Genetic code expansion enabled site-specific dual-color protein labeling: superresolution microscopy and beyond

Available online at www.sciencedirect.com

ScienceDirect Genetic code expansion enabled site-specific dual-color protein labeling: superresolution microscopy and beyond Ivana Nikic´ and Edward A Lemke Genetic code expansion is emerging as an important tool for manipulation and labeling of proteins in vitro and in vivo. In combination with click-chemistry it allows site-specific labeling of target proteins with small organic fluorophores. This is achieved by cotranslational incorporation of noncanonical amino acids (ncAAs) in target proteins via orthogonal tRNA/amino-acyl tRNA synthetase pairs. In a subsequent step, ncAAs are labeled with small dyes via clickchemistry. Small labeling tags and free choice of which fluorophore to use and where to put it into the protein are of particular importance for single molecule science and superresolution microscopy. Such genetically encoded click chemistry tools facilitate high resolution studies of protein function in live cells, viral imaging, as well as the improved design of antibody-drug conjugates. Address Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Corresponding author: Lemke, Edward A ([email protected])

Current Opinion in Chemical Biology 2015, 28:164–173 This review comes from a themed issue on Synthetic biomolecules Edited by Christian Hackenberger and Peng Chen For a complete overview see the Issue and the Editorial Available online 22nd August 2015 http://dx.doi.org/10.1016/j.cbpa.2015.07.021 1367-5931/# 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction Site-specific incorporation of noncanoncial amino acids (ncAAs) by genetic code expansion offers numerous novel possibilities for manipulation and labeling of proteins in vitro in a test tube, as well as in vivo in cell culture or at the whole organism level. NcAAs bearing various functional handles (for labeling, crosslinking, photoactivation, etc.) can be incorporated in bacteria, yeast, mammalian cells, plants and even animals, such as C. elegans and D. melanogaster [1,2,3]. Particularly interesting are clickable ncAAs which can be coupled to different probes via click-chemistry [4]. The biggest advantage of such labeling is that it can be done sitespecifically with single residue precision anywhere in a protein of interest (POI). Current Opinion in Chemical Biology 2015, 28:164–173

A prerequisite for the site-specific incorporation of a ncAA is the use of an amino-acyl tRNA synthetase (RS) and its cognate tRNA which are not cross-reactive with any of the host tRNA/RS pairs, a property called orthogonality. In that regard, Escherichia coli Tyr and Leu tRNA/RS pairs are commonly used in yeast and mammalian cells, Methanococcus jannaschii Tyr tRNA/RS pair in E. coli, and Methanosarcina pyrrolysine tRNA/RS pair in bacteria and eukaryotes [1]. Despite their different origins, all these orthogonal tRNA/RS pairs have in common that they can incorporate their substrate ncAA in response to a unique codon during POI translation. Instead of reprogramming sense codons, stop codons are most frequently used to target orthogonal tRNAs to one specific site in a POI, as this bypasses competition with naturally occurring tRNA/RS pairs. Due to its lower abundance in prokaryotes, the most widely used is the Amber (UAG) codon, but other stop codons can also be exploited. In addition to stop codons, blank quadruple codons can be used, as was done in bacteria [5,6] and even mammalian cells [7]. To encode a ncAA, the substrate binding pocket of the RS typically has to be engineered so that no cognate AA, but only the desired ncAA of choice is accepted. This frequently requires multi-step protein engineering and selection procedures [1]. As pyrrolysine is not a natural AA in bacteria and eukaryotes, the archaeal tRNA/RS pair that encodes for pyrrolysine (the 22nd natural amino acid) in Methanosarcina has shown great merits in the genetic code expansion field as it does not recognize natural AAs. Meanwhile, multiple tRNA/RS pairs targeting different codons exist that permit incorporation of a variety of clickable ncAAs. This can for example be used for single or dual-color labeling of proteins. The latter is already a routine tool for probing the structure and function of purified proteins with single molecule FRET1 (Fo¨rster resonance energy transfer) [2,8,9]. Also at the whole cell level, labeling via click-chemistry is gaining a lot of interest as it represents the smallest available, and thus in principle the least invasive, labeling tag. Only one amino acid is modified and in principle any fluorophore can be attached directly to the POI in living 1 While we limited ourselves to more in vivo and cell biology studies, additional applications of dual-labeling for fluorescence spectroscopy and dynamic structure determination experiments are discussed in the chapter by Haney, Wissner and Petersson in this volume in greater detail.

www.sciencedirect.com

Genetically encoded click chemistry Nikic´ and Lemke 165

cells. This is particularly useful for systems which cannot easily be labeled with larger labeling probes, such as viruses, as we will discuss later. In addition, the small size of the tag can permit increased labeling density – a parameter of crucial importance for superresolution microscopy (SRM) [10,11]. In that regard, we will discuss recently developed SRM compatible method for virus and protein labeling in living mammalian cells [12] and the potential to go beyond cellular imaging [13]. Since click-chemistry labeling is an extraordinary versatile tool, and in principle any probe with a suitable chemistry can be coupled to POIs, it also has a tremendous impact outside of the fluorescence field, which we will briefly discuss in another related application, where two ncAAs were used to generate and visualize antibody drug conjugates [14].

Click-chemistry as a dual-color labeling tool for SRM and FRET Labeling proteins modified with ncAAs via click-chemistry is a two-step process: in the first step, ncAAs bearing clickable functionalities are site-specifically incorporated into the POI by means of genetic code expansion technology. In a subsequent step, ncAAs are labeled via clickchemistry reactions. Until now, a plethora of ncAAs bearing azides, ketones, tetrazines, alkynes, and alkenes functionalities etc. in their side chains have been developed [1–3]. The type of the ncAA side chain will determine the choice of subsequent labeling reaction (please see Table 1 for a selection of those ncAAs for which we describe applications in greater detail in Figures 1–4): aliphatic alkynes react with azides in copper(I)-catalyzed azide–alkyne cycloaddition (CuACC), ketones with hydroxylamines, strained alkynes with azides in copper-free strain promoted alkyne–azide cycloaddition (SPAAC), and strained alkynes or alkenes with tetrazines in strain promoted inverse-electron-demand Diels–Alder cycloaddition (SPIEDAC) [1,15,16]. Of all these reactions, SPAAC and SPIEDAC proceed at physiological conditions, and are in principle particularly suitable for live cell labeling [17,18]. Another important feature of SPAAC and SPIEDAC for fluorescence science is their potential fluorogenic character. In a fluorogenic reaction, the dye switches from a dark to a bright state only upon successful conjugation to its substrate. This can for example be achieved by clever integration of the azide group in the dye scaffold [19,20] or the use of the intrinsic quenching property of tetrazines when coupled to dyes [21,22]. It is thus particularly beneficial if the required strained alkyne or alkene is genetically encoded, and not the ncAAs bearing azide or tetrazine side chain. This confers an additional advantage as strained ring amino acids are usually more stable during the long-term expression in cells than many azides and tetrazines [23]. Since imaging of more than one biological entity is very www.sciencedirect.com

common and demanded in biology, combinations of SPIEDAC and SPAAC reactions are even more attractive, since they can be orthogonal to each other and used for dual-color labeling [17,24,25]. However, to achieve dualcolor labeling of the same POI with two different ncAAs, we do not only need two orthogonal click-reactions but we also need two mutually orthogonal tRNA/RS pairs which can specifically incorporate two distinctly clickable ncAAs. The only system that has so far enabled incorporation of ring strained clickable ncAAs in mammalian cells is pyrrolysine tRNA/RS from Methanosarcina. It was even found that a particular double mutant of this pyrrolysine tRNA/RS pair makes the binding pocket promiscuous to recognize a variety of strained alkynes and alkenes. This omits need for tedious evolution/selection protocols and permits novel multi-color applications as discussed in the following [12,17,26]. The promiscuity of pyrrolysine RS was exploited by Lemke and colleagues to introduce two clickable ncAAs in a time-dependent (pulse-chase) manner. During expression of insulin receptor (IR), cells were first incubated with one ncAAI, which was then chased with a second ncAAII [12]. Such experimental design permits following different populations of the same protein, which can be of relevance for studies of protein trafficking, synthesis, sorting and fate. The result was two uniquely reactive ncAAs installed site-specifically in two different IR populations, differing by their age (Figure 1). Two populations of IR were selectively labeled with a combination of two fast SPIEDAC reactions (Figure 1b and c). These two reactions were found to be orthogonal to each other under tested conditions which was the first report of two fast genetically encoded SPIEDAC reactions (Figure 1d, kon,I = 1180 M 1 s 1 and kon,II = 700 M 1 s 1)[27]. The authors also tried a combination of SPAAC and SPIEDAC. However, not surprisingly, SPAAC was too slow for labeling (kon,SPAAC  1 M 1 s 1) and only possible when using high dye concentration and long incubation times which compromised viability of the cells [12,28]. This study was also the first to demonstrate the potential of click-chemistry based protein labeling for SRM (Figure 1c). As a follow-up, a recent study showed labeling of cytoskeletal proteins actin and vimentin inside mammalian cells [29], which due to their high abundance, high local protein concentration and distinct morphology in cells are frequently used to establish SRM imaging conditions. Intracellular labeling of low abundant proteins is more challenging and remains to be achieved. Labeling of two distinct proteins or two positions in a single protein would further expand the scope of click-chemistry labeling as it would for example enable Current Opinion in Chemical Biology 2015, 28:164–173

166 Synthetic biomolecules

Table 1 Overview of a subset of click-chemistry labeling reactions as used in the applications described in Figures 1–4. NcAA functionality

Dye functionality

Type of labeling reaction

Reference, used in Figures 1–4

Ketone

Hydroxylamine

Oxime ligation

[14]

DIBO-alkyne

SPAAC

[14]

Azide

SPAAC

[26]

SPIEDAC

[12,17,26]

SPIEDAC

[12,13,17]

O

NH2

O

H3C H2N

COOH

pAcF

Azide N3 O NH

O

H2N

COOH AzK

Strained alkyne

N3 O O

O NH

Tetrazine

NH

O

N N R H2N

H2N

COOH

COOH

BCN

SCO

Strained alkene O O

Tetrazine N N

O NH

H2N

O

COOH CP

N N

R N N

NH

H2N

COOH TCO*

dynamic studies of protein behavior through FRET. As mentioned, for this we need two tRNA/RS pairs encoding two distinct ring strained ncAAs for two orthogonal reactions, which has to date not even been achieved in E. coli. Instead, recently a combination of M. jannaschii Tyr tRNA/RS and pyrryolsine tRNA/RS was used to label two positions in a single protein for bulk FRET experiments by incorporating tetrazine and alkene-bearing ncAAs [30]. M. jannaschii Tyr tRNA/RS pair was used to incorporate a tetrazine bearing ncAA (which reacts with bicyclononyne – BCN dyes) and pyrrolysine tRNA/RS pair was used to incorporate alkene-bearing ncAA (a norbornene ncAA which reacts slowly with tetrazines: reaction rate kon,norbornene  1 M 1 s 1) into calmodulin. Calmodulin was then first labeled with BCN and then with tetrazine-containing dyes. In addition to sequential labeling, one pot labeling was also achieved where terminal alkyne Current Opinion in Chemical Biology 2015, 28:164–173

and cyclopropene bearing ncAAs were incorporated and then labeled with azide and tetrazine-dyes simultaneously [31]. The disadvantage of such one pot labeling strategy was the need for copper as a catalyst, which can be detrimental to many proteins and live cell labeling studies.

Click-chemistry as a virus labeling tool Another important application of genetic code expansion facilitated click-chemistry labeling is for virology studies. Labeling of viruses is important for visualization of viral life cycle: virus–host interactions/infection, internalization, replication, etc. All these studies are frequently hindered by the fact that direct virus labeling remains challenging. Due to their small and compact genomes, viruses often contain overlapping open reading frames (ORFs [32], Figure 2a) and they frequently use noncanonical translational mechanisms, such as ribosome frameshifting, leaky www.sciencedirect.com

Genetically encoded click chemistry Nikic´ and Lemke 167

Figure 1

(a)

transfect

ncAAI

ncAAII

label ncAAI

label ncAAII

(b) N N

N

N N

N

N

N O

O

O

O

+

(c)

+

(d)

<1

Azide

~1 Lys

Lys

BCN

118

H-Tet

00

804

680

1200 0 36 00 0 Lys

TCO*a ~0 M–1S–1 ≤1 M–1S–1

1μm

0

1μm

1μm

39

0

SCO

Me-Tet

Lys

TCO*e ∼100-1000 M–1S–1 >10000 M–1S–1

Current Opinion in Chemical Biology

Dual-color labeling of insulin receptor (IR) for superresolution microscopy (SRM). (a) Transfected HEK293T cells were sequentially incubated with two ncAAs (ncAAI and ncAAII) and labeled with two orthogonal SPIEDAC reactions. (b) Specific labeling of two distinct IR populations — ncAAI (TCO*) reacted with methyl-tetrazine and ncAAII (SCO) reacted with H-tetrazine. (c) Widefield (left) and ground state depletion with individual molecule return (GSDIM, right) images of IR. Below inset from right panel is shown. Scale bar is 1 mm. (d) Scheme showing different SPIEDAC and SPAAC reactions between strained alkynes (BCN, SCO), strained alkene (TCO*) containing ncAAs and azides (blue), H-tetrazines (green) and Methyl-tetrazines (red). Reactions rate are indicated (in M 1 s 1) and with thickness of lines for each reaction as measured in Ref. [27]. Dashed line means that no reaction was observed under tested conditions. Source: Panels b, c, and d adapted from Ref. [12].

scanning, internal ribosomal entry sites, etc. [33]. Such noncanonical translational mechanisms make labeling of viruses with standard genetic tools very challenging, since introduction of any kind of larger labeling tag will not only lead to the desired fusion protein, but likely also results in a scrambled sequence of other proteins within the overlapping ORFs (Figure 2b). It has been shown that GFP fusions to viral proteins can affect infectivity and behavior of viruses. For example gag and pol genes of HIV-1 overlap and a translational frameshift is required for the synthesis www.sciencedirect.com

of the Gag-Pol precursor protein. That is why making gaggfp fusion scrambles the Pol protein and why gfp-gag cannot be used in the context of formation of functional viral particles [34]. With only one amino acid change in form of ncAA, which can in principle be put anywhere in the POI, impact on other proteins encoded by alternative ORFs can be minimized (Figure 2c). Recently, Influenza viral like particles (VLPs) were labeled with SRM compatible dyes via click-chemistry Current Opinion in Chemical Biology 2015, 28:164–173

168 Synthetic biomolecules

Figure 2

(a)

compact genome

O OR R FA

F

ORF C

overlapping genes:

ORF A (0 frame)

ORF B (–1 frame)

B

(b) scrambeled protein B GFP fusion of protein A virus genome

(c) WT protein B Amber mutant of protein A virus genome

intensity [a.u.]

(d)

1 70nm 113nm

55nm 74nm

0 0

[nm]

1500

Current Opinion in Chemical Biology

Click-chemistry based labeling of viral proteins. (a) Viruses often contain small and compact genomes with overlapping ORFs. (b) When ORF A is fused to GFP (depicted in green), this will lead to the scrambled protein B. (c) In case of ncAA labeling, only one amino acid (depicted in white) is changed and that is why the frame of ORF B is not affected. (d) Widefield image of Influenza Virus-like particles shown on left and GSDIM images on the right, including the line profile (graph on the bottom right). Scale bar is 1 mm. Source: Panel d adapted from Ref. [12].

[12]. Analog to the aforementioned IR labeling, a combination of two SPIEDAC reactions allowed dual-color labeling (Figure 2d). Such dual-color labeling scheme could be of interest for studies of newly synthesized versus old proteins during virus replication, maturation, etc. Moreover, the Chen group used genetic code expansion to label the surface of live infectious Hepatitis D virus [35]. The authors used pyrrolysine tRNA/RS pair to Current Opinion in Chemical Biology 2015, 28:164–173

incorporate a set of pyrrolysine analogues which did not interfere with virus maturation and could be used for virus labeling with fluorescent dyes and biotin. Study of different mutations for placing the Amber codon and subsequent suppression showed that the infectivity can vary greatly — because of this it is important to test different labeling positions [35]. This is another reason why the versatility of site-specific labeling is very beneficial and represents one of the most promising approaches for virus labeling. www.sciencedirect.com

Genetically encoded click chemistry Nikic´ and Lemke 169

Labeling of antibody drug conjugates

protein expression in mammalian cells using the E. coli Tyr tRNA/RS pair and pyrrolyinse tRNA/RS pair to introduce p-acetylphenylalanine and azide containing ncAA (azido-lysine), respectively. After antibody production and purification, the p-acetylphenylalanine side chain was conjugated to the cytotoxic drug via oxime ligation and the encoded azido-lysine was labeled with the azide reactive Alexa Fluor 488-DIBO (dibenzocyclooctyne)-alkyne dye (Figure 3b) in a copper-free click reaction. Such dual labeling allows more-in-depth studies of ADCs’ mode of action, since fluorescent labeling allows for more quantitative uptake assays to be performed. In addition, such dual-function labeling could be exploited a for attaching two drugs, or one drug and positron emission tomography (PET) tracer for imaging in living organisms. While it was not necessary for the goal of the study by Xiao et al., an extension to SRM visualization would have easily been possible [14].

Another application where click-chemistry labeling is showing great promise is the production of antibody drug conjugates (ADC). ADCs are a class of targeted therapeutics, consisting of cytotoxic drugs linked to monoclonal antibodies recognizing tumor specific antigens [36]. This ensures targeting of the drug to the tumor cells (Figure 3a). Several factors determine the efficiency, pharmacokinetics, etc. of such therapeutics. The choice of antibody and the drug itself are important, but also appropriate labeling sites, linkers and conjugation approaches need to be considered. There are several ways to attach drugs to ADCs. Most widely used is labeling via lysine residues. However, such mechanisms lead to heterogeneity in terms of where and how many drug molecules are attached which can result in decreased efficiency due to heterogeneity of the ADCs (Figure 3a). By increasing the homogeneity improved pharmacokinetics and reduced side effects have been observed. In that sense, site specific labeling offers great hope which was recently exploited by the Schultz lab [37,38]. In 2013, Schultz and colleagues developed even more improved ADCs with dual functionalities [14]. They combined

Click-chemistry in higher organisms Since ncAAs are incorporated during protein translation in living cells, there is no conceptual reason why their incorporation could not be applied to whole organisms.

Figure 3

(a) tumor specific antigen

normal cell

tumor cell

(b) O N

O O

N N N

O N H

O

N

O

N H

O

dy e

N N N

dy e

O

Current Opinion in Chemical Biology

Labeling of ADCs. (a) ADCs are specifically targeted to tumor cells. Labeling via native amino acids (lysine, cysteine) leads to nonhomogenous populations of ADCs functionalized with different number of cytotoxic drug molecules. (b) The Schultz group used a combination of mutually orthogonal E. coli Tyr and pyrrolysine tRNA/RS pairs to incorporate ncAAs bearing ketone and azide, respectively [14]. In a subsequent step, ncAAs were labeled with cytotoxic drug via oxime ligation and with fluorescent dye via alkyne-azide cycloaddition. www.sciencedirect.com

Current Opinion in Chemical Biology 2015, 28:164–173

170 Synthetic biomolecules

Figure 4

tissue specific expression of tRNA/RS pair

proteome extraction

proteome labeling

in situ labeling

microscopy

Current Opinion in Chemical Biology

Whole animal genetic code expansion and labeling. Pyrrolysine tRNA/RS can be expressed tissue-specifically in D. melangoster. Pyrrolysine tRNA anticodon loop can be mutated to recognize sense codons which can be used for tissue specific proteome wide incorporation of ncAAs (shown on the left). Proteins from specific tissue can be identified by tetrazine labeling and used for mass spectrometry analysis (such experiment was done in Ref. [13] for ovarial tissue). The figure also shows a virtual experiment in which whole animal genetic code expansion could be used for tissue/cell specific in situ labeling and microscopy (shown on right).

Indeed, the genetic code of D. melanogaster [39], C. elegans [40] and plants (A. thaliana) [41] were expanded. Recently, the pyrrolysine tRNA/RS pair was also used to label proteins from specific tissues in D. melanogaster [13]. Since pyrrolysine RS does not recognize the anticodon loop of pyrrolysine tRNA, the tRNA was mutated to recognize sense codons. Like this, ncAAs could be incorporated proteome wide in a tissue specific way. This opens novel possibilities for proteomics studies as proteins synthetized in different tissues can easily be identified by tetrazine labeling (again, while not yet done, also conceptually possible at SRM level) and pulled down for mass spectrometry analysis (Figure 4). Current Opinion in Chemical Biology 2015, 28:164–173

Current limitations, future perspectives and potential Since ncAA are incorporated into target proteins during translation, there is no limitation in terms of localization of the target protein inside the cell. However, when it comes to labeling, some locations might be easier to label than the others. In that regard, labeling of surface proteins and abundant cytosolic proteins seems easier, while labeling of not so abundant intracellular proteins is still challenging. This is related to several challenges that Amber codon suppression and click-chemistry labeling bring, such as low efficiency of ncAA incorporation, delivery of ncAAs and dyes, potential biological interference and toxicity. www.sciencedirect.com

Genetically encoded click chemistry Nikic´ and Lemke 171

One of the main limitations for highly sensitive fluorescent applications like single molecule imaging and SRM is to achieve the required high signal to noise. A particular problem with intracellular click-chemistry labeling is that the cytoplasm can be full of free (non-specifically sticking or bound to tRNA or RS) ncAAs which can also react with the dyes, and need to be washed efficiently prior to labeling. That is why it is much easier to achieve labeling at the cell membrane than inside the cell. For intracellular labeling, new ncAAs which are more hydrophilic and can be washed out easily would help to achieve higher signal to noise. In addition, more improved and stable ncAAs which would even further increase labeling efficiency [27] are necessary, as well as more cell permeable and fluorogenic dyes [21,22,42,43]. Dyes that were used for insulin receptor labeling [12], such as sulfonated-Cy5 and Atto532 are negatively charged and do not penetrate the membrane of living cells. For intracellular labeling of cytoskeletal proteins [29], cell permeable silicon-rhodamine dyes were used [43], but there is much room left to further improve fluorogenicity of such dyes. Furthermore, the yield of Amber codon suppression needs to be increased. Since Amber is a naturally occurring stop codon there is a constant competition between orthogonal tRNA/RS pairs and translation termination factors. To reduce this and increase the yield of ncAA incorporation, attempts to knock-down components of the host translational termination machinery which can compete with Amber suppression seem to be very promising approach even in mammalian cells [44]. Another problem in mammalian cells is the existence of nonsense mediated mRNA decay (NMD) pathway, in which mRNAs containing premature stop codons get degraded. This also might reduce the yields of Amber codon suppression. To overcome this, several Amber mutants of POI need to be tested as different mutants are more or less prone to degradation via NMD. When it comes to dual-color labeling of two proteins or two positions in single protein in mammalian cells, the number of possible ncAAs to encode and to use in different reactions is constantly growing (Hoffmann et al. [27] and Wagner et al. [45] recently also showed that different diastereomers of a ncAA can have very different reactivity, see also Figure 1d). However, distinct orthogonal tRNA/RS pairs that can target distinct strained ncAAs to unique codons are much in need. In addition to stop codons, quadruple codons could be used [7]. If the level of unique quadruple codons suppression is more increased in mammalian cells, they should even be a more preferred choice than Amber codon for ncAAs incorporation. This is, because using naturally occurring stop codon, such as Amber, leads to incorporation of ncAAs not only in the engineered POI but in other proteins that contain the Amber codon as a natural stop codon. Even though around 20% of stop codons in mammals are www.sciencedirect.com

Amber, so far in labeling experiments, this ‘unwanted’ fraction does not seem to be a major ‘visible’ problem. However, care must be taken when aiming to visualize rare proteins or when Amber suppression is performed for long time periods, where during the whole cell cycle various proteins are transcribed. Such unwanted incorporation could lead to toxicity which still needs to be investigated in mammalian cells. The fact that genetic code expansion of whole animals was already achieved [13,39,40] gives hope that toxicity will not be the major issue. However with the aim of increasing Amber suppression yields, this might become a problem. While so far whole animal genetic code expansion was used mainly for proteomics studies, considering the potential that click-chemistry labeling has shown at the cell culture level, it could also be exploited for in vivo imaging (Figure 4). Labeling could be done with fluorescent dyes for SRM or probes for PET [46–48], magnetic resonance imaging (MRI) or ultrasound imaging [49] expanding the scope of click-chemistry labeling towards clinically relevant applications. With the resolution of SRM technology constantly growing, there is a pressing need for using small labels to facilitate as high labeling densities as possible while not obstructing biological function [50,51]. Click mediated genetic code expansion technology fulfills exactly these requirements and further developments will thus permit to ultimately utilize the full potential that SRM technology encodes for biomedical research.

Acknowledgements We thank the members of the Lemke, Schultz and Briggs groups for useful comments and discussions. I.N. acknowledges financial support by the European Commission Seventh Framework Program (FP7) through Marie Curie Actions (FP7-PEOPLE-IEF) and a European Molecular Biology Organization (EMBO) long-term fellowship. E.A.L. acknowledges funding by the Emmy Noether program, SFB1129 and SPP1623 of the Deutsche Forschungsgemeinschaft (DFG).

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Lang K, Chin JW: Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem Rev 2014, 114:4764-4806.

2.

Tyagi S, Lemke EA: Single-molecule, FRET and crosslinking studies in structural biology enabled by noncanonical amino acids. Curr Opin Struct Biol 2015, 32:66-73.

3.

Lemke EA: The exploding genetic code. Chembiochem 2014, 15:1691-1694.

4.

Hackenberger CP, Schwarzer D: Chemoselective ligation and modification strategies for peptides and proteins. Angew Chem Int Ed Engl 2008, 47:10030-10074.

5.

Neumann H, Wang K, Davis L, Garcia-Alai M, Chin JW: Encoding multiple unnatural amino acids via evolution of a quadrupletdecoding ribosome. Nature 2010, 464:441-444. Current Opinion in Chemical Biology 2015, 28:164–173

172 Synthetic biomolecules

6.

Wang K, Schmied WH, Chin JW: Reprogramming the genetic code: from triplet to quadruplet codes. Angew Chem Int Ed Engl 2012, 51:2288-2297.

7.

Niu W, Schultz PG, Guo J: An expanded genetic code in mammalian cells with a functional quadruplet codon. ACS Chem Biol 2013, 8:1640-1645.

22. Wu H, Yang J, Seckute J, Devaraj NK: In situ synthesis of alkenyl tetrazines for highly fluorogenic bioorthogonal live-cell  imaging probes. Angew Chem Int Ed Engl 2014, 53:5805-5809. More recent report on the improved synthesis pathways for tetrazine dye derivates. In addition, the authors made additional fluorescent probes (with up to 400-fold fluorescence turn-on) which were used for live cell imaging.

8.

Milles S, Lemke EA: Single molecule study of the intrinsically disordered FG-repeat nucleoporin 153. Biophys J 2011, 101:1710-1719.

23. Milles S, Tyagi S, Banterle N, Koehler C, VanDelinder V, Plass T, Neal AP, Lemke EA: Click strategies for single-molecule protein fluorescence. J Am Chem Soc 2012, 134:5187-5195.

9.

Milles S, Koehler C, Gambin Y, Deniz AA, Lemke EA: Intramolecular three-colour single pair FRET of intrinsically disordered proteins with increased dynamic range. Mol Biosyst 2012, 8:2531-2534.

24. Karver MR, Weissleder R, Hilderbrand SA: Bioorthogonal reaction pairs enable simultaneous, selective, multi-target imaging. Angew Chem Int Ed Engl 2011, 51:920-922.

10. van de Linde S, Heilemann M, Sauer M: Live-cell superresolution imaging with synthetic fluorophores. Annu Rev Phys Chem 2012, 63:519-540. 11. Banterle N, Bui KH, Lemke EA, Beck M: Fourier ring correlation as a resolution criterion for superresolution microscopy. J Struct Biol 2013, 183:363-367. 12. Nikic I, Plass T, Schraidt O, Szymanski J, Briggs JA, Schultz C,  Lemke EA: Minimal tags for rapid dual-color live-cell labeling and superresolution microscopy. Angew Chem Int Ed Engl 2014, 53:2245-2249. This is the first report on use of click-chemistry for superresolution microscopy compatible protein labeling. The authors applied a combination of two ultrafast and ortohogonal tetrazine cycloadditions with strained alkynes and alkenes for dual-color labeling of mammalian cells and viruses. 13. Elliott TS, Townsley FM, Bianco A, Ernst RJ, Sachdeva A,  Elsasser SJ, Davis L, Lang K, Pisa R, Greiss S et al.: Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal. Nat Biotechnol 2014, 32:465-472. The authors used genetic code expansion to label the proteome in a tissue specific manner during defined developmental stages of D. melanogaster. 14. Xiao H, Chatterjee A, Choi SH, Bajjuri KM, Sinha SC, Schultz PG:  Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells. Angew Chem Int Ed Engl 2013, 52:14080-14083. The authors used mutually orthogonal tRNA/RS pairs to incorporate two noncanonical amino acids (ncAAs) into the same antibody for subsequent expression in mammalian cells. These two ncAAs were used to produce fluorescently labeled herceptin-auristatin ADC by combing oxime-ligation and copper-free click-chemistry. 15. Blackman ML, Royzen M, Fox JM: Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels– Alder reactivity. J Am Chem Soc 2008, 130:13518-13519. 16. Milles S, Lemke EA: What precision-protein-tuning and nanoresolved single molecule sciences can do for each other. Bioessays 2013, 35:65-74. 17. Plass T, Milles S, Koehler C, Szymanski J, Mueller R, Wiessler M, Schultz C, Lemke EA: Amino acids for Diels–Alder reactions in living cells. Angew Chem Int Ed Engl 2012, 51:4166-4170. 18. Lang K, Davis L, Wallace S, Mahesh M, Cox DJ, Blackman ML, Fox JM, Chin JW: Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels–Alder reactions. J Am Chem Soc 2012, 134:10317-10320. 19. Herner A, Estrada Girona G, Nikic I, Kallay M, Lemke EA, Kele P: New generation of bioorthogonally applicable fluorogenic dyes with visible excitations and large stokes shifts. Bioconjug Chem 2014, 25:1370-1374. 20. Herner A, Nikic I, Kallay M, Lemke EA, Kele P: A new family of bioorthogonally applicable fluorogenic labels. Org Biomol Chem 2013, 11:3297-3306. 21. Meimetis LG, Carlson JC, Giedt RJ, Kohler RH, Weissleder R: Ultrafluorogenic coumarin-tetrazine probes for real-time biological imaging. Angew Chem Int Ed Engl 2014, 53:7531-7534. Current Opinion in Chemical Biology 2015, 28:164–173

25. Liang Y, Mackey JL, Lopez SA, Liu F, Houk KN: Control and design of mutual orthogonality in bioorthogonal cycloadditions. J Am Chem Soc 2012, 134:17904-17907. 26. Plass T, Milles S, Koehler C, Schultz C, Lemke EA: Genetically  encoded copper-free click chemistry. Angew Chem Int Ed Engl 2011, 50:3878-3881. This is the first report on genetical encoding of strained ring noncannoncial amino acids. 27. Hoffmann JE, Plass T, Nikic I, Valle Aramburu I, Koehler C, Gillandt H, Lemke EA, Schultz C: Highly stable transcyclooctene amino acids for live-cell labeling. Chemistry 2015 http://dx.doi.org/10.1002/chem.201501647. 28. Nikic I, Kang JH, Girona GE, Aramburu IV, Lemke EA: Labeling proteins on live mammalian cells using click chemistry. Nat Protoc 2015, 10:780-791. 29. Uttamapinant C, Howe JD, Lang K, Beranek V, Davis L, Mahesh M, Barry NP, Chin JW: Genetic code expansion enables live-cell and superresolution imaging of site-specifically labeled cellular proteins. J Am Chem Soc 2015, 137:4602-4605. 30. Wang K, Sachdeva A, Cox DJ, Wilf NM, Lang K, Wallace S, Mehl RA, Chin JW: Optimized orthogonal translation of unnatural amino acids enables spontaneous protein doublelabelling and FRET. Nat Chem 2014, 6:393-403. 31. Sachdeva A, Wang K, Elliott T, Chin JW: Concerted, rapid, quantitative, and site-specific dual labeling of proteins. J Am Chem Soc 2014, 136:7785-7788. 32. Chirico N, Vianelli A, Belshaw R: Why genes overlap in viruses. Proc Biol Sci 2010, 277:3809-3817. 33. Firth AE, Brierley I: Non-canonical translation in RNA viruses. J Gen Virol 2012, 93:1385-1409. 34. Muller B, Daecke J, Fackler OT, Dittmar MT, Zentgraf H, Krausslich HG: Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative. J Virol 2004, 78:10803-10813. 35. Lin S, Yan H, Li L, Yang M, Peng B, Chen S, Li W, Chen PR: Site specific engineering of chemical functionalities on the surface of live hepatitis D virus. Angew Chem Int Ed Engl 2013, 52:1397013974. This is the first study showing an application of genetic code expansion for labeling living and infectious viruses. 36. Panowksi S, Bhakta S, Raab H, Polakis P, Junutula JR: Sitespecific antibody drug conjugates for cancer therapy. MAbs 2014, 6:34-45. 37. Axup JY, Bajjuri KM, Ritland M, Hutchins BM, Kim CH, Kazane SA, Halder R, Forsyth JS, Santidrian AF, Stafin K et al.: Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci U S A 2012, 109:16101-16106. 38. Schumacher D, Hackenberger CP: More than add-on: chemoselective reactions for the synthesis of functional peptides and proteins. Curr Opin Chem Biol 2014, 22:62-69. 39. Bianco A, Townsley FM, Greiss S, Lang K, Chin JW: Expanding the genetic code of Drosophila melanogaster. Nat Chem Biol 2012, 8:748-750. 40. Greiss S, Chin JW: Expanding the genetic code of an animal. J Am Chem Soc 2011, 133:14196-14199. www.sciencedirect.com

Genetically encoded click chemistry Nikic´ and Lemke 173

41. Li F, Zhang H, Sun Y, Pan Y, Zhou J, Wang J: Expanding the genetic code for photoclick chemistry in E. coli, mammalian cells, and A. thaliana. Angew Chem Int Ed Engl 2013, 52:9700-9704.

46. Zeglis BM, Sevak KK, Reiner T, Mohindra P, Carlin SD, Zanzonico P, Weissleder R, Lewis JS: A pretargeted PET imaging strategy based on bioorthogonal Diels-Alder click chemistry. J Nucl Med 2013, 54:1389-1396.

42. Carlson JC, Meimetis LG, Hilderbrand SA, Weissleder R: BODIPY tetrazine derivatives as superbright bioorthogonal turn-on probes. Angew Chem Int Ed Engl 2013, 52:6917-6920. The authors report on the synthesis and characterization of extremely fluorogenic BODIPY-tetrazines which can be used for live cell imaging of mammalian cells.

47. Denk C, Svatunek D, Filip T, Wanek T, Lumpi D, Frohlich J, Kuntner C, Mikula H: Development of a (18) F-labeled tetrazine with favorable pharmacokinetics for bioorthogonal PET imaging. Angew Chem Int Ed Engl 2014, 53:9655-9659.

43. Lukinavicius G, Umezawa K, Olivier N, Honigmann A, Yang G, Plass T, Mueller V, Reymond L, Correa IR Jr, Luo ZG et al.: A nearinfrared fluorophore for live-cell superresolution microscopy of cellular proteins. Nat Chem 2013, 5:132-139. 44. Schmied WH, Elsasser SJ, Uttamapinant C, Chin JW: Efficient multisite unnatural amino acid incorporation in mammalian cells via optimized pyrrolysyl tRNA synthetase/tRNA expression and engineered eRF1. J Am Chem Soc 2014, 136:15577-15583. 45. Wagner JA, Mercadante D, Nikic I, Lemke EA, Grater F: Origin of orthogonality of strain-promoted click reactions. Chemistry 2015 http://dx.doi.org/10.1002/chem.201501727.

www.sciencedirect.com

48. Seckute J, Devaraj NK: Expanding room for tetrazine ligations in the in vivo chemistry toolbox. Curr Opin Chem Biol 2013, 17:761-767. 49. Zlitni A, Janzen N, Foster FS, Valliant JF: Catching bubbles: targeting ultrasound microbubbles using bioorthogonal inverse-electron-demand Diels-Alder reactions. Angew Chem Int Ed Engl 2014, 53:6459-6463. 50. Sengupta P, van Engelenburg SB, Lippincott-Schwartz J: Superresolution imaging of biological systems using photoactivated localization microscopy. Chem Rev 2014, 114:3189-3202. 51. Hell SW: Far-field optical nanoscopy. Science 2007, 316:1153-1158.

Current Opinion in Chemical Biology 2015, 28:164–173