The ncAAs 1–9 were purchased from Chem-Impex Inc. (Wood Dale, IL, USA). Polymerase chain reaction (PCR) was performed using the KOD Hot Start Polymerase kit (Merck, Bedford, MA, USA). The oligonucleotide synthesis and DNA sequencing service were provided by Genomics Inc. (Taipei, Taiwan). The sfGFP-10ams gene, amber mutations at 10 phenylalanine positions F8/F27/F46/F71/F100/F114/F130/F145/F165/F223, and human heavy chain ferritin Ftn gene synthesis with E. coli codon optimization were performed by Integrated DNA Technologies (IDT) Inc. The primer sequences in this study are listed in the Supplementary Materials. The construction of plasmids pET-sfGFP-27am, pET-sfGFP-2∼8ams, and sfGFP-10ams followed the general cloning protocols. The sfGFP-27am gene was generated using the sfGFP gene amplified by PCR from pET-pylT-sfGFP (Jiang et al., 2020a) and then prepared by overlapping PCR with two fragments (fragment 1: sfGFP-NdeI-F and 27am-R; fragment 2: 27am-F and sfGFP-SacI-R). The overlapped sfGFP-27am gene products and pET-pylT plasmid were double digested with restriction enzymes NdeI and SacI and then ligated using T4 DNA ligase. Similarly, pET-pylT plasmids harboring sfGFP with multiple TAG amber stop codons were constructed with multiple fragments by overlapping PCR. Briefly, the sfGFP-2ams gene with F27/F46 amber mutations was synthesized using three fragments: fragment 1, sfGFP-NdeI-F and 27am-R; fragment 2, 27am-F and 46am-R; and fragment 3, 46am-F and sfGFP-SacI-R. The sfGFP-3ams gene, with amber mutations at F8/F71/F100, was prepared using the following three fragments by overlapping the PCR method: fragment 1, NdeI-F8am-F and 71am-R; fragment 2, 71am-F and 100am-R; and fragment 3, 100am-F and sfGFP-SacI-R. The sfGFP-4ams gene with F27/F84/F100/F165 amber mutations was amplified by overlapping PCR using the following five fragments: fragment 1, sfGFP-NdeI-F and 27am-R; fragment 2, 27am-F and 84am-R; fragment 3, 84am-F and 100am-R; fragment 4, 100am-F and 165am-R; and fragment 5, 165am-F and sfGFP-SacI-R. The sfGFP-5ams gene with F27/F84/F100/F114/F165 amber mutations was prepared by two fragments (fragment 1: sfGFP-NdeI-F and 114am-R; and fragment 2: 114am-F and sfGFP-SacI-R), using pET-sfGFP-4ams as the template. The sfGFP-6ams gene with F27/F84/F100/F114/F165/F223 amber mutations was prepared by two fragments (fragment 1: sfGFP-NdeI-F and 223am-R; and fragment 2: 223am-F and sfGFP-SacI-R), using pET-sfGFP-5ams as the template. The sfGFP-7ams gene with the additional F8 amber mutation on the sfGFP-6ams gene was prepared by primers NdeI-F8am-F and sfGFP-SacI-R, using pET-sfGFP-6ams as the template. The sfGFP-8ams gene with S2 amber mutation on the sfGFP-7ams gene was prepared by primers NdeI-F2am-F and sfGFP-SacI-R, using pET-sfGFP-7ams as the template. The amber codon positions for sfGFP variants are listed in Supplementary Table S1.

For the construction of the pET-Ftn plasmid, the synthesized Ftn gene was inserted into the pET-pylT plasmid using the same approach as to that for the preparation of pET-sfGFP-27am. For the construction of pET-Ftn-F81am, pET-Ftn-K143am, and pET-Ftn-2ams, respectively, the Ftn-F81am, Ftn-K143am, and Ftn-2ams genes were synthesized from wild-type Ftn genes with F81 and/or K143 amber mutations by overlapping PCR. Briefly, the Ftn-F81am gene was generated by overlapping two fragments, fragment 1: Ftn-NdeI-F and F81am-R and fragment 2: F81am-F and Ftn-SacI-R, using the template pET-Ftn. The Ftn-K143am and Ftn-2ams genes were synthesized by the same approach using two fragments, fragment 1: Ftn-NdeI-F and K143am-R; and fragment 2: K143am-F and Ftn-SacI-R, using templates pET-Ftn and pET-Ftn-F81am, respectively. The plasmids pET-A-Ftn and pET-Ftn-A were also constructed by introducing genes encoding the N- or C-terminal AHNP peptide and linker (GGGGS)₃ to the Ftn gene to generate A-Ftn and Ftn-A genes. The A-Ftn and Ftn-A genes were synthesized with two fragments using pET-Ftn as the template: fragment 1 (N-AHNP-F and N-AHNP-R for A-Ftn construction; whereas C-ANHP-F and C-ANHP-R for Ftn-A) and fragment 2 (3XG4S-Ftn-F and 3XG4S-Ftn-R for A-Ftn, whereas Ftn-3XG4S-C-ANHP-F and Ftn-3XG4S-C-ANHP-R for Ftn-A). The plasmids pET-A-Ftn-F81am, pET-A-Ftn-K143am, and pET-A-Ftn-2ams were constructed utilizing the same approach, with the same primers for the construction of the A-Ftn gene and using pET-Ftn-F81am, pET-Ftn-K143am, and pET-Ftn-2ams as the templates, respectively.

MmPylRS variants, IFRSs, evolved from directed evolution (Wang et al., 2011) which charge p-iodo-l-phenylalanine (IF, 1), were generated with designated mutations on the active site (Table 1). Briefly, for the construction of the plasmid of pCDF-IFRS1, the IFRS1 gene product was generated by overlapping PCR with three fragments using the template pCDF-PylRS (Jiang et al., 2020b), fragment 1: EcoR1-F and IFRS1-MLS-R; fragment 2: IFRS1-MLS-F and IFRS1-SM-R; and fragment 3: IFRS1-SM-F and BamHI-R. The overlapped IFRS1 gene product and pET-pylT plasmid were then double digested with restriction enzymes EcoRI and BamHI and then ligated using T4 DNA ligase. pCDF-IFRS2 was constructed by modifying the IFRS1 gene of pCDF-IFRS1, and the overlapping PCR product was generated with two fragments, fragment 1: EcoR1-F and IFRS2-TA-R and fragment 2: IFRS1-TA-F and BamHI-R. For the construction of pCDF-AzFRS, the AzFRS gene product was generated with three fragments using pCDF-PylRS as the template, fragment 1: EcoRI-F and AzFRS-AM-R; fragment 2: AzFRS-AM-F and AzFRS-L-R; and fragment 3: AzFRS-L-F and BamHI-R. Using pCDF-AzFRS as the DNA template, different combinations of C-terminal mutations (listed in the Supplementary Materials), K431M, D433G, and A441S, were introduced through the designed primers to generate AzFRS-M, AzFRS-G, AzFRS-S, AzFRS-MG, AzFRS-MS, AzFRS-GS, and AzFRSc genes (Table 1).

To produce ncAA-encoded sfGFP proteins, the pET-sfGFP-27am and pCDF-AzFRS-MS co-transformed into E. coli BL21 (DE3), sequentially. After the transformation, the cell suspension was spread onto the agar plate containing ampicillin (Amp) (100 μg/ml) and streptomycin (Sm) (100 μg/ml). A single colony was chosen from the plate and then cultured in 1 ml Luria–Bertani (LB) medium overnight. The cultured cells were then transferred to 50 ml fresh LB medium and incubated at 37°C until OD₅₉₅ reached 0.6–0.8. Followed by centrifugation (10 min, 6,000 rpm, 4°C), the medium is changed to GMML medium. Protein expression was induced with the supplement of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and ncAA and incubated at 37°C for another 12 h. Cells were harvested, resuspended with lysis buffer (1× phosphate-buffered saline [PBS], pH 7.4), and then sonicated to lyse cells. Followed by centrifugation (60 min, 20,000 rpm, 4°C), the supernatant was collected. As sfGFP was designed with a 6× His tag, the supernatant collected was incubated with 0.5 ml Ni²⁺-NTA resin (Roche, Basel, Switzerland) for protein purification. Five milliliters of lysis buffer and 2.5 ml of washing buffer (1× PBS, 5 mM imidazole, pH 7.4) were used to wash out nonspecific proteins on the resin. The target protein was eluted from the resin by 2.5 ml of elution buffer (1× PBS, 200 mM imidazole, pH 7.4). The buffer of eluted fraction was changed to 1× PBS with Amicon Ultra-15 Centrifugal Filter Units (MWCO 10 kDa). For Ftn proteins, further purification by anion exchange column HiTrap Q HP (GE Healthcare, Chicago, IL, USA) was conducted with NaCl gradient elution buffer (50 mM Tris, 50 mM to 1 M NaCl, pH 8.0) and size exclusion chromatography (SEC) column HiLoad 10/300 Superdex 200 pg (GE Healthcare) with PBS buffer. Purified sfGFP and Ftn were analyzed by 12% SDS-PAGE with Instant Blue (Marvelgent Biosciences, Canton, MA, USA) staining. The Ftn assembling status was analyzed by native gel, 4%–15% Mini-PROTEAN^® TGX™ Precast Protein Gel (Bio-Rad, Hercules, CA, USA) with a native protein marker (Thermo Fisher Scientific, Waltham, MA, USA) in TG buffer (25 mM Tris–HCl, 192 mM glycine, pH 8.5) under 80 V for 6 h at 4°C. The gel was stained by Instant Blue for 15 min and destained by H₂O.

Whole cells were collected and lysed at 100°C with SDS loading dye for 15 min and then subjected to 12% SDS-PAGE analysis. The gels were stained with Instant Blue to visualize the target proteins with the expected molecular weight around 28 kDa corresponding to the protein size of sfGFP. The suppression efficiency of amber codons in producing sfGFP with the C-terminal His tag was visualized by Western blotting against the anti-6X His tag antibody. The Western blots were performed using Trans-Blot Turbo System (Bio-Rad) and RTA transfer kit. The antibodies used for immunoblotting were Anti-His (SignalChem, Richmond, Canada; H99-61M-100) and HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA; 7076P2). After SDS-PAGE analysis, the gel was immersed in the transfer buffer and then blotted onto the polyvinylidene fluoride (PVDF) membrane (25 V/1.3 A, 10 min). After the completion of the transfer process, the PVDF membrane was washed with PBST buffer for 5 min thrice. Next, the membrane was blocked with 5% skim milk for 1 h at room temperature. The membrane was then washed with PBST buffer for 5 min thrice (washing step). A primary antibody (1:1,000 dilution) was added and then incubated with the membrane for 1 h at room temperature following the washing step. Subsequently, an HRP-conjugated secondary antibody (1:5,000 dilution) was added and the membrane was incubated for another 1 h at room temperature. The membrane was then washed. Finally, the WesternBright ECL HRP substrate (Advansta, San Jose, CA, USA; K-12045-D50) was mixed and spread onto the membrane to visualize the band signals using ChemiDoc Imaging Systems (Bio-Rad) in bioluminescence detection mode.

The purified protein was diluted with 50% acetonitrile and 1% formic acid. An aliquot corresponding to one pmol of the pure protein was injected via an ESI source (Waters LockSpray Exact Mass Ionization Source) with a syringe pump (Harvard Apparatus, Holliston, MA, USA), and a flow rate of 5 μl/min was held throughout the analysis. The mass of intact proteins was determined using a Waters SYNAPT G2 HDMS mass spectrometer (Waters, Milford, MA, USA). The acquired spectra were deconvoluted to the single-charge state using the MaxEnt1 algorithm of the MassLynx 4.1 software (Waters).

To understand the suppression of MmPylRS variants, ncAA 1–9 screening assay was performed. The plasmids pET-sfGFP-27am and pCDF-PylRS were co-transformed into E. coli BL21 (DE3), sequentially. The cell suspension was spread onto the agar plate containing Amp (100 μg/ml) and Sm (100 μg/ml). The plate was incubated at 37°C overnight. Ten colonies were selected and then inoculated and cultured in 3 ml of LB medium at 37°C overnight. Five hundred microliters of cells was transferred to 25 ml of fresh LB medium and incubated at 37°C until OD₅₉₅ reached 0.6–0.8. The cells were harvested, washed twice with M9 salts, and suspended in M9 medium (M9 salts, 1% glycerol, 2 mM MgSO₄ and 0.1 mM CaCl₂) supplemented with 1 mM IPTG. Aliquots of 50 μl suspended cells were loaded in a 384-well plate supplemented with 1–9 ncAAs (1 mM) in designated wells. Cells were incubated in the plate reader (BioTek, Winooski, VT, USA) at 37°C for 12 h, with continuous monitoring of fluorescence emission intensity (λ _ex/λ _em = 535/595 nm) as well as OD₅₉₅. The control experiments used to measure background signals were performed for wells without adding IPTG and ncAAs, or wells adding IPTG only. The control signal (without adding ncAAs but IPTG) was subtracted from the fluorescence emission intensity of sfGFP obtained and then divided by OD₅₉₅ to generate the relative fluorescence intensity.

To synthesize Ftn-dye conjugates, purified Ftn-81-4, Ftn-143-4, Ftn-2am-4, A-Ftn-81-4, A-Ftn-143-4, and A-Ftn-2am-4 proteins were conjugated to fluorescence dyes or Dox by the SPAAC reaction. The DBCO-Cy3 and DBCO-Cy5 (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in 100% DMSO to the final concentration of 1 mM. The Ftn-81-4 protein (20 μl, 20 μM) in PBS buffer at pH 7.4 was mixed with 50 μl of DBCO-Cy3 and/or DBCO-Cy5 (80 μM, 1:5 M ratio) in the buffer of 90% PBS and 10% DMSO, and then the volume was adjusted to 500 μl. Reaction was incubated at 37°C, 300 rpm for 16 h. A-Ftn-81-TAMRA conjugates were synthesized by mixing A-Ftn-81-4 and DBCO-PEG4-TAMRA (Sigma-Aldrich) under the same condition. The Ftn-dye conjugates were further purified using the SEC column (HiLoad 10/300 Superdex 200 pg, GE Healthcare) and then subjected to SDS-PAGE, native PAGE, gel fluorescence imaging, and ESI-MS analysis. A-Ftn-143-Dox was synthesized by coupling A-Ftn-143-4 and dibenzocyclooctyne–PEG4–doxorubicin (DBCO–PEG4–DOX) using the same method. DBCO–PEG4–DOX was synthesized by NHS-amine coupling chemistry. DBCO–PEG4–NHS (Sigma-Aldrich) (50 μl, 1.5 mM, 30% DMSO in PBS) and doxorubicin hydrochloride (Dox, TCI) (50 μl, 2 mM, 30% DMSO in PBS) were mixed and reacted at 25°C for 4 h. The chemical reaction progress was monitored by normal-phase thin-layer chromatography.

To analyze the particle size by dynamic light scattering (DLS), Ftn variants were diluted to the final concentration of 0.5 mg/ml and the aggregates were removed from the solution by centrifuge. The protein samples were loading into the designated 1 ml disposable cuvettes, and the particle size of protein was measured by Malvern Zetasizer Nano ZS. Measurements were repeated three times, and the primary size distribution by intensity value was determined.

To prepare Ftn samples for transmission electron microscope (TEM) analysis, 50 μg/ml of protein was dissolved in 50 mM Tris buffer at pH 8.0. After the support film grid (formvar/carbon 400 mesh copper) discharged with 25 mA for 30 s by Emitech K100X Glow, 5 μl of protein was loaded onto the grid and incubated for 60 s. The sample was gently drained from the grid by filter paper. Distilled deionized water and 1% uranyl acetate (UA) were alternatively applied onto the grid, incubated for 30 s, and drained by filter paper three times for background cleaning. Images of Ftn proteins were collected on FEG-TEM, FEI Tecnai G2 TF20 Super TWIN. Microscopy was operated at an accelerating voltage of 120 kV.

The fluorescence emission spectra were determined by Fluorolog-3 (Jobin Yvon, Edison, NJ, USA). Proteins were diluted to 50 μg/ml in PBS at pH 7.4 and loaded into the designated 10-mm quartz cuvettes. Ftn-143-Cy3, Ftn-143-Cy5, and Ftn-143-Cy3/Cy5 were excited at 550 nm. The emission spectra were recorded from 560 to 700 nm with 1 nm per point scanning resolution.

Human breast carcinoma (BT-474) cells (BCRC, Taiwan) were cultured in 45% high-glucose DMEM (Corning, Tewksbury, MA, USA) and 45% low-glucose DMEM (Corning, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) at 37°C with 5% CO₂ in a humid atmosphere in petri dishes (Falcon). Cells were ready for the detachment when the confluency reached above 60%. BT-474 cells were rinsed with PBS twice and then trypsinized with 0.25% trypsin–EDTA (Gibco, USA). An appropriate volume of trypsin-EDTA was added into petri dishes. After gently shaking, cells were incubated with trypsin–EDTA at 37°C for 1 min. The detached cells were collected by pipetting over the cell layer with fresh complete medium. Twenty microliters of suspended cells was taken and mixed with trypan blue for cell density measurement. Cell density was determined by an automated cell counter (Invitrogen, Carlsbad, CA, USA). One-third of cells was added into a new petri dish and diluted to the appropriate volume with fresh complete medium. Poly-d-lysine (Sigma-Aldrich) coated coverslips were used for BT-474 cell attachment. Coverslips were incubated with 50 μg/ml poly-d-lysine for 2 h. Subsequently, the solution was removed and then the coverslips were rinsed with PBS and sterile water three times, alternatively. The coated coverslips were allowed to dry for at least 2 h before introducing cells and medium. Suspended cells (1 × 10⁵) were seeded on 18-mm coverslips within a 12-well plate and incubated for 24 h, allowing cells to attach on coverslips.

After cells were attached on coverslips, the culture medium was replaced by fresh DMEM containing Dox, fluorescence molecules, A-Ftn-dye, or A-Ftn-drug conjugates. The cells were incubated at 37°C for the designated time and washed twice with PBS solution. The cells were fixed on coverslips by incubating with 4% para-formaldehyde (PFA) (Sigma-Aldrich) for 10 min. PFA was removed and the coverslips rinsed twice with PBS. After equilibrating the coverslips with PBS, the cells were ready to be stained. The nucleus of cells was stained by DAPI (GeneCopoeia, Rockville, MD, USA). An appropriate volume of 300 nM DAPI was added into a culture plate with coverslips and incubated for 5–10 min. Then, DAPI was removed and the coverslips alternatively rinsed with PBS and sterile H₂O three times. The coated coverslips were allowed to dry for at least 5 min before mounting. The cells were mounted onto a slide with one drop or 10 μl of mounting medium, ProLong Gold Antifade Mountant (Invitrogen). The cells were observed under the corresponding channel by confocal microscopy (Leica SP5).

The cytotoxicity of the A-Ftn–DOX conjugate, A-Ftn-4, and free Dox, respectively, against Her2⁺ human breast cancer cells, BT-474, was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Thermo) assay. As the confluency of BT-474 reached above 60%, the cells were detached and 1 × 10⁴ cells were seeded onto a 96-well transparent flat plate per well with 100 μl of fresh medium. The cells were further incubated for 24 h to allow the cell to attach onto the plate. Subsequently, the free Dox, A-Ftn-4, and A-Ftn–DOX conjugates were added into designated wells with the final concentration ranging from 0 to 5 μM. After the incubation for 72 h, the medium was removed and replaced by 100 μl of 1.2 mM MTT in PBS per well, following the 4-h incubation. The MTT solution was carefully removed, and DMSO was added into wells to dissolve the purple formazan crystals. The absorption at 540 and 570 nm was measured by Synergy HT plate reader (Biotech).

To incorporate AzF (4), the evolved PylRS•tRNA^Pyl pair (Wang et al., 2011) is chosen from the MmPylRS plasmid library (Wang et al., 2010) to be fine-tuned in charging ncAA 1. The wild-type MmPylRS and three IFRS variants, IFRS1, IFRS2, and AzFRS (Table 1), are selected to explore the substrate recognition against nine para-substituted phenylalanine analogs, 1–9 (Scheme 1; Figure 2A), respectively. The mutations at the active site of these three MmPylRS variants cover two conserved mutation sites at N346 and C348, together with randomly distributed mutations at other sites (Table 1; Figure 2B). Both the pCDF vector containing the mutated PylRS gene and the pET vector containing tRNA^Pyl and the sfGFP-27am gene are co-introduced into E. coli. The read-through of the amber codon in response to ncAAs 1–9 (Scheme 1), respectively, can be indicated through the fluorescence emission resulting from the sfGFP-27am gene product. The screening results obtained from both IFRS1 and IFRS2 give the preference in charging IF (1) and MeOF (6) (Figure 2A), indicating that both IFRS1 and IFRS2 show high substrate specificity. All AzFRS variants share W417L/N346A/C348M mutation sites (Table 1). The screening results of AzFRS variants give the high preference when charging 6, whereas mild preferences are observed when charging 1–5. AzF (4) and p-propargyl-l-phenylalanine (PrF, 5), from which versatile functional groups can be derived for click reactions such as SPAAC and CuAAC, show noticeable signal enhancement when being charged by AzFRS.

To enhance the activity of MmPylRS variants, the engineering for the second sphere of the active site or the terminal sequence of MmPylRS is conducted. Although the second sphere or global sequence engineering (Suzuki et al., 2017; Sharma et al., 2018; Jiang et al., 2020a) of MmPylRS has been studied, the effects of MmPylRS CTD mutations on amber suppression efficiency remain unclear. Based on the available x-ray crystal structure, mutations at the CTD for rationally designed AzFRS have been introduced and studied (Figure 2C). Among the evolved MmPylRS variants, HarRS, which charges homoarginine with K431M/D433G/G444E mutations at HarRS CTD (Mukai et al., 2015), is found to be able to enhance the activity and selectivity for homoarginine incorporation. Likewise, in this study, to develop the hypothesized remodeled pi–pi interactions among Y242, H432, and F434 positions, K431M/D433G double mutations are chosen for the CTD mutation sites due to their strong hydrophobicity (Figures 2C,D). In addition, the A441S mutation is performed to affect the adjacent hydrogen bond network built by the main chain of the R439 residue and to indirectly participate in the hydrogen bond network between the side chain of R439 and the neighboring alpha helix (Figure 2C, positions 191–201), whose spatial movement is found in the DhPylRS CTD/tRNA^Pyl (cyan) co-crystal structure (Figures 2D,E). Those mobile helical regions (green and cyan helices on the left side of Figure 2E) are interfaced with the tRNA D-loop region based on the superimposed structures of the DhPylRS CTD/tRNA^Pyl co-crystal (cyan, Figure 2E) and the MmPylRS CTD (green, Figure 2E).

Upon the introduction of three mutations, K431M/D433G/A441S, on MmPylRS to evolve into the AzFRS variant, another three variants with a single mutation at the AzFRS CTD are prepared to examine its effect: AzFRS-M, AzFRS-G, and AzFRS-S (Table 1). Through monitoring the fluorescence emitted from the sfGFP-27am gene product, it is noted that, compared with AzFRS, AzFRS-M and AzFRS-G give the 1.4- and 1.6-fold enhancement in the fluorescence emission in charging MeOF (6), respectively, and give the 1.6- and 1.3-fold enhancement in the fluorescence emission in charging AzF (4), respectively (Figure 3A). Compared with AzFRS, AzFRS-S yields 2- to 4.3-fold enhancement in the fluorescence emission when charging 1–6, which shows the improved activity toward those ncAAs. To examine the likelihood whether a combination of those mutations can further elevate the activity of AzFRS, AzFRS variants with double mutations, i.e., AzFRS-MG, AZFRS-MS, and AzFRS-GS, as well as the AzFRS variant with triple mutations, i.e., AzFRSc, are prepared and tested with the same approach. Interestingly, D433G mutation does not seem to contribute to the enhanced activity. The AzFRS-MG variant merely shows a neutral effect, whereas the AzFRS-GS variant even shows a negative effect. Relative to AzFRS, AzFRS-MS and AzFRSc variants yield a 4-fold increase in the fluorescence emission when charging 4. Based on these results, AzFRS-MS is chosen in charging 4 in the subsequent studies.

The amber suppression efficiencies of AzFRS, AzFRSc, and AzFRS-MS are evaluated through the generation of sfGFP-2ams, sfGFP-3ams, sfGFP-4ams, sfGFP-5ams, sfGFP-6ams , sfGFP-7ams, sfGFP-8ams, and sfGFP-10ams gene products (Amiram et al., 2015) (Figure 3B; Supplementary Figures S1, S2). With the similar endogenous protein background, the whole-cell SDS-PAGE and Western blotting analysis of full-length sfGFP indicate an improvement in the suppression efficiency. AzFRS and AzFRS-MS can overly express sfGFP-2ams, sfGFP-3ams, sfGFP-4ams, sfGFP-5ams, and sfGFP-6ams gene products, with the given protein amount obtained from AzFRS-MS which is greater than that obtained from AzFRS (Figure 3B; Supplementary Figure S1). Furthermore, the full-length sfGFP-7ams, sfGFP-8ams, and sfGFP-10ams gene products can be expressed after introducing K431M/A441S double mutations to AzFRS CTD (Figure 3B; Supplementary Figure S1). Although AzFRSc shows slightly better amber read-through efficiency than AzFRS-MS through the observation of sfGFP-27am gene product generation, AzFRSc does not generate the full-length sfGFP-3ams and sfGFP-8ams gene products (Supplementary Figure S2). Therefore, AzFRS-MS is selected for the characterization and its Ftn-4 protein production is examined. To further confirm the ncAA screening results of AzFRS-MS, sfGFP-1∼9 are respectively expressed and purified and then subjected to ESI-MS analysis. The observed mass of all nine sfGFP proteins generated by AzFRS-MS matches the calculated mass of the full-length sfGFP (Table 2; Figure 3C; Supplementary Figures S3–S11).

The Ftn consists of 24 subunits and can self-assemble into a spherical structure with the outer diameter of 12 nm. The sites for the chemical moiety loading, i.e., a fluorophore or a drug with the molecular weight less than 1,000 Da, are selected based on the x-ray crystal structure of Ftn (PDB code: 2FHA, Figure 4A). The residues F81 pointed outward (labeled in blue in Figure 4A) and K143 pointed inward (labeled in red in Figure 4A) on Ftn are chosen to estimate drug loading efficiency based on the penetration of small molecules into the Ftn cavity and the SPAAC reaction. Ftn-F81am, Ftn-K143am, and Ftn-2ams gene products supplemented with AzFRS-MS and AzF (4) produce Ftn-81-4, Ftn-143-4, and Ftn-2ams-4, respectively, and these products are characterized by ESI-MS spectrometry. The measured molecular weight of those products matches their corresponding calculated molecular weight (Table 2; Supplementary Figures S12–14). It is found that, for Ftn-81-4 and Ftn-2ams-4, azide reduction occurs, followed by the conversion to an amino group (Table 2).

DBCO-Cy3 and DBCO-Cy5 (molecular weight: 980.28 and 992.28 Da, respectively) are used to probe the labeling efficiency of the SPAAC reaction in loading DBCO-dye (Figure 4B). The quantitative labeling of Ftn-81-4 with DBCO-Cy5 is conducted with the 1:5 M ratio. The SDS-PAGE analysis shows that Ftn is not being labeled after DBCO-Cy5 treatment while Ftn-81-4 gives the bioorthogonality upon DBCO-Cy5 treatment as a clear band can be visualized (Figure 4C). For Ftn-81-4, after 4 h treatment with DBCO-Cy5, a species with about 1.0 kDa mass shift is noted from the deconvoluted ESI-MS spectrum, which indicates that Cy5 is labeled through the azido group on Ftn-81-4. With the reaction time of 16 h, Ftn-81-4 (found molecular weight: 22,220 Da; calculated molecular weight: 22,220 Da) is found to be completely converted into Ftn-81-Cy5 (found molecular weight: 23,207 Da; calculated molecular weight: 23,207 Da). A slight azide reduction on Ftn-81-4 turning into an amino group is observed (22,173 Da); in addition, an oxygen adduct of Ftn-81-Cy5 (23,223 Da) is found (Table 2; Figure 4D; Supplementary Figures S12, S15).

In consideration of structural disruption and disassembling, DBCO-Cy3 labeling on Ftn, Ftn-81-4, Ftn-143-4, and Ftn-2am-4 for 4 h is tested; the corresponding labeling efficiency and assembling ratio are then analyzed by SDS-PAGE and native PAGE (Figure 4E). It is noted that Ftn is not being labeled after the treatment with DBCO-Cy3 and remains assembled but shows less protein amount after treating with DBCO-Cy3, which might be resulted from the aggregation. Relatively, Ftn-81-4, Ftn-143-4, and Ftn-2am-4 proteins give shifted fluorescent bands on SDS-PAGE and clear fluorescent bands of the assembled protein on native PAGE upon being labeled (Figure 4E). On the SDS-PAGE, Ftn-2am-4 gives two bands that represent single- and double-labeled Cy3 adducts.

With the successful SPAAC reaction in the DBCO-Cy5 and DBCO-Cy3 labeling on Ftn-81-4 Ftn-143-4, and Ftn-2am-4, respectively, the capacity for the incorporation of different and multiple molecules/drugs is subjected to be evaluated. As the inner diameter of Ftn is 8 nm, Cy3 and Cy5 dyes inside Ftn deem to be a suitable fluorescence resonance energy transfer (FRET) pair (Roy et al., 2008) to probe adjacent Cy3/Cy5 within a 70-Å distance. Ftn-143-4 is selected and treated with DBCO-Cy3 (λ _ex/λ _em = 555/570 nm) and DBCO-Cy5 (λ _ex/λ _em = 640/664 nm) dyes at the 1:2.5:2.5 M ratio for 16 h. FRET of the Ftn-143-Cy3 and Ftn-143-Cy5 mixture (1:1; as the control experiment) and Ftn-143-Cy3/Cy5 are measured (Wu and Brand, 1994a; Wu and Brand, 1994b) to ensure that both Cy3 and Cy5 molecules can be co-incorporated into one cage (Figure 4F). After the excitation at 550 nm, the mixture of Ftn-143-Cy3 and Ftn-143-Cy5 (1:1) gives an emission peak at 572 nm but lacks the emission peak at 664 nm, which supports this control system. In contrast, after the excitation at 550 nm on Ftn-143-Cy3/Cy5, a low emission peak at 569 nm is observed but an extra high emission peak at 673 nm arises, which demonstrates the successful FRET and proves that both Cy3 and Cy5 are co-incorporated into the Ftn cavity (Figure 4F).

To equip Ftn with a moiety targeting Her2⁺ breast cancer cells, Ftn is fused with AHNP through a non-structural triple GGGGS peptide at one of the two termini (Figure 5A). Since the fused AHNP may affect the self-assembling of Ftn, leading to twisted cavities, the structure analysis of the purified A-Ftn and Ftn-A is conducted using native PAGE, DLS, and TEM, respectively. Native PAGE analysis on A-Ftn and Ftn-A shows that they have similar assembled sizes and patterns, but their sizes are slightly larger than Ftn (Figure 5B). DLS analysis indicates that A-Ftn shares a similar outer diameter with Ftn (13.8 and 13.3 nm, respectively) (Figure 5C); TEM analysis shows that they both share a spherical shape (Figure 5D). However, DLS analysis shows that Ftn-A possesses a larger outer diameter of 18.0 nm (Figure 5C) and the TEM image shows that Ftn-A is irregular in its shape (Figure 5D). Thus, the A-Ftn construct is selected to target the HER2 receptor as it is more structurally suitable.

A-Ftn-81-4 reacts with DBCO-PEG4-TAMRA (Figure 6A) at 1:5 M ratios for 4 h to form A-Ftn-81-TAMRA. SDS-PAGE analysis is performed after the reaction, which gives a fluorescent band with a larger size, supporting that A-Ftn is being labeled (Figure 6B). The effect of A-Ftn on the targeting of the HER2 receptor is evaluated using the BT474 breast cancer cell line. BT474 cells are treated with A-Ftn-81-TAMRA for 1 h and then observed by confocal microscopy. The fluorescence images obtained from confocal microscopy analysis indicate that A-Ftn-81-TAMRA can be found in the cytosol of BT474 cells, suggesting that A-Ftn-81-TAMRA can enter through the cell membrane following the recognition of AHNP by HER2 receptors (Figure 6B).

Upon the successful demonstration for the capability of A-Ftn entering into BT474 cells, a chemotherapeutic drug, Dox, is loaded into A-Ftn to test the cell targeting and the drug release (Figure 1). The DBCO-PEG4-DOX is synthesized through the NHS-amine coupling between DBCO-PEG4-NHS and Dox. Using the SPAAC reaction, A-Ftn-81-DOX, A-Ftn-143-DOX, and A-Ftn-2am-DOX are synthesized by mixing A-Ftn-81-4, A-Ftn-143-4, and A-Ftn-2am-4 with DBCO-PEG4-DOX, respectively, for 16 h (Figure 7A). SDS-PAGE analysis shows that all three A-Ftn proteins are successfully conjugated with DOX and yield an almost complete conversion into A-Ftn–DOX products (Figure 7B). ESI-MS spectrometry is employed to characterize A-Ftn-81-4 and A-Ftn-81-DOX, respectively. The results show that the protein mass matches the corresponding calculated protein mass, leading to the confirmation of quantitative labeling with the reaction time of 16 h (Table 2, Supplementary Figures S14, S15).

Furthermore, an A-Ftn-143-DOX conjugate is chosen for the evaluation of the capability in entering BT474 cells. The short-term (1 h) and long-term (72 h) treatment of A-Ftn-143-DOX with BT474 cells are examined, with the free Dox as the control treatment. MTT cell viability assay is conducted to examine the cell toxicity for BT474 cells after the treatment with free Dox, A-Ftn-143-4, and A-Ftn-143-DOX, respectively, for 72 h. A-Ftn-143-DOX gives a compatible trend as free Dox at the concentration from 0.5 to 5 μM while A-Ftn-143-4 presents a negligible toxicity effect at this concentration range (Figure 7C). For the short-term (1 h) treatment of free Dox with BT474 cells, it is noted from the merged DAPI and Dox images that free Dox is found in the nucleus (Figure 7D). Nevertheless, for the short-term (1 h) treatment of A-Ftn-143-DOX with BT474 cells, Dox is mainly distributed in the cytosol of BT474 cells (Figure 7E), which suggests the elongated drug release. The slight Dox signal is observed in the nucleus, which resulted from free Dox molecules released from A-Ftn-143-DOX. After the long-term (36 h) treatment, free Dox released from A-Ftn-143-DOX is found to reside in the nucleus (Supplementary Figure S18).