A rationally evolved, orthologous MbtRNAPyl-optbearing mutations at the base of the acceptor and T-stems improved the incorporation of certain non-canonical α-amino acid substrates into proteins expressed in BL21 (DE3) and Top10 E. coli,28and mutations at the identical sites in an evolved EctRNASecincreased the incorporation of selenocysteine at TAG codons when grown in a derivative strain of C321.ΔA.376 B24-055-2WO

[0046] To evaluate whether these mutations could also improve the incorporation of (S)-β2-OH 3, we transplanted the 4 or 6 mutations found in MbtRNAPyl-optand EctRNASecrespectively into the MatRNAPylbody to generate two new chimeric tRNAs: MatRNAPyl-opt1and MatRNAPyl-opt2encoded in a pMega-MaPylRS plasmid backbone. However, in plate reader-based expression assays of C321.ΔΑ.exp E. coli transformed with pET22b-sfGFP-3TAG and either pMega- MaPylRS encoding either MatRNAPyl-opt1or MatRNAPyl-opt2, we observed severe attenuation of the sfGFP F528 / OD600signal for established MaPylRS substrates (S)-α-NH21 and (S)-α-OH 2 and complete loss of signal over background for (S)-β2-OH 3. Of the cell strains and tRNA chimeras tested, expression of sfGFP-3TAG in C321.ΔΑ.exp using WT MatRNAPyland supplemented with 0.1 mM (S)-β2-ΟΗ 3 yielded the best F528 / OD600signal over background.

[0047] To confirm that (S)-β2-OH 3 was being introduced into sfGFP-3TAG, we isolated sfGFP-3TAG from a preparative growth of C321.ΔΑ.exp cells transformed with pMega- MaPylRS and pET22b-sfGFP-3TAG and supplemented with 0.1 mM (S)-β2-OH 3, and characterized the product using SDS-PAGE and LC-HRMS. SDS-PAGE of cultures supplemented with (S)-β2-OH 3 produced a major protein product of ~28 kDa that ran similarly to proteins expressed in the presence of (S)-α-NH21 and (S)-α-OH 2. The deconvoluted mass spectrum of purified sfGFP-3TAG expressed in the presence of (S)-β2-OH 3 included a major peak at 27840.69 Da, corresponding to the expected molecular mass of sfGFP with (S)-β2-OH 3 at position 3 but lacking residues 1-2, as anticipated due to ester hydrolysis. A second, smaller peak at 27725.47 Da was also observed, corresponding to the mass of sfGFP with Gln at position 3; in this case residue 2 is retained. SDS-PAGE Thus, although (S)-β2-OH 3 and (R)-β2- OH 4 are excellent substrates for PylRS in vitro, with activities that equal that of α-NH21, only (S)-β2-OH 3 is incorporated into proteins in cells, and with lower efficiency than anticipated on aaRS activity in vitro (vide infra).

[0048] To evaluate if a protein containing an intact β2-ester could be isolated, we designed three additional sfGFP expression plasmids in which a TAG codon was inserted in place of or between residues E213 and K214. These two residues can function as the N- and C-termini of an sfGFP variant that assembles from two independent polypeptides.38We reasoned that these sites would therefore be well-suited to accommodate an internal β-ester without disrupting either the sfGFP fold or chromophore maturation. C321.ΔΑ.exp cells were co-transformed with pMega- MaPylRS and a pET22b plasmid encoding sfGFP with an in-frame TAG codon at position 213 (pET22b-sfGFP-213TAG), position 214 (pET22b-214TAG), or between positions 213 and 214 (pET22b-213-TAG-214). Growths were supplemented with 0.1 mM 2, 3, or 4 as described previously, and both F528and OD600were monitored as a function of time. Again, although the rate of increase in F528was greater for growths containing (S)-α-OH BocK 2 than those 7 B24-055-2WOcontaining (S)-β2-OH 3 or (R)-β2-OH 4, by 24 h all growth curves had reached saturation and hence this time point was used for comparisons of F528 / OD600. We observed a robust increase in the F528 / OD600signal of growths expressing sfGFP-213TAG, sfGFP-214TAG, or sfGFP-213- TAG-214 in the presence of (S)-α-OH 2 and a modest increase in F528 / OD600when cultures expressing sfGFP-213-TAG-214 were supplemented with (S)-β2-OH 3.

[0049] sfGFP variants were isolated from of C321.ΔΑ.exp cells transformed with pMega-MaPylRS and either sfGFP-213TAG, or sfGFP-213- TAG-214 and supplemented with BocK 1, (S)-α-OH 2, or (S)-β2-OH 3. The products were characterized by SDS-PAGE, with and without base treatment (pH 10.5) as well as by LC- HRMS. SDS-PAGE analysis of sfGFP-213-TAG-214 isolated from C321.ΔΑ.exp cultures supplemented with (S)-α-ΟΗ 2 and not subjected to base treatment shows the presence of two bands, one that migrates with the MW expected for intact sfGFP (~28 kDa) and one that migrates with the MW expected for the ester hydrolysis product (~24 kDa). SDS-PAGE analysis of an analogous sample subjected to base treatment led to almost complete loss of the 28 kDa band and an increase in the intensity of the 24 kDa, consistent with the presence of a base-labile ester bond. SDS-PAGE analysis of sfGFP-213-TAG-214 grown in the presence of (S)-β2-OH 3 also yielded two bands at ~24 and 28 kDa prior to base treatment. Base treatment led to partial loss of the 28 kDa band and an increase in intensity of the 24 kDa band, suggesting that a fraction of the sample contained a base-labile ester linkage. Only a truncated product could be isolated from growths programmed with sfGFP-213TAG and supplemented with (S)-α-OH 2; in the case of an analogous growth supplemented with (S)-β2-OH 3, both full length and truncated protein was observed and the ratio was unaffected by base treatment. This observation implies that an ester linkage at position 213 of sfGFP is hydrolytically labile.

[0050] The successful internal incorporation of (S)-β2-OH 3 into sfGFP-213-TAG-214 was confirmed by LC-HRMS. sfGFP isolated from growths programmed with sfGFP-213-TAG-214 and supplemented with 0.1 mM (S)-β2-OH 3 contained two sfGFP variants: one contained a single residue of 3, the other a single residue of Gln. As predicted by gel analysis, sfGFP isolated from growths programmed with sfGFP-213-TAG and supplemented with (S)-β2-OH 3 revealed the presence of sfGFP with Gln at position 213 as well as a pair of protein fragments whose approximate molecular weights (~4 kDa and ~23.6 kDa) correspond to those expected if sfGFP was severed at position 213. However, the exact mass of the large (23625.43 Da) fragment was 18 Da less than that predicted on the basis of sequence alone. It is possible that ester hydrolysis in this case is promoted by the Asn residue at position 212, which induces self- cleavage of the ester in a manner analogous to that used by asparagine lyase self-cleaving enzymes, the product of which would undergo a dehydration and loss of water.398 B24-055-2WO

[0051] MaPylRS supports the in vivo synthesis of sfGFP containing two β2-HA monomers

[0052] Having identified that (S)-β2-OH 3 could be introduced into sfGFP at position 3 as well as between positions 213 and 214, we next asked whether this monomer could be introduced at both positions simultaneously. C321.ΔA.exp cells were transformed with pMega-MaPylRS as well as a plasmid encoding sfGFP with TAG codons at position 3 as well as between E213 and K214 (pET22b-sfGFP-3TAG-213-TAG-214), and grown in the presence of 0.1 mM (S)-β2-OH 3 or 0.1 mM (S)-α-OH 2. LC-HRMS analysis of the isolated sfGFP generated in the presence of (S)-β2-OH 3 confirmed the introduction of this monomer at both positions, albeit with significant Gln contamination at one or both positions. However, expression of sfGFP-3TAG-213-TAG- 214 with 0.1 mM (S)-β2-OH 3 in defined media lacking Gln25,40led to only two products: one contained (S)-β2-OH 3 at position 3 and glutamine at position 213-214 (27969.1 Da); the other contained (S)-β2-OH 3 at both positions.

[0053] Metadynamics simulations probe enantioselectivity of the PTC with respect to β2- OH-monomers

[0054] For more than thirty years, the level of non-canonical α-amino acid incorporation at a stop codon has been used as a proxy for aaRS activity in vivo.41This proxy fails for the β2- backbone monomers studied here: Although MaPylRS acylates tRNAPylwith both (S)-β2-OH 3 and (R)-β2-OH 4 at levels comparable to (S)-α-ΝΗ21 and (S)-α-ΟH 2 in vitro, only (S)-β2-OH 3 is introduced into protein in cells. We turned to a previously reported metadynamics workflow42to learn more about how (S)-α-NH21, (S)-α-OH 2, (S)-β2-OH 3 and (R)-β2-OH 4 are accommodated within the ribosomal A-site when loaded on tRNAPyl. The simulations made use of a reduced ribosome model (RRM) containing fMet-tRNAfMetin the P site and either 1-acyl- tRNAfMet, 2-acyl-tRNAfMet, 3-acyl-tRNAfMet, or 4-acyl-tRNAfMetin the A site. Each 100 ns metadynamics simulation was initiated using two distinct monomer poses, performed in duplicate, and the results averaged. One pose aligned the A-site nucleophile with the α-amine of the A-site Met in the 2.1 Å cryo-EM model used to build the RRM;42this pose placed the α-OH (or β2-OH) nucleophile as close as possible to the P-site carbonyl. The second pose was generated by rotating the psi (ψ) angle of the β2-OH monomer by 180°; this pose placed the β2- OH nucleophile as far as possible from the P-site carbonyl.

[0055] Previous results suggest that reactivity within the PTC is related to two distinct parameters: the Nα-C3p2distance between the A-site nucleophile (Nα) and the P-site carbonyl electrophile (Csp2), and the Bürgi-Dunitz attack angle αBD. Monomers that react readily within the PTC populate a conformational space characterized by a Nα-C3p2distance of <4 Å and a αBDvalue between 76 and 115o.42Examination of plots showing αBDas a function of the Nα–Csp2distance reveal minima that differentiate highly reactive ((S)-α-NH21, (S)-α-OH 2), moderately 9 B24-055-2WOreactive ((S)-β2-OH 3), and non-reactive ((R)-β2-OH 4) monomers. The free energy surface for an RRM containing 1-acyl-tRNAfMetin the A-site is defined by an Nα-C3p2distance of 3.9 ± 0.2 Å and αBDof 92.3o± 13.5oaveraged for all final poses within 1 kcal / mol of the energy minimum. Both of these values are comparable to those reported for an RRM with Met- tRNAfMetin the A-site (Nα-C3p2= 3.7 Å and αBD= 76o).42Simulations with 2-acyl-tRNAfMetin the A-site yielded comparable values (Nα-C3p2= 3.9 ± 0.1 Å and αBD= 69.8 ± 3.6o), which is fully consistent with the high reactivity of (S)-α-OH 2 in vivo.

[0056] The free energy surfaces for an RRM containing 3-acyl-tRNAfMetor 4-acyl-tRNAfMetare defined by different parameters. For the RRM containing 3-acyl-tRNAfMetin the A-site, we observe low energy poses characterized by Nα-C3p2distances of 4.5 ± 0.3 Å and αBDvalues of 72.1o± 9.8o. The Nα-C3p2distance for 3-acyl-tRNAfMetfalls outside the Nα-C3p2distance range for monomers predicted to be highly reactive in the ribosome and suggests that the relatively low incorporation of (S)-β2-OH 3 is due in part to poor sampling of conformations within the PTC that support rapid bond formation. The free energy surface of an RRM containing 4-acyl- tRNAfMetis defined by a similar averaged Nα-C3p2distance and αBDbut with much smaller standard deviations (Nα-C3p2= 4.8 ± 0.1 Å and αBD= 88.8o± 1.7o). These differences suggest that 3-acyl-tRNAfMetcan achieve Nα-Csp2 distances that permit modest incorporation of (S)-β2- OH 3 into a ribosomal product whereas 4-acyl-tRNAfMetcannot. This analysis provides one plausible explanation for the observed difference in in vivo reactivity between (S)-β2-OH 3 and (R)-β2-OH 4.

[0057] We next examined the overall structure of the PTC during each trajectory to learn more about how the ribosome itself accommodates alternative backbones within the A-site. We calculated the average distance between the C1’ atoms of all rRNA bases within 5 Å of either acyl-tRNAfMetin the P- or A-site of RRMs containing 1-acyl-tRNAfMet, 2-acyl-tRNAfMet, 3-acyl- tRNAfMet, or 4-acyl-tRNAfMet. The distances calculated from the trajectories with 2-acyl- tRNAfMet, 3-acyl-tRNAfMet, or 4-acyl-tRNAfMetin the A-site were then subtracted from analogous distances seen with 1-acyl-tRNAfMetto generate pairwise interaction analysis (PIA) plots that show how these distances vary in a monomer-dependent fashion.43,44The PIA plots reveal that the distances between PTC C1’ atoms in the RRM containing 3-acyl-tRNAfMetare similar to those observed in the RRM containing 2-acyl-tRNAfMet. In both cases, the largest differences are increases in distances involving A2504 (located within H89) and C2063 (part of the universally conserved A2450-C2063 non-Watson-Crick base pair). By contrast, the distances between PTC C1’ atoms in the RRM containing 4-acyl-tRNAfMetare very different, with many distances substantially lengthened and shortened, including those involving U2504, G2061, A2062, and A2602. Moreover, examination of the average distance between the P-site 10 B24-055-2WOcarbonyl and U2585 shows that the binding of 1-acyl-tRNAfMet, 2-acyl-tRNAfMet, 3-acyl- tRNAfMetresults in movement of U2585 away from the carbonyl, the induced fit expected for reactive monomers, whereas the binding of 4-acyl-tRNAfMetdoes not induce this movement. U2585 is a universally conserved nucleotide that shields the P-site carbonyl from premature hydrolysis by solvent nucleophiles and its movement away from the P-site carbonyl accompanies accommodation of reactive nucleophiles in the A-site.45These differences in RRM structure provide a second explanation for the observed enantioselective preference in in vivo between (S)-β2-OH 3 and (R)-β2-OH 4. Translation factor and / or ribosomal engineering,21,46,47or other approaches,48may be needed to achieve more robust levels of β-linkages within proteins produced in cells.

[0058] β2-amino acid / α-amino acid hybrid heteropolymers.

[0059] In analogous experiments we substituted β2-amino acids for the β2-hydroxy acids to demonstrate and validate β2-amino acid / α-amino acid hybrid heteropolymers. We similarly demonstrate that MaFRSA accepts β2-amino acids (β2-AAs) as substrates in vitro, supports in vivo synthesis of a protein containing a single β2-AA, and supports the in vivo synthesis of sfGFP containing two β2-AA monomers.

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Claims

CLAIMS 1. A method of heteropolymer synthesis comprising incorporating a β2-backbone substrate using an orthogonal aminoacyl-tRNA synthetase to form a β2-hydroxy acid (β2-HA) or β2-amino acid (β2-AA) / α-amino acid (α-AA) hybrid heteropolymer.

2. A method of claim 1, wherein the synthetase is a pyrrolysyl-tRNA synthetase (PylRS).

3. A method of claim 1, wherein the synthetase is a pyrrolysyl-tRNA synthetase (PylRS), that is Methanomethylophilus alvus (MaPylRS).

4. A method of claim 1, wherein the synthetase is a pyrrolysyl-tRNA synthetase (PylRS) mutant with an altered substrate spectrum.

5. A method of claim 1, wherein the synthetase is a pyrrolysyl-tRNA synthetase (PylRS) mutant with an altered substrate spectrum, selected from: MaPylRS N346A / C348A, MaPylRS N346A / C348L, MaPylRS N346A / C348K, MaFRS1 (N166A / V168L), MaFRS2 (N166A / V168K), and MaFRSA (N166A / V168A).

6. A method of claim 1, wherein the aminoacyl-tRNA is MatRNAPylpair.

7. A method of claim 1, wherein the substrate is an (S)-β2-amino acid.

8. A method of claim 1, wherein the substrate is an (S)-β2-hydroxy acid.

9. A method of claim 1, wherein the substrate is an (S)- β2-hydroxy acid or (S)-β2-amino acid, with (R) or (S) stereochemistry at the backbone substituted carbon.

10. A method of claim 1, wherein incorporation of the a β2-hydroxy acid (β2-HA) or β2-amino acid (β2-AA) substrate provides a heteropolymer with a functionality compared with a corresponding nonengineered protein, that is a functionality of improved utility in or as a biosurfactant, therapeutic, bioplastic, or and drug encapsulation / delivery system.

11. A method of claim 1, wherein the synthesis is in cellulo.

12. A heteropolymer incorporating a β2-backbone substrate to form a β2-hydroxy acid (β2-HA) or β2-amino acid (β2-AA) / α-amino acid (α-AA) hybrid heteropolymer. 15 B24-055-2WO

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