ncAAs for Copper-Catalyzed Click Reaction

I tested three different ncAAs amenable to copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC): two azide derivatives of phenylalanine and lysine, pAzpa and AZK, respectively, as well as the alkyne derivative of lysine, PRK. The chemical formulas of these compounds are shown in Figure 3-5.

pAzpa (p-azido-L-phenylalanine)

The non-canonical amino acid pAzpa has been added to the genetic code of Escherichia coli using the Methanococcus jannaschii tyrosyl-tRNA synthetase and a mutant tyrosine amber suppressor tRNA (Chin et al., 2002). It has also been used in Saccharomyces cerevisiae with the help of the tyrosyl-tRNA synthetase and Amber suppressor tRNA from E. coli (Chin et al., 2003). The incorporation of p-azido-L-phenylalanine and p-(propargyloxy)-L -phenylalanine has been achieved in E.coli using the optimized pEvol system comprising the tyrosyl-tRNA synthetase and tRNA pair from Methanocaldococcus janaschii (Young et al., 2010).

pAzpa has been among the first clickable ncAAs incorporated in mammalian cells (Liu et al., 2007) using a mutant E. coli aaRS and a Bacillus stearothermophilus suppressor tRNA.

In the same paper, a propargyloxy (alkyne) derivative of phenylalanine was also employed but its incorporation efficiency was lower than for pAzpa. This compound is not commercially available, so I did not test it for my project.

In this study, I have used the plasmid system pcpAzpaRS described by Yokoyama and collaborators to incorporate pAzpa in mammalian cells. The preliminary results from Figure 3-7 indicate that the incorporation of pAzpa was successful (see the GFP signal which is present only if the Amber stop codon was suppressed). Note that in the control where no ncAA was provided, there is no GFP signal, even though the cells were also transfected under the same conditions as for the pAzpa (Figure 3-7). This attests the fidelity of the incorporation of the ncAA and no other endogenuous amino acid. In terms of the click labeling efficiency, the results indicate nonspecific background in all cells, both in the control and the sample incubated with pAzpa (see Figure 3-7). What is more, the click signal is above background only for the cell with high expression levels SNAP-25-GFP, while the cells with low SNAP-25-GFP levels cannot be distinguished from the rest of the cells.

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65 This improper signal-to-noise ratio is a result of the relative lability of the azido group of pAzpa, which delivers low labeling levels, on one hand, and of the hydrophobicity of Atto647N, on the other hand (Kolmakov et al., 2010; Hughes et al., 2014). As a next step, I tried to optimize the click reaction by using other fluorescent dyes that generate a lower background, such as Star635P or Alexa647.

The pAzpa compound has several major disadvantages: it requires a 80% acetic acid solution to get dissolved as a dark yellow solution (maximum concentration is 0.242 M) and it is also highly unstable in solution. The saturated pAzpa solution in 80% acetic acid has to be freshly prepared and used within 5 minutes. Upon longer storage at RT or -20°C, it gets degraded and forms an amorphous precipitate. Additionally, pAzpa is a derivative of a bulky and hydrophobic amino acid – phenylalanine – and poses problems when substituting other smaller and more hydrophilic amino acids. If a hydrophobic residue is replaced, then the azido group of pAzpa might be secluded in a hydrophobic pocket and the labeling reaction could be impaired. Therefore, due to the above reasons and especially due to the high Figure 3-7 pAzpa click reaction results in low signal-to-noise levels

BHK cells were transfected with the pEGFP-N1 SNAP-25-GFP F84TAG and the pcpAzpaRS vectors.

The ncAA pAzpa (lower panel) was provided in the cell medium for approximately 18 hours, while the cells were allowed to express the protein of interest. In the control, no ncAA was added even though the cells were cotransfected under the same conditions. Before fixation, the cells were incubated for 2 hours with normal medium to remove the pAzpa excess. The click reaction was carried out with 2 µM Atto647N-alkyne and the nuclei were revealed with DAPI. The fluorescence intensity for the DAPI, GFP and click signal in both the pAzpa and the control were identically scaled. In the rightmost panels, overlays of the three different channels can be seen. Scale bar applies to all images and is 40 µM.

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instability of the pAzpa, I resorted to finding better ncAA candidates, as can be seen in the rest of this chapter.

AZK (azido-L-lysine) and PRK (propargyl-L-lysine)

The two aliphatic ncAAs azido-L-lysine (AZK) and propargyl-L-lysine (PRK) have been genetically encoded by bacteria using the Methanosarcina barkeri pyrrolysyl-tRNA and suppressor tRNA^Pyl (Nguyen et al., 2009; Milles et al., 2012). In this study, I used the optimized plasmid pCMV PylRS/tRNA^Pyl WT containing the Methanosarcina mazei bioorthogonal tRNA^Pyl and the wild-type (WT) pyrrolysyl-tRNA synthetase (Plass et al., 2011).

The Western blotting results from Figure 3-2 indicate that both of these ncAAs are well incorporated in VAMP2-GFP but the efficiency of the incorporation is slightly higher for AZK compared with PRK.

As a next step, I wanted to investigate not only the incorporation efficiency, but also the suitability to undergo labeling reactions for these lysine derivatives. For this, I resorted to fluorescence microscopy. I expressed VAMP2-GFP R125TAG and the bioorthogonal RS/tRNA pair in BHK cells in the absence (Figure 3-8 A) or in the presence of ncAAs (Figure 3-8 B-C). I subjected the fixed and permeabilized cells to CuAAC labeling with Atto647N derivatives. As can be seen in Figure 3-8, AZK and PRK are both effectively incorporated. However, only PRK is specific and efficient in the copper-catalyzed click reaction (compare the click signal panels with the VAMP2-GFP panels in Figure 3-8 C).

The reason behind this could be the high sensitivity of the azide group to undergo reduction, resulting in a non-reactive byproduct (Milles et al., 2012). It is plausible that after the incorporation of AZK into the protein of interest (in this case VAMP2-GFP), the clickable azide moiety was inactivated. The lack of reactivity of the azide after the incubation at 37°C for approximately one day was further proven by its poor labeling with the strained aza-dibenzobicyclooctyne (DIBAC) derivative of KK114 (data not shown). The latter reaction was first shown to effectively label enzymes in copper-free cycloaddition (Debets et al., 2010) and was later applied for the in vivo labeling of cells treated with L -azido-homoalanine (Saka et al., 2014a).

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67 Therefore, out of the three ncAAs suitable for copper-catalyzed click reaction, only PRK is well incorporated and reacts with the complementary fluorescent dyes in a specific and efficient manner. A summary of the results for the ncAA testing and optimization can be found in Table 3-1. As reported in Section 3.2.3, I sought to further optimize the labeling of PRK samples using more hydrophilic dyes at higher concentrations to ensure that all alkyne groups are saturated during the click labeling reaction.

Figure 3-8 AZK and PRK are well incorporated into VAMP2, but only PRK reacts efficiently in CuAAC

VAMP2-GFP R125TAG was expressed in BHK cells in the absence of any ncAA (A) or in the presence of AZK (B) or PRK (C). Approximately 18 hours post-transfection the cells were washed for 2 hours in normal medium, then fixed and permeabilized. Copper-catalyzed click reaction was performed with 2 µM Atto647N-alkyne (A-B) or 2 µM Atto647N-azide (C). Note that the images for each of the channels were scaled identically. Nuclei were revealed using DAPI staining. Scale bar, 40 µm.

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