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Combining Sense and Nonsense Codon Reassignment for SiteSelective Protein Modification with Unnatural Amino Acids Zhenling Cui,†,# Sergey Mureev,†,# Mark E. Polinkovsky,‡ Zakir Tnimov,§ Zhong Guo,† Thomas Durek,† Alun Jones,† and Kirill Alexandrov*,† †

Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia StemProtein, 6350 Nancy Ridge Drive, San Diego, California 92121, United States § Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom ‡

S Supporting Information *

ABSTRACT: Incorporation of unnatural amino acids (uAAs) via codon reassignment is a powerful approach for introducing novel chemical and biological properties to synthesized polypeptides. However, the site-selective incorporation of multiple uAAs into polypeptides is hampered by the limited number of reassignable nonsense codons. This challenge is addressed in the current work by developing Escherichia coli in vitro translation system depleted of specific endogenous tRNAs. The translational activity in this system is dependent on the addition of synthetic tRNAs for the chosen sense codon. This allows site-selective uAA incorporation via addition of tRNAs pre- or cotranslationally charged with uAA. We demonstrate the utility of this system by incorporating the BODIPY fluorophore into the unique AGG codon of the calmodulin(CaM) open reading frame using in vitro precharged BODIPY-tRNACysCCU. The deacylated tRNACysCCU is a poor substrate for Cysteinyl-tRNA synthetase, which ensures low background incorporation of Cys into the chosen codon. Simultaneously, p-azidophenylalanine mediated ambercodon suppression and its post-translational conjugation to tetramethylrhodamine dibenzocyclooctyne (TAMRA-DIBO) were performed on the same polypeptide. This simple and robust approach takes advantage of the compatibility of BODIPY fluorophore with the translational machinery and thus requires only one post-translational derivatization step to introduce two fluorescent labels. Using this approach, we obtained CaM nearly homogeneously labeled with two FRET-forming fluorophores. Single molecule FRET analysis revealed dramatic changes in the conformation of the CaM probe upon its exposure to Ca2+ or a chelating agent. The presented approach is applicable to other sense codons and can be directly transferred to eukaryotic cell-free systems. KEYWORDS: in vitro protein translation, sense codon reassignment, synthetic tRNA, site-specific protein modification, dual protein labeling, single molecular FRET

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thetase (tRNAPyl/PylRS) of the Methanosarcina family.2−4 Yet the efficient codon reassignment still poses a significant challenge as out of three termination codons naturally devoid of cognate tRNAs, only the amber-codon (UAG) is efficient and displays low level of amino acid misincorporation. Suppression of UGA opal, UAA ochre codons as well as of quadruplet codons were explored for directing cotranslational uAA incorporation5−7 but high levels of Trp misincorporation at opal codon5 and poor overall suppression of ochre codon6 significantly limit their practical use. Effective frameshift suppression requires modification of the ribosomal decoding center as otherwise the yield of polypeptide with uAA is significantly reduced due to competition with endogenous

he universal nature of the genetic code’s translation into protein sequence was considered indisputable after its elucidation in 1960s.1 However, the discovery that selenocysteine2 and pyrrolysine3 are genetically encoded by UGA and UAG codons respectively demonstrated plasticity of the genetic code and inspired the use of nonsense codons for amino acid encoding. Subsequent work by several groups established genetic code reassignment techniques which enabled incorporation of unnatural amino acids (uAAs) into polypeptides endowing the latter with novel chemical and biological activities.2,3 To date more than 150 uAAs have been cotranslationally incorporated into polypeptides in Escherichia coli mainly through engineered archaeal orthogonal tRNA/ aminoacyl tRNA synthetase (o-tRNA/aaRS) pairs derived from tRNATyr/tyrosyl-tRNA synthetase of Methanocaldococcus jannaschii (tRNATyr/TyrRS(Mj)) or tRNAPyl/pyrrolysyl-tRNA syn© 2016 American Chemical Society

Received: August 30, 2016 Published: December 14, 2016 535

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ACS Synthetic Biology tRNAs.7 Several approaches to achieve multisite specific uAA incorporation by either using two quadruplets suppression8 or by combining amber suppression with quadruplet or ochre suppression9,10 have been reported. However, the limited number of available nonsense codons and the low efficiency of quadruplet suppression call for the development of new strategies for multisite installation of uAAs. The degeneracy of the genetic code potentially enables orthogonalization of the redundant sense codons and their reassignment to uAAs.3 This was attempted by several groups with partial success.6,11−14 An early example demonstrated reassignment of UUU codons with 80% efficiency to naphthylalanine(Nal) by using engineered yeast tRNAPheAAA and PheRS mutant in Phe-auxotrophic strain.15 It is expected that the codons decoded through wobble recognition would be easier to reassign by an o-tRNA with Watson−Crick basepairing. The reassignment of UUU (Phe), AAG (Lys), CAU (His), AAU (Asn) codons through wobble recognition was evaluated using GFP reporters only achieving the maximum suppression efficiency of 6%.16 The attempt to reassign AGU (Ser) to 3-iodo-phenylalanine led to its 65% incorporation into position 208 of GFP but no incorporation into position 72 pointing to strong contextuality.17 Due to the low abundance of the corresponding tRNA isoacceptors in E. coli, AGG(Arg) codon was assumed to be the easiest one to reassign. However, an early attempt of AGGcodon reassignment to Nε-tert-butoxycarbonyl-L-lysine (BocK) using tRNAPylCUU in the BL21(DE3) strain was unsuccessful.6 Subsequently AGG reassignment to various uAAs mediated by TyrRS(Mj)12 or PylRS13,14 aminoacylation systems were achieved through multiple genetic knockouts, gene complementation, promoter tuning, codon replacements as well as Arg-depleted media to counteract the competing endogenous Arg-tRNA. These efforts point to strong competition of native aa-tRNAs with the uAA-(o-tRNA) for codon recognition against even the least abundant Arg-tRNA thus representing a major hurdle for quantitative uAA incorporation. The latter becomes particularly important for multisite protein labeling where otherwise partial suppression at each codon results in a mixed protein population. Hence, effective suppression of competition with the endogenous tRNA is critical. Recently, Suga’s group demonstrated the replacement of redundant isoacceptors for three wobble-restricted codon boxes with synonymous orthogonal counterparts charged with uAAs in the context of a fully reconstituted E. coli in vitro translation system. This enabled production of a peptide containing a library of 20 canonical AAs extended with 3 uAAs.18 Favorable competition of o-tRNAs for the reassigned codons with their endogenous counterparts that copurify with the cognate aaRSs and result in incorporation of native AAs,19 is achieved by maintaining ultrahigh o-tRNA/aaRS ratio. If applied to protein translation such high tRNA excess may affect the yield and the fidelity of the protein synthesis. Sense codon reassignment using S30 cell extract20 would reduce the technical and economic barrier for adoption and provide a platform for rapid generation of protein probes for EPR, NMR, cryoEM and single-molecule fluorescent spectroscopy.21 Förster resonance energy transfer carried out at singlemolecule resolution (smFRET) allows precise measurement of the distance between two dyes enabling rapid analysis of protein structure, folding and conformational rearrangements.22 Current strategies for site-selective incorporation of a FRETforming dye pair into proteins rely on the cotranslational

introduction of two uAAs that provide functionalities for the subsequent orthogonal conjugation with the dyes. One approach relies on simultaneous incorporation of azide and alkyne functionalities followed by their reciprocal and consecutive copper-catalyzed click chemistry coupling.5 The main drawbacks of this approach are the utilization of reciprocally compatible reactive groups and requirement for copper(I) which often induces protein aggregation and precipitation. The former limitation requires the removal of the unreacted group before initiating the next conjugation step. To overcome the problem of cross-reactivity a number of elegant methods have been developed. Incorporation of mutually nonreactive uAAs such as the tetrazine-/norbornene9 or azide-/ketone- systems10,23 is one method. Alternatively, “one-pot” sequential dual-labeling is achieved by either finetuning of the reactivity between strain-promoted alkynes and alkenes with tetrazine derivatives24 or combining the tetrazine/ alkene with azide/alkyne chemistry.25 Although the “one-pot” approach minimizes purification and handling steps, it requires multiple preparatory steps and precise timing to accommodate fine differences of the conjugation kinetics. Therefore, a straightforward strategy for smFRET probe preparation is needed. In this study, we present a generic approach for reassignment of sense codons to uAA in the in vitro translation system. It is based on a generally applicable approach for removal of tRNA of choice from the in vitro translation reaction by DNAhybridization chromatography. This approach could be potentially applied to a range of tRNAs allowing reassignment of multiple nonidentical codons. In order to simplify the introduction of fluorescent uAAs into the chosen positions we developed a chemo-enzymatic method that combines cotranslational incorporation of a fluorogenic uAA with post translational modification of a second orthogonal group with another fluorophore. The developed approach to the production of the calmodulin proteins furnished with FRET-forming fluorescent groups.

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RESULTS Freeing AGG Codon for uAA Incorporation. The main obstacle for in vitro sense codon reassignment is the presence of the native tRNA isoacceptors in the cell lysate. We recently demonstrated near-complete depletion of native arginine isoacceptors specific for the AGG codon from E. coli in vitro translation system.19 Such system failed to support translation of GFP ORF harboring a single AGG-codon that could be rescued by addition of the synthetic wild type tRNAArgCCU. Semisynthetic tRNA mixtures used in this approach offer great flexibility in composition but are laborious in preparation. Therefore, we decided to test an alternative approach where the target tRNA is selectively depleted from the total native tRNA mixture by DNA-hybridization chromatography. In E. coli the AGG codon is decoded by two isoacceptors, tRNAArgCCU and tRNAArgUCU.19 The DNA oligonucleotides complementary to the sequence spanning the D-arm down to the anticodon loop or variable loop up to the acceptor stem of tRNAArgUUc and tRNAArgCCU, respectively, were used for their chromatographic depletion from the total E. coli tRNA mixture. The translation efficiency of tRNA mixtures before and after tRNAArgCCU/UCU depletion was tested by their ability to support translation of GFP templates with one or six AGG codons (Supplementary Table S3). 536

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ACS Synthetic Biology The use of the total tRNA mixture depleted of AGGdecoding isoacceptors resulted in the negligible background translation of the 1AGG-template and no detectable translation for the 6AGG-template (Figure 1A, B). Depending on the

Using this assessment criteria, the depletion efficiencies for the total tRNA range from 89% to 94% (Figure 1B) when calculated based on 1AGG-template. The depletion efficiencies were almost 100% when calculated based on 6AGG-template (Figure 1A). In order to test whether this approach can be applied to other codons, we performed depletion of the isoacceptor decoding AGC (Ser) codon (Figure 1C). This isoacceptor is relatively abundant in E. coli accounting for approximately 2.18% of the total tRNA. We show that tRNA fraction depleted for this isoacceptor cannot support translation of templates with 2 consecutive AGC codons, while expression of templates with a single codon was strongly reduced. The depletion efficiencies of AGC-tRNA isoacceptor were approximately 100% and 60% when calculated based on 2AGC-and 1AGCcodon templates, respectively. Reassigning AGG Codon to AzF. After demonstrating that the AGG codon can be freed of its native tRNA decoder and made available for reassignment, we set out to identify the best tRNA-charging systems that could support this process. We took advantage of amber-codon suppression to test the ability of the available o-tRNA/aaRS pairs (MjTyr system and Pyl system) in the in vitro translation reaction (Supplementary Figure S1 and S2). Our findings demonstrated that the orthogonal MjTyr system mediated high level of p-azido-Lphenylalanine (AzF, 5 on Supplementary Figure S1a) incorporation at the amber-codon. AzF features a bioorthogonal azide group that can be derivatized by a range of reagents. MjTyrRS has a shortened anticodon-binding domain27 that relies mainly on G34 for tRNA recognition.28 Converting G to C in the first anticodon position in MjtRNATyr impairs its aminoacylation efficiency by approximately 100 fold.29 However, it still performs well in amber-codon suppression (Supplementary Figure S1c,d,e) and30,31. Mutating all three anticodon nucleotides (GUA to CAU) was reported to cause >800-fold reduction in aminoacylation efficiency.29 Hence mutation of the anticodon to AGG-codon suppressor is expected to require much higher o-tRNA/enzyme concentrations to overcome the reduction in affinity. We set out to evaluate the possibility of reassigning AGG codon to AzF by combining the lysate lacking isoacceptors for AGG codon with MjTyr system (o-tRNAAzF2CCU/AzFRS(Mj)/ AzF). O-tRNAAzF2CCU was constructed based on pAzPhe2.32 It was chosen due to the highest efficiency in supporting AzF incorporation among four tRNA variants tested in our study (Supplementary Figure S1c). To test the AGG-suppression under various codon contexts, we introduced a single AGGcodon at the position 1 or 151 of two codon-biased GFP templates designated A and B. In these templates the remaining arginines were encoded by the CGN family codons (Supplementary Table S3). The depletion efficiency of native AGG isoacceptors for A_151R, A_1R, B_151R, B_1R were found to be 75%, 80%, 93% and 84%, respectively (eq 1). For 151R and 1R, supplementation of the reaction with otRNAAzF2CCU resulted in 4.2 and 5.2-fold increase in GFP fluorescence for A subsets and 18.4 and 7.2-fold increase for B subsets (Figure 2). This indicates that more than 80% of GFP protein was modified with AzF. Mass spectroscopy analysis of the translation products demonstrated that most of the protein (>90%) had AzF incorporated at the AGG-defined position with a minor fraction harboring Arg at this site (Supplementary Figure S4b,c).

Figure 1. Translation efficiency of GFP-coding templates with testcodons in the tRNA-depleted E. coli lysate reconstituted with native total or isoacceptor-depleted E. coli tRNA. (A) Translation efficiency of GFP-coding templates with six AGG arginine codons in the presence of native or tRNAArgCCU/UCU depleted E. coli tRNA. The tRNA concentrations are indicated. (B) As in A but using GFP template with only one AGG codon. (C) Translation efficiency of GFP-coding templates with one or two consecutive AGC serine codons in the presence of native total or tRNASerGCU depleted E. coli tRNA at 19 μM. “Total tRNA” represents the native total tRNA mixture before depletion and “Depleted tRNA” represents the total tRNA mixture after depletion of the isoacceptors. “Depleted tRNA +t7tRNA” represents depleted tRNA supplemented with 5 μM of synthetic t7tRNAArgCCU or t7tRNASerGCU. The error bars represent standard deviations of three experiments.

number of AGG-codons in the coding templates, supplementation of the reactions with t7 polymerase-transcribed tRNAArgCCU (t7tRNAArgCCU) restored translation to the level similar or higher to that achieved with the native total tRNA mixture. According to previous report,26 when the E. coli cells grow at rate of 0.4 doubling per hour the proportion of tRNAArgCCU and tRNAArgUCU in the native total mixture is 0.65% and 1.34%, respectively. This corresponds to 0.1 μM and 0.25 μM concentration of the 19 μM native total tRNA mixture. The low abundance of AGG-decoding isoacceptors of the native tRNA mixture is responsible for the lower translational level of GFP-coding template with six AGG codons, compared to the one supplemented with 5 μM t7tRNAArgCCU (Figure 1A). We chose to use 5 μM of synthetic tRNAArgCCU in our experiments to ensure that this tRNA is present in large excess and is not limiting when templates with both 1 and 6 AGG codons are used. We defined tRNA depletion efficiency as 1 − (RFU(Depleted tRNA)/RFU(Depleted tRNA + t7tRNAArg CCU))

(1) 537

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reporter group to the enzymatically aminoacylated tRNA via the reactive sulfhydryl group of cysteine36 or ε-amino group of lysine.37 We chose to use cysteine due to small size of the aliphatic spacer-arm as well as the fact that cysteinyl-tRNA synthetase (CysRS) is robust enzyme that lacks the editing domain38,39 and, therefore, does not hydrolyze chemically precharged tRNA. We performed in silico modeling using crystal structure on EF-Tu complex with tryptophanyl tRNA40 to assess the ability of the former to accommodate a dye in the place of tryptophan side chain. The spacer separating BPFL ring from the aminoester backbone was adjusted to 5-mer as in BPFL-tRNACys conjugate (Supplementary Figure S5). In the resulting model BPFL occupies the pocket in a slightly different orientation as tryptophan without steric clashes (Supplementary Figure S6). We concluded that BODIPY-FL (BPFL) was sufficiently small to fit in the available binding pocket of the EF-Tu and hence could be efficiently incorporated during protein synthesis. To test this experimentally, the synthetic tRNACys variants harboring either CUA or CCU anticodons were aminoacylated in vitro and conjugated to BPFL. HPLC analysis demonstrated the formation of BPFL-tRNACys products with ∼15−20% final yield (Figure 3A, B). Next we introduced the purified BPFLtRNACysCCU into the “parental” and the tRNA-depleted lysate. Reconstitution of the latter lysate and native total tRNA retained more than 80% activity of its parental lysate as judged either by Western blotting with anti-GFP antibody (lane 1 and 2, Figure 3C) or the total GFP fluorescence signal (Supplementary Figure S7). The yield of labeled protein in the depleted system supplemented with BPFL-tRNACysCCU increased 1.4 times while the relative labeling efficiency (BPFL/Anti-GFP ratio) was 7-fold higher, compared to parental lysate (lane 8 and 5, Figure 3C). The yield of labeled protein is estimated to be 18 μg/mL, which is corresponding to 20% suppression efficiency (lane 1 and 8, Figure 3C). The BPFL-tRNA less favorably competes with the native tRNA for decoding the AGG codon in “parental” lysate due to the presence of native AGG-decoding isoacceptors.41 The further reduction of BPFL/Anti-GFP ratio in the reconstituted system of depleted lysate with 19 μM native tRNAs (lane 6, Figure 3C) or 5 μM synthetic tRNAArgCCU (lane 7, Figure 3C) indicate that the more abundant of the AGG-decoding tRNA isoacceptors in these systems compete effectively to the BPFL incorporation. Almost no GFP was produced in the depleted translation system supplemented with total tRNA mixture lacking isoacceptors for AGG (lane 4, Figure 3C). The expression of GFP could be restored by the addition of BPFL-tRNACysCCU (lane 8, Figure 3C) indicating that virtually all expressed polypeptide contained BPFL-fluorophore. We concluded that BPFL-charged tRNA was compatible with the host translational machinery including both the elongation factor and the ribosome. Importantly the tRNACysCCU discharged from uAA during translation undergoes only negligible cotranslational recharging with cysteine (Supplementary Figure S8). The latter is due to the reduced kcat/Km of endogenous CysRS toward transplanted tRNACysCCU39 that fails to compete with the endogenous wt tRNACysGCA. This ensures low level of Cys misincorporation in the developed system. Next we compared amber and AGG codon-mediated BPFL incorporation efficiency using four templates, (B_1X(151X) and B_1R(151R)) that harbor either single amber- or AGGcodon at the first and 151st position of GFP ORF

Figure 2. AGG reassignment to AzF in the context of four GFPencoding ORFs. (A) Translation efficiency of GFP-coding template A with single AGG codon at the 1st or 151st positon. Template A encoding eGFP with optimized codon composition (Supplementary Table S3) were used for expression in the cell-free reactions containing tRNA-depleted lysate, AGG-isoacceptors depleted tRNA, AzFRS and AzF with or without o-tRNAAzF2CCU. The final concentrations for otRNAAzF2CCU, AzFRS, AzF were 10 μM, 10 μM and 1 mM, respectively. (B) as in A but using template B with single AGG codon in the ORF (B_1R and B_151R) (Supplementary Table S3). Template B encodes sGFP with predominantly the unique codons for each type of amino acid.

Reassignment of AGG Codon to BODIPY-FL uAA Derivative. The successful reassignment of AGG to AzF by MjTyr o-system provided us with the orthogonal codon that may be combined with UAG codon for two-site uAAs incorporation. The recently developed bioorthogonal reaction between strained alkenes/alkynes and tetrazine displays rate constants several orders of magnitude higher than earlier versions of click-reactions.33 We reasoned that genetic incorporation of strained alkenes/alkynes would be an ideal strategy to achieve fast and selective protein labeling with tetrazine fluorophores. However, we failed to achieve efficient suppression with either large cycloaliphatic alkynes or alkenes (Supplementary Figure S1a,b). Propargyl-L-lysine (PrK) is a reasonably efficient substrate of Pyl system (Supplementary Figure S1a,b). However, the cross-reactivity between PrK and AzF requires iterative use of copper-catalyzed click chemistry5 which would complicate the labeling procedure. In order to circumvent the problem of potential crossreactivity we decided to devise an alternative approach based on the direct incorporation of a fluorescent uAA into one reassigned codon, followed by a click-chemistry based conjugation of the second fluorophore to uAA incorporated into the second reassigned codon. This approach is expected to be simpler than the previously published dual labeling procedures.5,9 Bulky fluorophore-bearing amino acid analogues cannot be efficiently accommodated by aaRS, precluding their cotranslational incorporation. Supplementation of tRNAs precharged with fluorescent amino acid provides an alternative approach. Although several strategies were developed for precharging tRNA with the uAAs, all of them are technically challenging. One involves ligation of chemically prepared aminoacylated dinucleotide to a truncated tRNA lacking the 3′-CA dinucleotide.34 In an alternative approach a tRNA acylation ribozyme termed flexizyme was developed to in vitro charge uAAs on tRNAs.35 Although this approach is in principle straightforward, it relies on multistep chemical synthesis and the charging efficiency varies for different uAAs (Supplementary Figure S3). Further approach is based on coupling of the 538

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Figure 3. Purification of BPFL-tRNACys conjugates and analysis of their suppression efficiencies of AGG and UAG codons. (A) HPLC purification of BPFL-conjugated tRNAs. The tRNACys bearing CUA anticodons charged with Cys by CysRS subsequently reacted with the BPFL iodoacetamide group. Ribonucleic acid and BPFL were detected at 254 and 490 nm, respectively. (B) As in A but using tRNACys bearing CCU. (C) SDS-PAGE analysis of in vitro translated GFP labeled with BPFL. The BPFL incorporation was detected by fluorescence scanning of the gel after eliminating GFP fluorescence by boiling. The total protein was detected by Western blotting with anti-GFP antibody. The relative density of each band was calculated using ImageJ. The sGFP_T2 template harboring one AGG codon was expressed with or without addition of BPFL-tRNACysCCU in the normal lysate or tRNA depleted lysate supplemented with indicated tRNA mixtures. “Depl. lysate” denotes the tRNA-depleted lysate. “Depl. lysate +depl. tRNA” denotes tRNA depleted lysate supplemented with the total tRNA fraction after depletion for AGG-decoding isoacceptor. “t7tRNAArgCCU” denotes synthetic tRNAArgCCU. The native tRNA mixtures were added to the cell free reaction at 19 μM while synthetic tRNAArgCCU was added to at 5 μM. (D) Comparison of BPFL incorporation mediated by amber or AGG codon suppression. The translation reactions for AGG suppression were reconstituted with tRNA-depleted lysate and AGG isoacceptors-depleted tRNA mixture and programmed by template B with single AGG at the 1st or 151st position of GFP ORF with or without BPFL-tRNACysCCU supplementation. Similar reactions were performed for amber suppression but using normal S30 lysate, the template harboring single amber-codon and BPFL-tRNACysCUA suppressor.

becomes more straightforward. We tested this approach by in vitro translating calmodulin (CaM) protein with AzF as a bioorthogonally reactive uAA and a fluorophore-bearing BPFL-Cys incorporated at first and 149th position via amber and AGG codon reassignment, respectively. TAMRA-fluorophore (TA), that form a FRET pair with BPFL, was then introduced into the target protein via copper-free click chemistry resulting in double labeled CaM-TA1-BPFL149. Fluorescence gel scanning confirmed successful installation of two FRET probes in CaM protein (Figure 4A). Analysis of the emission spectra of the solution of the purified, double-labeled CaM revealed two distinct peaks (Figure 4B) corresponding to emission of BPFL at 512 nm and TAMRA at 575 nm. To confirm formation of a FRET pair we used the same concentration of single-labeled CaM-TA1 as a reference. When both samples were excited at 488 nm, the emission fluorescence at 575 nm of double labeled protein was much stronger than that of the protein labeled with TAMRA only, indicating the energy transfer from donor fluorophore BPFL to acceptor fluorophore TAMRA in dually labeled CaM.

(Supplementary Table S3). The translation reactions were supplemented with purified BPFL-tRNACys and primed with the respective templates (Figure 3D). AGG suppression with BPFL-tRNACysCCU was more robust as it gave 1.4 and 3.4 times higher BPFL incorporation efficiency (fluorescence/total protein ratio) on the first and 151st position respectively, than amber suppression. This indicates low competition from the native tRNA isoacceptors for the AGG-codon compared to that of release factor-mediated translation termination on amber-codon. Combination of AGG and Amber-Codon Reassignment for Dual Protein Labeling. As alluded to in the introduction, the current strategies for site-specific dual protein labeling with a FRET-forming dye pair rely on incorporation of two bio-orthogonal groups followed by subsequent conjugation reactions delivering the FRET forming fluorophores.9,24 The requirement for the careful control of reactivity of the groups at the uAAs and fluorophores makes these approaches technically challenging. Since BPFL could be incorporated into a protein sequence cotranslationally, post-translational installation of the second fluorophore through the genetically encoded uAA 539

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Figure 4. Site-specific dual labeling of calmodulin (CaM) for smFRET analysis. (A) Fluorescence scan of SDS-PAGE loaded with mono- and duallabeled CaM. Dual labeled CaM protein was prepared in cell-free translation system reconstituted from tRNA-depleted lysate and AGG isoacceptorsdepleted tRNA mixture. CaM-coding template harboring single amber and AGG codon, respectively, at the 1st and 149th position of its ORF was used (Supplementary Table S3). The gel was scanned in two emission wavelengths. (B) Fluorescence emission spectra of single- and dual- labeled CaM excited at 488 nm. The concentration of TAMRA-labeled protein was adjusted to make sure its emission fluorescence was the same as that of dual labeled protein when excited at 543 nm. (C) Structural representation of CaM in Ca2+-free (PDB: 1CFD) and Ca2+-bound form (PDB: 1PRW) indicating the positions of the fluorescent labels. (D−F) smFRET histograms recorded for dual-labeled CaM under different conditions: (D) In 50 mM Tris-HCl, 150 mM NaCl buffer without Ca2+, (E) with 1 mM Ca2+, (F) with 10 mM EDTA. The solid lines represent Gaussian fits of the data in two-emission wavelength.

fluorescence events not involving FRET may be due to a defective or missing acceptor. They increase in prevalence because a higher chance of meeting the fluorescence threshold for a countable event. When Ca2+-chelating agent EDTA was added to the sample to 10 mM, the original FRET histogram was reproduced (Figure 4F), representing a return of the CaM conformation to the apo-form. Overall the smFRET data indicates that the fluorophores in the 1st and 149th position of CaM are positioned closer in the Ca2+-bound form than the Ca2+-free form in solution.

Previous structural studies give a slightly ambiguous view of the relative orientation of the N- and C-terminal domains of CaM.42,43 According to biochemical studies, samples in solution display a variety of conformational sub-states with various intraand inter-domain orientations in order to accommodate a broad range of targets.44 smFRET and ensemble FRET studies indicated that the more compact state is highly populated by the holo-form of CaM while the apo-form is generally more extended in solution due to repulsion between the N- and Cterminal domains.42,45 Our data (Figure 4) demonstrate that the binding of Ca2+ results in a conformational transition that brings the subunits of CaM closer together, thereby decreasing the distance between positions 1 and 149. In the FRET studies, the zero peak can largely be attributed, as customary, to direct excitation of the donor fluorophore combined with failure to achieve energy transfer in individual CaM proteins for a multitude of reasons. The higher-FRET peak, centered at 0.5, indicates the Ca2+-free (Figure 4D) conformation. Addition of Ca2+ to 1 mM eliminates the second peak corresponding to the apo-form. Concomitantly an emergent peak centered at ∼0.79 (Figure 4E), which reflects a more compact structure of the holo-form, as was suggested previously.42,45 In addition, Hellstrand, et al. observed that the fluorescence signal intensity decreases upon compaction of CaM due to quenching of the fluorophores by their proximity.42 This phenomenon is reflected in our data by the larger relative size of the zero peak in the holo-form (Figure 4E). The unquenched donor

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DISCUSSION In this report we describe a new strategy for reassigning sense codon to uAA in the E. coli in vitro translation system lacking selected tRNAs. This is based on a novel procedure for removing chosen tRNA isoacceptors from the total tRNA mixture by DNA-hybridization chromatography. Compared to our previously reported reconstitution approach based on semisynthetic tRNA complement,19 the tRNA-depletion method is more straightforward and efficient. We were able to achieve almost complete depletion of the native isoacceptors for AGG codon making it available for reassignment. In addition to the rare AGG-decoding isoacceptors, we achieved successful depletion of a medium abundant tRNA isoacceptor decoding AGC (Ser) codon. The depleted system for this isoacceptor could not support translation of templates 540

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ArgRS and delivered Arg to the CGG codons in Mycoplasma capricolum. In contrast, Leu tRNAPylGAG did not display unspecific charging in E. coli without the cognate uAA. This is consistent with the findings that LeuRS does not rely on the anticodon for substrate recognition.54 From this perspective the tRNAs for Leu, Ser and Ala codons are the best choice for reassignment as their aaRSs do not rely on the anticodon interaction.54 However, the successful reassignment of the rare AGG codon in vivo13,14 and in vitro suggested an efficient charging system or precharged uAA-tRNACCU could bypass the hurdles to avoid mischarging by the endogenous aaRS. We used the developed methodology to site-selectively duallabel CaM protein with two FRET-forming fluorophores. The successful reassignment of AGG sense codon to uAAs using our newly developed technique demonstrates the feasibility of creating a potentially large number of reassignable orthogonal codons for reassignment with diverse chemistries in the S30 based E. coli cell-free translation system. Recently, Suga’s group showcased successful division of codon boxes for Val, Arg and Gly where G-ending codons in unsplit codon boxes were reserved for native amino acids while C-ending ones were used for uAA incorporation.18 The fully reconstituted in vitro translation system (PURE) enabled manipulation of all the tRNA species. Application of our approach to division of unsplit codon family boxes in the most widely used and inexpensive S30 based cell free translation system, represents an alternative labeling approach applicable not only to peptides but also to full length proteins. Importantly, this approach can be directly transferred to eukaryotic cell-free systems. This sets our approach apart from those based on reconstituted translation systems that create almost unmanageable complexity when applied to eukaryotic systems. Our recent analysis demonstrates that the E. coli cell-free system could express only 10% of human cytosolic proteins in nonaggregated form while eukaryotic systems performed several fold better.55 This stresses the need for eukaryotic expression systems tailored for incorporation of the uAA into defined positions.

with 2 consecutive AGC codons while expression of templates with a single codon was dropped by 60%. Although the reduction was not as significant as for AGG-codon, we assume the residual amount of AGC-decoding isoacceptor would not prevent efficient uAA incorporation. This assumption is based on the success of reassignment of three redundant codons (GUC, CGC, GGC) in a special designed PURE translational system18 that has higher residual amount of native tRNAs than our depleted system.19 We have not proceeded further with this codon in the present study due to inefficient charging of the suppressor tRNA with AzF using AzFRS from Methanocaldococcus jannaschii. During preparation of our manuscript, Lee et al. demonstrated the success of free Arg-codons for uAA incorporation using Colicin D, a specific Arg-tRNAase, in the S12 lysate.46 The reassignable codons accessible with this strategy are confined by the specificity and efficiency of the available tRNAases while the use of DNA-hybridization allows predictable reassignment of a larger codon pool.47 Both of these methods demonstrate the feasibility of reassignment of orthogonal sense codons in an in vitro translation system. We took advantage of the open nature of cell-free translation systems to test the ability of the most widely used Pyl and MjTyr suppression systems to incorporate uAAs into nonsense and sense codons. We were particularly interested in some strained alkenes/alkynes due to the high rate constants in bioorthogonal reaction. However, none of these uAAs demonstrated good incorporation efficiency in our study (Supplementary Figure S1b). In contrast, n-propargyl-lysine (PrK) could be decoded by PylRSAF,48 which featured two mutations (Y306A and Y384F), with relatively high efficiency. Although this result revealed an unexpected inconsistency with the in vivo data on UAG suppression where large cycloaliphatic alkynes/ alkenes were incorporated efficiently,24,49 earlier reports demonstrated that PylRS was less efficient, with more than 1000-fold reduction in the kcat value50 as compared to canonical aaRS systems. This may reflect substrate-induced interdomain dynamics of the aaRS enzymes where amino acid changes within the substrate-binding pockets may affect overall aminoacylation efficiency. Further the eight-membered ring of the strained alkynes/alkenes may cause compatibility issues with EF-Tu.51 Comparison of four engineered o-tRNAs previously reported for the MjTyr system identified otRNAAzF2CUA as the most efficient one. The MjTyr system demonstrated less context-dependence in amber codon suppression while the extent of Pyl-mediated suppression was variable. This is consistent with previous findings that some gene contexts are more favorable than others.52,53 We demonstrate that AGG-codon could be reassigned to BPFL fluorophore modified cysteine by using BPFLtRNACysCCU. Preparation of this reagent involves several steps. However, once the precharged tRNA is prepared, the dual labeling procedure becomes much more straightforward and does not require balancing the selectivity of two labeling reactions. The deacylated tRNACysCCU is a poor substrate for protein translation due to the non-native anticodon and the limited concentration of endogenous CysRS and thus undergoes only negligible cotranslational recharging with cysteine preventing its misincorporation into the reassigned codon. Orthogonality of sense codon reassignment can be compromised due to the prominent contribution of the anticodon to specificity of many aaRSs. Previous report11 showed that heterologous tRNAPylCCG was charged by native

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MATERIALS AND METHODS Materials. The unnatural amino acid, p-azido-L-phenylalanine (AzF), was purchased from SynChem. TAMRA DIBO Alkyne were purchased from Life Technologies Australia Pty Ltd. BODIPY FL Iodoacetamide was from Molecular Probes. Ethanolamine−sepharose was prepared by coupling ethanolamine on epoxy-activated sepharose 6B (GE Healthcare). Eight grams of epoxy-activated sepharose 6B were washed extensively by water (∼200 mL per gram matrix) followed by incubation with 1 M ethanolamine at room temperature overnight with gentle agitation. Following the incubation, the matrix was washed by copious amount of water to remove the traces of ethanolamine. The resulting ethanolamine-conjugated sepharose matrix (∼20 mL) was stored as 20% (v/v %) slurry in buffer A (100 mM NaOAc (pH 5.2), 0.25 mM EDTA) with 2 mM NaN3. tRNA Production. The sequences of tRNAs used in this study are shown in Supplementary Table S1. The t7 transcripts for the tRNA species were prepared as described before.19 In brief, each DNA template was assembled from 5 or 6 oligos (Supplementary Table S2) by 3-step PCR. The PCR products were purified by ethanol precipitation and used as templates for runoff transcription by T7 RNA polymerase. The transcribed tRNAs were purified by affinity chromatography using ethanolamine−sepharose matrix.19 Specifically, the transcrip541

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After aminoacylation, the reaction mixture was diluted into 1× buffer A for ethanolamine-sepharose purification followed by conjugation reaction with BODIPY FL Iodoacetamide (BPFL-IA). The conjugation reaction was performed in dark in 50 mM Tris-HCl (pH 8.5), 80 μM cysteinylated-tRNA, 75% DMSO, 1 mM BPFL-IA and 100 mM NaCl. Conjugated product (BPFL-tRNA) was precipitated by ethanol and dissolved in a minimal volume of tRNA buffer (0.5 mM MgCl2, 0.5 mM NaOAc pH 5.0). The sample was then adjusted to 1× HPLC buffer (0.1 M TEAA, 1% ACN) followed purification by HPLC on POROS R1 10 μm column (Applied Biosystems) using buffers both containing 0.1 M TEAA and either 1% (buffer C) or 90% of ACN (buffer D); 9 min run duration with 1−20% linear gradient was applied. After HPLC purification, the BPFL-tRNA fraction was precipitated by ethanol, resuspended in tRNA buffer and stored at −80°. In Vitro Protein Translation Assay. The tRNA-depleted lysate is prepared by affinity chromatography as described.19 DNA templates corresponding to GFP and CaM-coding ORFs were constructed as described in Supplementary Methods. The sequence information on all the open reading frames used here is provided in Supplementary Table S3. The cell-free translation reactions for GFP production were performed following the standard protocol20 with the Mg2+ concentration optimized at 10 mM and 0.35 volume of depleted lysate (same as that for the standard S30 lysate). Any additional modifications to the reaction setup were given in the description of the corresponding experiments. GFP production was monitored on a fluorescence spectrometer with automatic plate reading module (Synergy) at 30° for 3−5 h at 485 nm excitation and 528 nm emission wavelengths. Protein Labeling. Expression of CaM with AzF and BPFL in cell-free translation reaction: The pOPINE-CaM template, with UAG and AGG codon located at position 1 and 149 of CaM ORF respectively (Supplementary Table S3), were supplemented at 80 ng/μL in the cell-free translation reaction containing 19 μM depleted tRNA mixture, 10 μM otRNAAzF2CUA, 10 μM AzFRS, 1 mM AzF, 10 μM BPFLtRNACysCCU in addition to the standard reagents.20 Two separate reactions were performed to prepare single labeled CaM protein. To express CaM-BPFL, 10 μM BPFLtRNACysCCU was supplemented to the corresponding translation reaction while 10 μM tRNATyrCUA was added for decoding UAG codon to Tyr. To obtain CaM-TAMRA, AzF was incorporated into protein sequence through the amber codon with 10 μM o-tRNAAzF2CUA, 10 μM AzFRS and 1 mM AzF while 5 μM synthetic tRNAArgCCU was supplemented for decoding AGG codon to Arg. The AzF was then modified by TAMRA DIBO through copper-free click chemistry as described below. The translation reaction was performed at 32° for 4 h. Purification of labeled CaM on Affinity Clamp resin: 20 μL 50% affinity clamp matrix (Supplementary Methods) preblocked with 0.1 mg/mL of BSA in PBS was added to the clarified translation sample. The resin was incubated with the sample for 30 min at room temperature, washed with PBSTriton (0.1%) buffer following overnight incubation with 60 μM TAMRA-DIBO in PBS at room temperature to allow TAMRA conjugation with the protein. After conjugation the resin was extensively washed with 20 mM Hepes-KOH pH 7.6, 0.5 M NaCl, 0.1 mg/mL BSA, 0.1% Triton buffer alternated by washes with just 0.1 mg/mL BSA, 0.1% Triton in water. Before elution via cleavage with PreScission protease the resin was

tion reaction was terminated by addition of 0.5 volume of 10× concentrated buffer A and equal volume of 20% matrix followed by addition of 2.5 volumes of water. The slurry was incubated for 1 h at 4° followed by extensive washing with at least 10 volumes of buffer A. The tRNA was eluted twice into one volume of buffer B, 2 M NaOAc (pH 5.2), 0.25 mM EDTA, 2.5 mM Mg(OAc)2, and precipitated by ethanol. Total tRNA Preparation. Total tRNA mixture was prepared from E. coli BL21(Gold) strain by modified Zubay’s method.56 In brief, the total tRNA was extracted from overnight culture. The cell pellet (20 g) was resuspended in 40 mL of 10 mM Mg(OAc)2, 1 mM Tris (pH 7.4) buffer. The nucleic acids were then extracted by 34.4 mL of liquefied phenol with vigorous agitation for 2 h in the cold room. The extraction was repeated again by adding 10 mL liquefied phenol. The aqueous phase was then collected by centrifugation at 18 000g for 30 min. RNA was precipitated with 0.05 volume 4 M potassium acetate and 2 volume of 100% ethanol for overnight at −20°. The precipitate was collected by a 10 min centrifugation at 5000g and resuspended in 20 mL of 1 M ice-cold NaCl. After stirring the precipitate vigorously for 1 h in cold room, the supernatant was collected and extraction was repeated. The supernatant from the two NaCl extractions was combined and precipitate by addition of 2 vol ethanol. After two repeats the crude tRNA fraction was dissolved in water. Specific tRNA Depletion in Total tRNA Mixture. The specific tRNA species were depleted by DNA-hybridization chromatography from the total mixture as described.19 The DNA oligos complementary to the sequence spanning the Darm down to the anticodon loop (tRNAArgUCU) or variable loop up to the acceptor stem (tRNAArgCCU) or anticodon stem to variable loop (tRNASerGCU) of the target tRNAs (Supplementary Table S2) were designed and synthesized together with 3′amine group by Integrated DNA Technologies (IDT). DNA oligos were immobilized on NHS-activated sepharose (GE) according to the manufacturer’s protocol with 15 nmole of oligonucleotide used per 100 μL of settled resin. 200 μL of 19 μM unfractionated tRNA total mixture was mixed with the same volume of 2× hybridization buffer (20 mM Tris-HCl (pH 7.6), 1.8 M NaCl, 0.2 mM EDTA) and then subjected for hybridization with the resin-immobilized oligonucleotides. For each target tRNA, 125 μL of settled resin was used. The tRNA mixture suspended with the resin was heat-denatured at 65° for 10 min, slowly cooled down to room temperature with agitation followed by collection of the nonhybridized tRNAfraction devoid of targeted isoacceptors by centrifugation. Twenty μL of 1× hybridization buffer used to wash the resin were combined with the main fraction of tRNAs and ethanol precipitated. tRNA Aminoacylation and Fluorophore Conjugation. The synthetic cysteine tRNAs with CCU or CUA anticodons were prepared by t7 transcription as described above. Cysteine (Cys) was charged on these tRNAs by recombinant cysteinyltRNA synthetase (Supplementary Methods). First, 50 mM Cys was preincubated with 50 mM TCEP at 37° for 15 min in order to convert it to fully reduced form. Meantime, tRNAs at 50 μM concentration were denatured at 78° and refolded in the presence of 5 mM Mg2+ slowly at room temperature to maintain their maximal activity. The aminoacylation reaction was then performed in 100 mM Hepes-KOH (pH 8.0), 2 mM Cys, 2 mM TCEP, 10 mM MgCl2, 5 mM KCl, 5 mM KOAc, 0.1 mM CTP, 4 mM ATP, 10 μM t7tRNACys variant, 5 μM CysRS, 25% DMSO at 37° for 1 h. 542

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house Ltd that holds a license for sense codon reassignment technology patent described in this paper.

equilibrated with the cleavage buffer containing 50 mM TrisHCl pH 8, 150 mM NaCl, 1 mM DTT. Subsequently 40 μL PreScission protease (0.1 mg/mL) in cleavage buffer was added to the matrix followed by overnight incubation at 16°. The protein was eluted by centrifugation and the beads were additionally eluted with the same volume of cleavage buffer. The two eluates were combined. SmFRET Measurements. SmFRET measurements were carried out using a Zeiss LSM710 microscope equipped with the ConfoCor3 module and BiG detectors. Excitation was achieved by focusing 488 nm Argon-ion laser light into 16 μL sample solution using a water immersion objective (Apochromat 1.2 NA, 40×; Zeiss). The fluorescence emission was separated from excitation light using a 488/561 dichroic mirror, collected using the same objective, spatially filtered through the 35 μm pinhole followed by splitting into donor and acceptor detectors using a second dichroic mirror and 500−550 nm and 570−610 nm band-path filters for donor and acceptor emissions, respectively, mounted into the same filter cube unit (Zeiss). Experiments were performed in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50 μg/mL BSA, either in the presence of 1 mM Ca2+ or 10 mM EDTA, loaded on a homemade silicone polymer plate (SYLGARD) attached to a 70 × 80 mm Coverglass No.1.5 (ProSciTech). Double labeled CaM was used at ∼100 pM concentrations to ensure that the fluorescence signal originated from single molecules transiting through the focal volume. The FRET efficiency histograms were generated by using Zen10 software where a two-channel data collection mode was used for simultaneous recording of donor and acceptor signals for 30 min (in 10 rounds by 3 min each) with a bin time of 1 ms. The leakage of donor emission into the acceptor channel was estimated in a separate experiment with 1 μM of donor fluorophore and calculated as 12% to correct the signals before FRET analysis. A threshold was set at 20 counts for the sum of donor and acceptor signals to filter the background noise out. The FRET efficiency was calculated as (IA − 0.12 × ID)/(IA + 0.88 × ID) and plotted as a histogram. IA and ID are light intensities in acceptor and donor channel, respectively. 0.12 is the leakage fraction from Donor emission in the acceptor channel. Gaussian function was used to fit the data by Origin software (OriginLab Corp.).

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ACKNOWLEDGMENTS We thank Dejan Gagoski for discussion on protein labelling and microscopy setup. We are grateful to Hadi Ahmad Fuaad for allowing access to their HPLC facility. We acknowledge the support from ARC grant DP1094080, and NHMRC program grant APP1037320 to K.A.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00245. Details on plasmid construction, recombinant production of aaRSs, chemical synthesis of uAAs and precharging on tRNAs, mass spectrum, structural modeling (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Zhenling Cui: 0000-0002-5664-8303 Author Contributions #

Z.C. and S.M. contributed equally.

Notes

The authors declare the following competing financial interest(s): Kirill Alexandrov has equity in Molecular Ware543

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