Lentiviral strategy for the genetic incorporation of unnatural amino acids (Uaas) into proteins in adult rat neural stem cells (HCN-A94 cells). (a) Diagram depicting three strategies for expressing the orthogonal tRNA/synthetase pair and the reporter gene GFP(TAG). AUAG amber stop codon was introduced at a permissive site, Tyr182, in the GFP gene. Strategy 1: mPGK promoter for TyrRS, CMV promoter for GFP(TAG), and H1 promoter for tRNA expression in three separate lentiviral vectors. The H1-tRNA cassette was repeated 4-times in tandem in the coding region. Strategy 2: Promoters for TyrRS and GFP(TAG) were identical to those used in Strategy 1; a single copy of H1-tRNA was inserted into the unique Nhe I site of the 3′LTR of lentiviral vectors expressing TyrRS and GFP(TAG). Strategy 3: the mPGK promoter was used for driving the transcription of TyrRS and GFP(TAG), which were connected by the SKIP peptide linker for equivalent translation. Four repeats of the H1-tRNA cassette were encoded in the other lentiviral vector. (b) Incorporation of Tyr at the TAG position in GFP by using the orthogonal E. coli pair in HCN-A94 cells. GFP fluorescence images are shown to the left and overlaid with DIC images to the right. Scale bar, 20 μm. (c) Western blot analysis of GFP expression in HCN-A94 cells before and after differentiation using different lentiviral strategies. Differentiation was initiated 24 hr after lentiviral infection. All cell samples were grown for 7 days post infection and then lysed for Western analyses. Quantification of blot intensity (GFP/actin) is indicated at the bottom. Lane 6 is wild type GFP expressed by a lentiviral vector as a positive control. (d) Structure of the Uaa p-benzoylphenylalanine (Bzo). (e) Incorporation of the Uaa Bzo into GFP in HCN-A94 cells. Bzo-specific and GFP(TAG) reporter genes were delivered by using strategy 2 into HCN-A94 cells. GFP fluorescence was detected in cells only when Bzo was added in the growth media.

Tyr and Bzo incorporation does not affect the differentiation program of HCN-A94 cells into neuron-like progenies. (a) Time sequences of lentiviral infection and neural differentiation are shown at the top. Fluorescence images of cells were recorded on different days to monitor the differentiation process. Imaging parameters were the same for all samples, and images are displayed using the same intensity scale. Scale bar, 20 μm. Transgenic genes delivered by lentiviral vectors into HCN-A94 cells are indicated to the left. The orthogonal tRNA/synthetases pairs were delivered using strategy 2 as described. (b) Immunocytochemical analysis of HCN-A94 cells expressing GFP and cells expressing GFP(TAG) together with the Bzo-specific . An antibody against the stem cell-specific marker SOX2 was used. Scale bar, 10 μm. (c) Percentage of SOX2 postive cells relative to GFP positive cells was similar between HCN-A94 cells expressing GFP (white bars) and HCN-A94 cells expressing GFP with Bzo incorporation by the (dark bars). Error bars represent s.d. (d) Immunocytochemistry confirmed the neural differentiation of HCN-A94 cells when Bzo was incorporated into GFP. An antibody against the neural marker Tuj-1 was applied to HCN-A94 cells expressing GFP(TAG) and the in the presence of Bzo on day 6 after differentiation. Scale bar, 10 μm.

Incorporation of Uaa DanAla into the voltage-sensitive domain (VSD) of CiVSP. (a) Cartoon depicting the topology of the VSD of the membrane protein CiVSP. The C-terminal phosphatase domain of CiVSP was replaced by a red fluorescent protein mKate. Sequence alignment of the S4 residues of CiVSP and Kv1.2 shows the conserved voltage sensitive basic residues, which are represented by + signs along S4 in the cartoon. (b) Structure of Uaa DanAla. (c) Three-dimensional model of the VSD of CiVSP generated using the rat Shaker potassium channel Kv1.2 as a structural template. Side chains corresponding to the six voltage-sensing basic residues in S4 are highlighted in cyan. Phe234 and Gln208 are highlighted by green coloring. Four lipid molecules are shown to indicate the likely membrane embedded region of the VSD. (d) Fluorescence images of HEK293 cells expressing VSD-mKate (top), VSD-mKate(F234TAG) and with DanAla in the growth media (middle), VSD-mKate(F234TAG) and without DanAla (bottom). In the absence of DanAla, the VSD-mKate(F234TAG) gene expressed a truncated form localizing throughout the cell. When DanAla was added, full-length VSD-mKate(F234TAG) was expressed and correctly localized to the membrane as for the wild type VSD-mKate.

(a) Sensing current traces recorded from HEK293T cells expressing VSD-mKate and its DanAla mutants. The membrane was clamped at −60 mV and a series of voltage pulses with voltages ranging from −60 mV to 110 mV in 10-mV increments were applied sequentially over a period of 2 seconds for each pulse. Online compensation for cell capacitance was applied during recording of these traces. The inserts highlight the time scale in expanded format. (b) Sensing charge as a function of membrane potential (Q-V curve) for VSD-mKate and its DanAla-containing mutants. Solid lines are single Boltzmann fits.

DanAla genetically incorporated into the VSD of CiVSP optically reports membrane depolarization of neurons differentiated from HCN-A94 cells. (a) Modified lentiviral vector for enhancing the expression of VSD-mKate(TAG) in HCN-A94 cells. The CMV promoter was replaced with the CAG promoter to drive expression of the VSD-mKate(TAG) gene, and the 3′UTR sequence from the rat tubulin was added. The orthogonal pair was delivered using strategy 2 described in Fig. 1a. (b) Fluorescence images of VSD-mKate(F234TAG) and VSD-mKate(Q208TAG) expressing neurons differentiated from HCN-A94 cells grown in the presence (top) and absence (bottom) of DanAla. Scale bar, 10 μm. (c) Immunostaining of HCN-A94 cells incorporating DanAla into VSD-mKate(F234TAG) on day 6 after differentiation with Tuj-1 specific antibody. Similar immunostaining results were obtained for HCN-A94 cells incorporating DanAla into VSD-mKate(Q208TAG). The presence of mKate fluorescence on cell membrane and Tuj-1 signal indicate that DanAla incorporation into VSD-mKate did not alter HCN-A94 differentiation into neuronal cells. Scale bar, 10 μm. (d) DanAla fluorescence change upon membrane depolarization induced by high K⁺ shock. The duration of K⁺ shock is indicated by a black bar. Each frame encompassed 500 ms. Top panels show DanAla channel fluorescence changes in cells with DanAla incorporated into VSD-mKate at position 234 (left) and position 208 (right); bottom panels depict the DanAla channel fluorescence change in cells in the same imaging field without DanAla incorporation as the negative control. Error bars represent s.e.m.; n = 6 for site 234; n = 5 for site 208.