Optimizing the Labeling of PRK

As PRK was the non-canonical amino acid that provided the most reliable labeling in click reactions among all the tested compounds, I decided to pursue this research line and optimize its reaction with fluorescent dyes.

For PRK, the best incorporation in the proteins occurs at a final concentration of 250 µM in the cell medium (data not shown). In addition, it is beneficial to use media in the absence of antibiotics as this reduces cell death caused by the transfection procedure. The next step was to optimize the click reaction by adjusting the dye hydrophilicity and concentration during the click reaction.

To identify better dye candidates, I compared the labeling efficiencies of Atto647N-azide, KK114-azide, and Star635P-azide in CuAAC. While KK114 and Star635P are very soluble in water and in PBS, Atto647N is rather hydrophobic and requires organic solvents for solubilization (Wurm et al., 2012). As a result, the highest amount of Atto647 derivatives (e.g. azide or alkyne) I could use with a fairly good signal-to-noise ratio was 2 µM. When

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75 applied in higher concentrations, the Atto647N strongly binds to membranes thereby causing unspecific labeling (Hughes et al., 2014).

In order to make sure that all the proteins that had incorporated PRK were fully labeled, I resorted to the new generation coumarin dyes KK114 and Star635P which have comparable brightness and stability, while at the same time, being more biocompatible due to their excellent solubility under physiological conditions and low unspecific interactions with biological material (Kolmakov et al., 2010).

In Figure 3-12, a direct comparison between Atto647N and KK114 is made for the copper-catalyzed labeling of PRK incorporated in SNAP-25-GFP. Even though the concentration of KK114-azide used for the click reaction is more than twice that of the Atto647N-azide, the background for the click signal is minimal (compare the click signal panels in Figure 3-12 A and B). The Atto647N dye binds considerably even to cells which were not incubated with the ncAA (control; upper panels in Figure 3-12 A), attesting for its unspecific binding to cellular material. In addition, for the PRK treated samples there is considerable Atto signal in cells that do not express GFP (i.e. non-transfected). In contrast, the click signal provided by KK114-azide is highly specific with almost no background in the control or in the non-transfected cells (see Figure 3-12 B).

In Figure 3-12, note that the higher signal in the GFP signal in the control could indicate a bleed-through of Atto fluorescence into this channel.

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In view of the above, I ruled out Atto647N as a click stain and decided to further improve the click reaction using hydrophilic dyes. Figure 3-12 clearly shows that for these identically scaled images, the intensity of the Atto staining is higher than the KK114 one (compare the click signal images in Figure 3-12 A versus B). However, the two fluorophores themselves have comparable brightness (Kolmakov et al., 2010). Therefore, only insufficient labeling could be responsible for this observation.

Figure 3-12 Labeling specificity Atto647N-azide and KK114-azide

PRK was incorporated into SNAP-25 M219TAG by BHK cells. The samples were fixed, permeabilized, and subsequently labeled using CuAAC with 2 µM Atto647N-azide (A) or 5 µM KK114-azide (B). Control samples represent cells that were transfected in the absence of any ncAA (upper panels), while PRK was added in the medium the cells were transfected for the lower panels.

All corresponding channel were identically scaled for comparison purposes. Scale bars, 40 µm.

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77 As a next step, I therefore optimized the dye concentration for the click reaction. I used the KK114 dye only for initial experiments and then I switched to the commercially available Star635P-azide. The latter synthetic fluorophore is designed to have an even lower fluorescent background and offer a better contrast in cellular stainings (Kolmakov et al., 2012). Figure 3-13 illustrates the labeling parameters for an optimal click staining: 30 minutes incubation time with 50 µM Star635P-azide. The click signal reaches saturation already at 25 µM (data not shown), indicating that the reaction is complete. Also, by analyzing cell expressing GFP-tagged α-synuclein and at the same time labeled in a copper-catalyzed click reaction with Star635P, it is evident that this reaction offers a very accurate and proportional labeling compared with the GFP signal (see Figure 3-13 A). The very good correlation between the GFP (i.e., the ncAA-containing protein) and the click signal can be visually assessed in Figure 3-12 B. The transfected cell shows comparable GFP and click signal, whereas the surrounding non-transfected cells are not labeled, as should be the case.

Figure 3-13 Optimal labeling parameters using PRK and Star635P-azide

The click labeling reaction was optimized and the best conditions are shown. BHK cells expressing α-synuclein-GFP G141TAG and the bioorthogonal PylRS/tRNA for PRK incorporation were fixed and permeabilized then clicked with 50 µM Star635P-azide for 30 minutes at room temperature. A. The graph shows the intensity of the Star635P-azide fluorescence plotted against the GFP fluorescence intensity. There is clear linear correlation of the Star635P fluorescence with the fluorescence of the α-synuclein-GFP. This corroborates with the fact that the reaction is not only specific, but also very efficient. The symbols depict fluorescence intensity averages derived from 172-1902 cells from a single typical experiment. B. The images show cells that have been allowed to incorporate PRK into α-synuclein-GFP and were later click labelled with Star635P-azide and DAPI stained. The GFP expressing cell shows strong labeling with Star635P. Scale bar, 40 µm.

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