Xi
Chen
ab and
Yao-Wen
Wu
*ab
aChemical Genomics Centre of the Max Planck Society, Otto-Hahn-Str. 15, 44227 Dortmund, Germany
bMax-Planck Institute for Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany. E-mail: yaowen.wu@mpi-dortmund.mpg.de
First published on 23rd February 2016
Over the years, there have been remarkable efforts in the development of selective protein labeling strategies. In this review, we deliver a comprehensive overview of the currently available bioorthogonal and chemoselective reactions. The ability to introduce bioorthogonal handles to proteins is essential to carry out bioorthogonal reactions for protein labeling in living systems. We therefore summarize the techniques that allow for site-specific “installation” of bioorthogonal handles into proteins. We also highlight the biological applications that have been achieved by selective chemical labeling of proteins.
Recent years have seen tremendous progress in chemical protein labeling. There are already some excellent reviews on bioorthogonal reactions and selective chemical labeling of proteins.2–4,6,12–18 In this review, we compare the features of reported bioorthogonal reactions and chemical tagging approaches, e.g. the reaction rate or time, reaction conditions, reagents, etc. This could be helpful to identify the most suitable labeling reaction for a particular application. Moreover, we highlight the applications of established chemical labeling techniques to tackle biological problems.
Reactant A | Reactant B | Product | k (M−1 s−1) or t | Features | Reference(s) |
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t: reaction time. (A) RT: room temperature; ABAO: aminobenzamidoxime; KAHA: α-ketoacid-hydroxylamine; ACL: aldehyde capture ligation. (B) PBS: phosphate buffered saline; TBTA: tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; CuAAC: copper-catalyzed azide alkyne cycloaddition; SPAAC: strain-promoted azide alkyne cycloaddition; BCN: bicyclononyne. (C) TCO: trans-cyclooctene; ED: electron-donating. (D) TQ-ligation: thiovinyl ether o-quinolinone quinone methide ligation; oQQM: o-quinolinone quinone methide; EW: electron-withdrawing. (E) BIAN: bis-imine of acenaphthenequinone and mesitylamine; dba: dibenzylidene acetone. (F) TCEP: tris(2-carboxyethyl)phosphine; Dha: dehydroalanine; MSH: O-mesitylenesulfonylhydroxylamine; CBT: cyanobenzothiazole; NCL: native chemical ligation; MESNA: 2-mercaptoethanesulfonate; MPAA: 4-mercaptophenylacetic acid. | |||||
A: Condensation through carbonyls | |||||
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0.006 (pH 7, RT) | Oxime ligation: p-phenylenediamine (p-PDA) or aniline, etc. as a catalyst | Dirksen (2006)20 |
0.29 (pH 7, RT, 10 mM p-PDA) | Wendeler (2014)21 | ||||
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PBS (pH 6.5, RT) 0.0087, or 0.46 (1 mM 5MA) | Hydrazone ligation: aniline or 5-methoxyanthranilic acid (5MA), etc. as a catalyst; ∼85% yield in 5 h | Crisalli (2013)22 | |
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18 h (pH 6.5, 37 °C) | Pictet-Spengler reaction: formation of a stable C–C bond; suitable for N-terminal protein labeling | Sasaki (2008)23 | |
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10 (pD 4.5) | Pictet-Spengler ligation: ligation is faster under lower pH conditions; formation of a stable C–C bond | Agarwal (2013)24 | |
2 (pD 6) | |||||
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4.17 (pH 6) | Hydrazino-Pictet-Spengler ligation: ligation proceeds fastest under pH 6; formation of a stable C–C bond | Agarwal (2013)25 | |
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40 (pH 4.5) | ABAO ligation: formation of a fluorescent dihydroquinazoline (λmax 490 nm); quantitative labeling in 1 h | Kitov (2014)26 | |
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1.61 (pH 7.4, RT, 10 μM CuSO4, air) | Under air; requires Cu(II) or Zn(II) catalyst; quantitative labeling in 2 h | Ji (2014)27 | |
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24 h (pH 7, RT) | Mukaiyama-Adol condensation: 11–55% conversion; stable | Alam (2010)28 | |
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4 h (≥10% H2O in DMSO, 60 °C) | KAHA ligation: formation of a native amide bond; 56–76% yield | Bode (2006)29 |
Pattabiraman (2012)30 | |||||
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1.65 (DMF, RT); 40 h [10% DMF in PBS (pH 7)] | ACL: selective N-terminal protein labeling is possible at pH 7; ∼70% labeling of ubiquitin | Raj (2015)31 |
B: Click reactions through azides | |||||
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0.003 (PBS) | Staudinger-Bertozzi ligation: phosphine is prone to oxidation | Saxon (2000)32 |
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0.0074 (H2O) | Traceless Staudinger ligation: needs transient protection of phosphine by BH3; 80% yield at pH ≥ 7.5 | Nilsson (2001)33 | |
Tam (2007)34 | |||||
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6–24 h (H2O, RT) | Staudinger-phosphite ligation: phosphite is stable against air oxidation; up to 90% yield | Serwa (2009)35 | |
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∼3 [50 μM Cu(I) + 50 μM TBTA, DMSO![]() ![]() ![]() ![]() |
CuAAC: ligand for copper catalyst is required; up to 94% yield | Rostovtsev (2002)36 | |
Presolski (2010)37 | |||||
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up to 0.0024 (H2O, 25 °C) | Oxanorbornadiene cycloaddition: metal-free, but is slow | van Berkel (2007)38 | |
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0.0024–0.076 | SPAAC: cyclooctynes are susceptible to thiols | Agard (2004)39 | |
Baskin (2007)40 | |||||
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0.057–0.96 | SPAAC: cyclooctynes are lipophilic; nonspecifically stick to serum proteins and cellular membranes | Ning (2008)41 | |
Jewett (2010)42 | |||||
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0.29; 2–2.9 (with electron-deficient azide) | SPAAC: BCN is readily accessible; the reaction rate is accelerated using an electron-deficient azide | Dommerholt (2010/2014)43,44 | |
C: Inverse electron-demand Diels–Alder cycloadditions (DA INV ) | |||||
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210–∼8.6 × 105 (PBS, 37 °C) | TCO is lipophilic and isomerizes over time; 100% conversion in 5 min | Blackman (2008)45 |
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437–2672 | BCN is readily accessible; almost quantitative conversion | Lang (2012)46 | |
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0.12–9.46 | Norbornene is stable | Devaraj (2008)47 | |
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0.03–27 | Cyclopropene is small and stable; ED groups increase reactivity; quantitative labeling | Patterson (2012)48 | |
Elliott (2014)49 | |||||
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0.39 (DMSO![]() ![]() ![]() ![]() |
Used for affinity based protein profiling | Engelsma (2014)50 | |
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0.017–0.041 | Terminal olefin only reacts with highly reactive tetrazines | Niederwieser (2013)51 | |
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0.12–0.58 (THF![]() ![]() ![]() ![]() |
Tertiary isonitrile is required; the reaction is slow | Stöckmann (2011)52 | |
D: Other dipolar cycloadditions | |||||
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39 (MeCN![]() ![]() ![]() ![]() |
Strain-promoted alkyne-nitrone cycloaddition (SPANC): some nitrones are prone to hydrolysis; up to 95% yield | Ning (2010)53 |
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0.043 (CDCl3, RT) | Diazo-strained alkyne cycloaddition: the reaction rates are similar to the equivalent reactions of azide | McGrath (2012)54 | |
Josa-Culleré (2014)55 | |||||
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0.054 (21 °C, 55% MeOH in H2O) | Strain-promoted sydnone-BCN cycloaddition: quantitative reaction in organic solvent and in aqueous buffer | Wallace (2014)56 |
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0.25 (PBS/EtOH) | Quadricylane ligation: quadricyclane is stable; the product is light sensitive | Sletten (2011)57 |
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N.A. | Nitrile oxide-norbornene cycloaddition: nitrile oxide may cross react with nucleophiles; mostly used for labeling of nucleic acids | Gutsmiedl (2009)58 |
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0.0015–0.028 (37 °C, 1![]() ![]() ![]() ![]() |
TQ ligation: oQQM generated in situ; EW-groups on oQQM accelerate the reaction | Li/Dong (2013)59 |
Zhang (2015)60 | |||||
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0.79 (MeCN![]() ![]() ![]() ![]() |
Tetrazole-alkene photo-click reaction: irradiation wavelength is tetrazole dependent; nitrile imine generated in situ; up to quantitative yield | Wang (2008/2009)61,62 |
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2 min (PBS, RT): give 41% labeling of lysozyme | Azirine ligation: azirine generates a reactive nitrile ylide intermediate in situ | Lim (2010)63 | |
E: Transition metal-catalyzed coupling/decaging reactions | |||||
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30 min (ligand, pH8, 37 °C) | Suzuki–Miyaura coupling: boronic acids are moderately toxic; >95% conversion | Chalker (2009)64 |
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30 min (Cu ion, ligand, RT) | Sonogashira coupling: Mg(II) inhibits the binding of Pd(II) to the protein | Kodama (2007)65 | |
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2–5 h (pH 8, 30% tBuOH/H2O, RT to 37 °C) | Olefin cross metathesis: requires ruthenium catalyst; up to 90% yield | Lin (2008)66 |
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24 h (Pd(OAc)2, BIAN, O2, pH 7, RT) | Aqueous oxidative Heck reaction: reaction opens to air; up to full conversion if excessive catalyst and boronic acid are used | Ourailidou (2014)67 | |
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allyl2Pd2Cl2 or Pd(dba)2 |
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180 min (10 μM Pd(dba)2, 37 °C) | Useful for in vivo protein activation | Li (2014)68 |
F: Labeling at cysteine residue(s) | |||||
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<30 min (PBS, RT) | TCEP should be added to maintain cysteine resides in a reduced form | Kim (2008)69 |
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5 min (pH 7.5, RT, 5![]() ![]() ![]() ![]() |
X = I, Br, Cl, OTf; quantitative labeling. Cy = cyclohexyl- | Vinogradova (2015)70 | |
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90 min (pH 9.0, 4 °C or RT) | Dha can be derived from cysteine by MSH (pH8.0, 4 °C, 20 min); >95% labeling | Bernardes (2008)71 |
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9.19 (PBS) | CBT ligation: side reactions with thiols of CBT | Ren (2009)72 |
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pH 7.0 | NCL: thiol catalysts are used, e.g., MESNA, MPAA | Dawson (1994)73 | |
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After reduction of disulfide: 2 h (pH 7.8, 4 °C) | S–S intercalation: reaction under mildly basic pH; formation of a stable product | Shaunak (2006)74 |
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TCEP, 1 h; then pH 8, RT, 3 h | Acetone crosslinking: the introduced ketone allows oxime ligation; cross linking also stabilizes helical structures | Assem (2015)75 |
Aside from oxime ligation and hydrazone ligation, aldehydes and ketones can undergo a Pictet-Spengler reaction with β-arylethylamines,78 which has been used for protein labeling at the N-terminus.23 To date, several modified versions have been introduced, including Pictet-Spengler ligation24 and hydrazino-Pictet-Spengler ligation.25 Additionally, proteins engineered with an N-terminal aldehyde tag can be labeled via the Mukaiyama-Adol condensation using silyl ketene reagents with the formation of a stable C–C bond.28 KAHA (α-ketoacid-hydroxylamine) ligation allows the condensation between an α-ketoacid and hydroxylamine or 5-oxaproline to form a native amide bond. This ligation has been a valuable alternative to native chemical ligation (NCL)73 to join two unprotected peptide fragments in peptide synthesis.29,30 Owing to the rapid association between aldehydes and amines, an aldehyde has been elegantly employed as an amine-capture auxiliary in aldehyde capture ligation (ACL). In ACL, a C-terminal selenobenzaldehyde ester can interact with the N-terminus of a peptide/protein. A native amide bond linkage is formed following a Se→N acyl shift. The ACL has been used for site-specific N-terminal modification of ubiquitin (Table 1A).31
A recently introduced reaction is ABAO (2-aminobenzamidoxime) ligation. ABAO combines an aniline moiety for iminium-based activation of the aldehyde with a nucleophilic group at the ortho-position to the amine for intramolecular ring closure. In addition to the rapid condensation reaction kinetics (up to 40 M−1 s−1), the condensation forms a fluorescent dihydroquinazoline derivative, making it possible to develop fluorogenic aldehyde-reactive probes.26 Alkyl aldehydes can also efficiently couple with aryl diamines under mild conditions (RT, neutral aqueous solution) in the presence of Cu(II) or Zn(II) ions via an oxidative condensation process.27 This reaction forms stable benzimidazole linkages and has been utilized to label the T4 lysozyme protein with an aldehyde dye (Table 1A).
With respect to labeling biomolecules in live cells or organisms, carbonyl compound-related condensations have not been widely used. This is because the catalysts are usually toxic, and endogenous ketones and aldehydes, e.g. glucose and pyruvate, would interfere with the labeling reaction. Nevertheless, ketones and aldehydes are generally not present on the cell surface. Therefore, carbonyls serve as useful chemical handles for labeling biomolecules on the cell surface using hydrazide or aminooxy probes.20,79–83
CuAAC is a hallmark of bioorthogonal chemistry that was reported independently by Sharpless36 and Meldal85 in 2002. Its application as a bioorthogonal reaction revolutionized our ability to modify and manipulate proteins.86,87 CuAAC becomes popular mainly due to the following reasons: (1) the azide and alkyne groups are highly specific toward each other but remain inert to other chemically active molecules in live systems; (2) the reaction produces a regioselective 1,4-triazole product which is stable and inert; (3) CuAAC exhibits fast reaction kinetics (∼3 M−1 s−1 in the presence of 50 μM Cu(I) and 50 μM TBTA)37 and various ligands have been developed to stabilize Cu(I) and further increase the reaction speed.
Cu ions catalyze the production of reactive oxygen species, leading to cytotoxicity.88 This limits the application of CuAAC in living systems. By choosing an appropriate ligand, CuAAC can be biocompatible with minimal cytotoxicity while showing an increased reaction rate.89 A panel of these ligands is summarized in Scheme 1A. Copper-chelating azides bring the Cu(I) ion into close proximity and thereby significantly increase the reaction rate (Scheme 1B).90 The ligand BTTP (3-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]propanol) stabilizes the Cu(I) ion while speeding up the click reaction (Scheme 1A). In addition, its complex with Cu(I) is cell permeable and non-cytotoxic, facilitating CuAAC in live E. coli cells.91 Using this reaction, an environment-sensitive fluorogenic fluorophore (alky-4DMN) was site-specifically introduced into HdeA in both the periplasm and cytoplasm of E. coli. HdeA, an acid-stress chaperone that adopts pH-dependent conformational changes, was genetically encoded with an azide-carrying unnatural amino acid ACPK at residue 58 within its pH-responsive region. The resulting hybrid pH indicator enables compartment-specific pH measurement to determine the pH gradient across the E. coli cytoplasmic membrane (Scheme 1C).91
Fluorogenic azide probes display substantial fluorescence enhancement upon cycloaddition reaction and therefore confer labeling with minimal background (Fig. 1A). Based on photo-induced electron transfer (PET) mechanism, highly fluorogenic blue-emissive azidomethyl substituted anthracene (A) and its analogues have been generated.92 Substitution at the 3 or 7 position of coumarin has a strong impact on its fluorescence properties. Guided by this principle, 3-azido substituted coumarins (B) show an 80-fold increase in fluorescence intensity upon the cycloaddition reaction.93 The quantum yield (Φ, QY) of a probe after a click reaction can be calculated by density function theory (DPF), allowing rational design of fluorogenic azide probes.94 Using this approach, the green-emission azido-fluorescein (C) has been designed, which exhibits a fluorescence enhancement of 29 to 34-fold upon cycloaddition with different alkynes.95 Fluorogenic green- to far red-emitting CalFluors enable sensitive detection of biomolecules under no-wash conditions (D, E, F).96
An important application of CuAAC lies in the target identification of biologically active small molecules in biomedical research and drug discovery.97 A bioactive compound is typically derivatized with a photo-crosslinking moiety (e.g. diazirine) and a terminal alkyne, which is denoted as an affinity-based probe (AfBP) (Fig. 1B). By photo-crosslinking, the AfBP probe in situ captures the protein target and forms a stable protein–ligand complex in the cell. Subsequent labeling of the target proteins using azide probes is followed by separation via gel-electrophoresis and determination via mass spectroscopy.97 Among many bioorthogonal groups, a terminal alkyne is a well suited tag as it is small with minimal interference of protein–ligand interactions, chemically inert, and can be easily modified via CuAAC.
Although the cytotoxicity of Cu(I) can be reduced by using ligands, copper-free click chemistry is more straightforward. Strain-promoted azide–alkyne cycloaddition (SPAAC) and other copper-free click reactions38 without the requirement for a metal catalyst have been developed (Table 2A). The first SPAAC reaction for protein labeling was reported by Bertozzi and coworkers in 2004 using a strained cyclooctyne.39 However, the reaction rate is slow (k = 2.4 × 10−3 M−1 s−1), which is comparable to Staudinger ligation (k = 3 × 10−3 M−1 s−1). To date, new variants of strained alkynes have been developed with improved properties, such as enhanced specificity, reduced lipophilicity98 and increased reaction rates (Table 2B).
There are several ways to improve strained alkynes.99 The first strategy is to modulate the electronic properties by introducing electron-withdrawing (EW) groups, e.g. fluorine, near the triple bond.100 Examples include MOFO101 and DIFO.40 The second approach is to fuse the cyclooctynes with rigid aromatic rings, leading to an enhancement of reactivity by increasing ring strain. The ring-fused cyclooctynes, including DIBO,41,102 DIBAC,103 COMBO104 and BARAC,42 show a 25–400-fold increase in the reaction rate.105 However, synthesis of these cyclooctynes is usually laborious. Bicyclononyne (BCN), a cyclopropane-fused cyclooctyne, shows relatively fast reaction kinetics (0.3–1 M−1 s−1) toward azides and can be facilely prepared in only three steps.43 Using an electron-deficient azide, e.g. 4-azido-1-methylpyridin-1-ium iodide, the cycloaddition reaction rate with BCN can be further increased to 2–2.9 M−1 s−1.44 By introducing both EW groups and ring strain, difluorobenzocyclooctyne (DIFBO) shows only a moderate increase of reactivity (0.22 M−1 s−1) with a significant reduction in stability.106 The third strategy is to shorten the ring size as exemplified by the 7-membered tetramethylthiazacycloheptyne (TMTH),107 cyclohexyne and cyclopentyne.108 However, these compounds have poor stability (Table 2B). The copper-free click reactions have been used for antibody-free western blot analysis,109 visualization of glycosylation on cell surfaces110 and protein labeling inside live cells.111
Tetrazines are highly reactive so that they can also readily react with strained alkynes, isonitriles52 and even terminal alkenes. Among these reactions, the reaction between tetrazine and BCN or cyclopropene has proven to be the most useful. Tetrazine reacts with BCN in a rapid fashion with a reaction rate constant of 3000 M−1 s−1.46 In comparison to TCO that requires complicated synthetic procedures, BCN is readily available through organic synthesis. Compared to BCN and TCO groups, cyclopropene (Cpp) is much smaller, and therefore has been employed as a “minimalist” tag for live-cell imaging and affinity-based protein labeling.114
Alkenes and alkynes display different reactivity toward tetrazine (Table 3B).116 sTCO features a cyclopropane ring, which brings additional strain and increases the reaction rate of the cycloaddition up to 160-fold in comparison to TCO.116 The carbamate bond near the trans-double bond in TCO* reduces the chance of nucleophilic attack. Thus, TCO* shows better stability than TCO with only 2-fold reduction in reactivity toward tetrazine.117 The reaction rate of BCN with tetrazine is about 10 times slower than that of TCO.46 Norbornene is ca. 10000 times less reactive than TCO, as it is more bulky and exhibits less ring strain than TCO.46,47 Other alkenes, including Cpp,48,49,114 acylazetine50 and terminal alkene,51 are less reactive, and therefore can react only with highly reactive tetrazines.
Different tetrazines show varied reactivity toward strained alkenes (Table 3C). Studies have been conducted to evaluate the reactivity and stability of tetrazines.115 The introduction of EW substituents can substantially increase the reaction rate. However, it is a double-edged sword. Increase in the reactivity of tetrazine often results in the reduction of stability and lifetime in the serum. ED substituents on the other hand decrease the reactivity of tetrazine.115 Besides electronic effects, steric effects also play a crucial role. In general, there is a trade-off between reactivity and stability. The rate constants of tetrazine reactions with TCO as well as their stability are summarized in Table 3C.
Green- and red-emitting fluorophores display electronic interactions with tetrazine chromophores that have absorption maxima at 500–525 nm. As a consequence, tetrazine-conjugated fluorophores often show reduced fluorescence. After a cycloaddition reaction, tetrazine is deconjugated and loses its quenching capability. Hence, many tetrazine-fluorophores feature fluorogenic properties, such as BODIPY-FL, Oregon Green 488, BODIPY-TMR, VT680,118 TAMRA, and fluorescein.119 These probes show a moderate turn-on ratio (up to 20-fold) (Fig. 2A). By adapting through-bond energy transfer (TBET) for fluorescence quenching, Weissleder and coworkers developed green-emitting BODIPY-tetrazine probes with up to 1600-fold turn-on ratio120 and blue-emitting coumarin-tetrazine probes with up to 11000-fold fluorescence enhancement.121 The tetrazine moiety is attached at the meta-position to the fluorophore on a rigid phenyl ring (Fig. 2A). Under these conditions, the tetrazine group is perpendicular to the fluorophore moiety, leading to the collinear alignment of two dipoles.121
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Fig. 2 (A) Fluorogenic tetrazine-fluorophores; (B) dual protein labeling using orthogonal DAINV pairs for super-resolution microscopy (SRM). |
To achieve dual- or multi-labeling of proteins, mutually orthogonal reactions are desirable.122 By fine-tuning the DAINV reactions, “selectivity enhanced” DAINV reactions have been applied to sequential dual-color labeling of insulin receptors (IRs) and virus-like particles (VLPs) on the cell surface of HEK293T cells, facilitating dual-color super-resolution microscopy (Fig. 2B).117
In addition to CuAAC, SPAAC and DAINV, which are popularly used for protein labeling, other dipolar cycloaddition reactions are summarized in Table 1D, including strain-promoted alkyne-nitrone cycloaddition (SPANC),53 diazo-strained alkyne cycloaddition,54,55 nitrile oxide-norbornene cycloaddition,58 quadricylane ligation,57 TQ-ligation,59,60 strain-promoted sydnone-BCN cycloaddition,56 and plenty of photo-triggered cycloaddition reactions, such as the tetrazole-alkene photo-click reaction61,62 and azirine ligation.63
Terminal olefins can undergo an oxidative Heck reaction with boronic acid in the presence of Pd(OAc)2/BIAN catalysts. This reaction has been used for the site-specific labeling of 4-oxalocrotonate tautomerase (4-OT).67 Olefin metathesis is the redistribution of fragments of alkenes by scission and regeneration of carbon–carbon double bonds.117,118 Hence, in order to avoid undesired cross coupling products, a terminal olefin and a more reactive thiovinyl ether are required.126 Water soluble ruthenium catalysts have been developed to mediate the reaction in aqueous solution.126,127
Chemical protein labeling not only makes proteins visible but also renders them controllable. Recently, palladium catalysts have been used to manipulate protein function in cells. Pd-mediated cleavage of the propargyl carbamate group leads to the generation of a free lysine residue. The protected lysine analogue can be genetically and site-specifically incorporated into a protein using an unnatural amino acid (UAA) mutagenesis technique (discussed in the later section). This strategy enables protein activation in living cells by decaging the lysine residue located at the active site of a protein, and has been utilized to elucidate the virulence mechanism of a bacterial type III effector protein in its host cells (Fig. 3A).68 A ruthenium-catalyzed cleavage reaction has been used for the cleavage of allyl carbamate to unmask caged Rhodamine 110 (R110) inside living cells (Fig. 3B).128
Adjacent cysteines are usually oxidized to form disulfide bonds under non-reducing conditions. In these cases, the solvent accessible disulfide bond can be first gently reduced and subsequently “intercalated” by mono-sulfone reagents. This approach permits the site-specific PEGylation of a variety of therapeutic proteins, including human interferon α-2b and antibody fragments.74 A more recent approach employed 1,3-dichloroacetone (DCA) to introduce a reactive ketone tag, enabling subsequent oxime ligation (Table 1F).75
N-terminal cysteine displays unique reactivity. Proteins carrying an N-terminal cysteine can undergo native chemical ligation with thioester probes and chemoselective ligation with aldehydes to form thiazolidines.132 N-terminal cysteine can also specifically react with cyanobenzothiazole (CBT) derivatives at a fast reaction rate (9 M−1 s−1).72 The reaction of CBT compounds with D-cysteine is highly biocompatible and has been used for bioluminescent imaging of protease activity in live mice.133
Palladium-tolyl complexes using 2-dicyclohexylphosphino-2′,6′-diisopropylbiphenyl (RuPhos) as the ligand have been developed to mediate efficient and highly selective cysteine conjugation reactions under biocompatible reaction conditions.70 At pH 7.5, the rate of a palladium-medicated reaction is comparable to that of the maleimide reaction. This bioconjugation strategy has demonstrated its broad utility for making stapled peptides, site-specific labeling of proteins with a coumarin fluorophore, and the preparation of antibody-drug conjugates (ADCs) (Scheme 2).70
Tag name (size in AA) | Probe | Enzyme | Cellular labeling | Features | Reference(s) |
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AA: amino acids. (A) BG: O6-benzylguanine; BC: O2-benzylcytosine; BG-FL: BG-fluorescein conjugate; BC-FL: BC-fluorescein conjugate. (E) ACP: acyl carrier protein; PPTase: 4′-phosphopantetheinyl transferase; CoA: coenzyme A; PCP: peptide carrier proteins. | |||||
A. Metal chelation based peptide tags | |||||
His-tag (6): HHHHHH |
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No | Cell surface/intracellular | Ni(II) is toxic to cells and quenches fluorescence; intracellular labeling is possible using cell-penetrating multivalent chelator carrier complexes | Guignet (2004)152 |
Wieneke (2014)153 | |||||
His-tag (6): HHHHHH | Zn(II) complex: HisZiFit | No | Cell surface | Zn(II) is non-toxic; the zinc complex is membrane-impermeant | Hauser (2007)154 |
D4 tag (4): (DDDD)n, n = 1–3 | Multinuclear Zn(II) complexes: Zn(II)-DpaTyrs probe | No | Cell surface | Spectroscopic change upon chelation; D4 tag/Zn(II)-DpaTyrs pair is orthogonal to the His tag/Ni(II)-NTA pair | Ojida (2006)155 |
B. Self-labeling peptide tags | |||||
CCXXCC (6) (tetracysteine) |
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No | Intracellular | Fluorogenic; non-specific reactions and interactions to endogenous thiols; biarsenical probes may be cytotoxic | Griffin (1998)156 |
Gaietta (2002)157 | |||||
SSPGSS (6) (tetraserine) |
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No | Cell surface | Fluorogenic; reduced background staining; cytotoxic; off-target labeling of endogenous tetraserine motif; RhoBo is cell permeable | Halo (2009)158 |
HyRe tag (11): HKSNHSSKNRE |
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No | Cell surface | Neutral pH; imine byproduct formation at pH <6 | Eldridge (2011)159 |
SpyTag (13) | SpyCatcher protein domain (138 AA) | No | Cell surface | k = 1.4 × 103 M−1 s−1; high yield under diverse conditions of pH, temperature and buffer; formation of a stable amide bond linkage; not traceless | Zakeri (2012)160 |
E3 tag (21): (EIAALEK)3 | K3 peptide (21 AA): (KIAALKE)3 | No | Cell surface | E3/K3 coiled-coil template induces proximity-driven rhodium(II)-catalyzed modification or acyl transfer reaction (t1/2 = 10 min) | Chen (2011)161 |
Reinhardt (2014)162 | |||||
C. Ligand binding domains (LBDs) | |||||
FKBP’ (108) (FKBP12_F36 V) |
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No | Intracellular | Specific binding between FKBP’ and SLF’; non-covalent | Clackson (1998)163 |
Marks (2004)164 | |||||
eDHFR (159) |
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No | Intracellular | Tight and specific binding between eDHFR and TMP; non-covalent/covalent; non-cytotoxic; affinity conjugation is rapid (t1/2 = 1.8 min) | Miller (2005)165 |
Liu (2014)166 | |||||
PYP-tag (125) |
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No; PYP tag is derived from a purple bacteria | Intracellular | PYP-tag covalently binds to the thioester derivative of cinnamic acid/coumarin; fluorogenic; up to k = 3950 M−1 s−1 | Hori (2009/2013)167,168 |
D. Self-labeling enzymatic domains | |||||
SNAP tag (182) |
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hAGT (mutated human O6-alkylguanine-DNA alkyltransferase) | Intracellular | BG: k = 0.3 × 104 M−1 s−1; BG-FL: k = 2.8 × 104 M−1 s−1; removal of guanine after labeling | Keppler (2003)169 |
CLIP tag (182) |
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Intracellular | BC-FL: k = 0.1 × 104 M−1 s−1. SNAP-tag and CLIP-tag possess orthogonal substrate specificities; removal of cytosine after labeling | Gautier (2008)157 | |
BL-tag (263) |
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Non-catalytic β-lactamase variant | Cell surface | k = 7.8 × 104 M−1 s−1, E166N mutant of β-lactamase; fluorogenic labeling | Mizukami (2009)170 |
Halo tag (290) |
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Dha (mutated haloalkane dehalogenase) | Intracellular | k = 2.7 × 106 M−1 s−1; covalent; probe easily accessible by synthesis | Los (2008)171 |
Cutinase (197) |
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A small globular serine esterase of 22 kDa | Cell surface | Cutinase is labeled by the covalent inhibitor, p-nitrophenyl phosphonate (pNPP) | Bonasio (2007)172 |
E. Enzymatic modifications | |||||
ACP (77)/S6(12)/A1(12)/PCP(80)/ybbR tag(11) | Coenzyme A (CoA) derivatives | PPTase (AcpS or Sfp) | Cell surface | AcpS/Sfp catalyzes the transfer of a CoA-activated probe to the Ser residue of the tag. kcat = 5.5 × 10−4–0.32 s−1, kcat/Km = 5.3–4550 M−1 s−1 | George (2004)173 |
Yin (2005)174 | |||||
Zhou (2007)175 | |||||
AP tag (15): GLNDIFEAQ![]() |
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Biotin ligase (BirA) | Cell surface | Ketobiotin is a substrate for BirA and is incorporated into the Lys residue of the peptide tag; labeling time >20 min | Chen (2005)83 |
Q-tag (7): PKPQQFM | Cadaverine derivatives | Transglutaminase (TGase) | Cell surface | TGase mediates the formation of an amide bond between the amine of cadaverine and the Glu residue of the Q-tag | Sato (1996)176 |
Lin (2006)177 | |||||
LAP tag (13–22) |
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Lipoic acid ligase (LplA)/LplA mutant | Cell surface/intracellular | k cat = 0.048 s−1 for the conjugation of azido lipoic acid; coumarin addition by W37VLplA: kcat = 0.019 s−1, kcat/Km = 3.8 × 102 M−1 s−1; W37ILplA: kcat = 0.016 s−1, kcat/Km = 0.6 × 102 M−1 s−1 | Fernandez-Suarez (2007)178 |
L![]() |
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Formylglycine generating enzyme (FGE) | Cell surface | FGE catalyzes the transformation of a Cys of the peptide tag to a formylglycine | Carrico (2007)79 |
LPX![]() |
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Sortase A (SrtA) | Cell surface | SrtA cleaves the peptide at the Thr-Gly site, and attaches a labeled polyglycine peptide | Popp (2007)179 |
CAAX (4) |
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Protein farnesyltransferase (PTFase) | Cell surface | CAAX is also present in endogenous proteins; an alkyne group is introduced for click labeling | Wollack (2009)180 |
Split intein [IntC (35) or IntN (25)] | Another half of split intein [IntN (100) or IntC (104)] | Reconstituted split inteins | Cell surface/intracellular | Intein-mediated protein trans splicing removes intein after reaction (traceless); yield 55–90% and k = 0.9–4.7 s−1 using Npu DnaE intein and AceL-TerL intein | Giriat (2003)181 |
Schütz (2014)182 | |||||
TITS![]() |
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AnkX from Legionella pneumophila | In vitro | k cat/Km = 0.9–5 × 102 M−1 s−1, covalent labeling can be removed by Lem1 | Heller (2015)183 |
Tub-tag (14): VDSVEGEGEEEGEE |
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Recombinant tubulin tyrosine ligase (TTL) | In fixed cell | Yield up to 99% with moderate enzyme concentrations and short reaction times | Schumacher (2015)184 |
F. Incorporation of UAA as a “minimalist” tag | |||||
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BCN-RS/tRNACUA | Cell surface/intracellular | UAA bearing BCN or TCO can be site-specifically incorporated to proteins in live cells for labeling with tetrazine dyes | Lang (2012)46 |
Prominent applications of self-labeling peptide tag approaches involve visualization of newly synthesized proteins and tracking of protein trafficking in live cells. This is readily achieved via the “pulse-chase” technique. The old populations of proteins were pulse-labeled by green-emitting FlAsH, while the newly synthesized proteins were chased by red-emitting ReAsH. Consequently, old and new copies of an individual protein were labeled using two colors. In one example, this approach was used to elucidate the mechanism of connexin assembly and turnover in HeLa cells.191 In another example, the approach was employed to study AMPA receptor (AMPAR) trafficking. Regulation of AMPA receptor (AMPAR) trafficking is important for neural plasticity. GluR1 and GluR2 are two AMPAR subunits that play a key role in the activity-dependent trafficking of the AMPARs during long-term potentiation (LTP) and depression (LDT). In order to examine the trafficking and synthesis of GluR1 and GluR2, a tetracysteine motif (EAAAREACCRECCARA) was attached at the C-termini. ReAsH-EDT2 was first applied and after 6–8 h, FlAsH-EDT2 was applied to cells expressing tetracysteine-tagged GluR1 or GluR2 (Scheme 4B). In this case, the red ReAsH-EDT2 labels all preexisting GluR1/2 subunits, while the green FlAsH-EDT2 labels those AMPRAR subunits synthesized during the 6–8 h chase period. The measurements suggested that both GluR1 and GluR2 are synthesized in dendrites and that an activity blockade enhances the dendritic synthesis of GluR1 but not GluR2.195
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Scheme 4 Dual-color “pulse-chase” labeling of neurons using ReAsH and FlAsH for the visualization of the synthesis and trafficking of AMPA receptors. |
Because of the non-covalent binding, the labeling via the FKBP’ or eDHFR tag is reversible. In order to achieve a stable labeling, the affinity conjugation approach has been introduced. A cysteine mutation is introduced in the proximity of the TMP binding site on eDHFR, which can be specifically labeled by TMP-acryloyl probes due to a proximity-induced effect.196 The reaction of a mildly reactive acryloyl group with other thiols in the cell is minimal under certain conditions.197 Based on the affinity conjugation principle, a rapid and fluorogenic TMP-AcBOPDIPY probe is developed with a half-life of less than 2 min for covalent labeling (Fig. 4A).166 Intracellular proteins fused with eDHFR_N23C were rapidly labeled by the TMP-AcBOPDIPY probe under no-wash conditions (Fig. 4B). In addition, the chemical probe displays a superior dynamic range in fluorescence lifetime imaging microscopy (FLIM) for intracellular FRET studies.
Recently, a more versatile “tagging-then-labeling” approach has been realized, enabling efficient introduction of bioorthogonal groups into proteins for bioorthogonal labeling in live cells. The TMP-AcAz ligand incorporates an azido group to proteins fused with the eDHFR tag (Fig. 4C). Subsequently, strain promoted cycloaddition reactions using DBCO- or BCN-conjugates facilitate protein labeling with various probes inside live cells (Fig. 4D).111
The eDHFR tag has been used for live-cell imaging of protein–protein interactions (PPIs) between the first PDZ domain of ZO-1 (fused with eDHFR) and the C-terminal YV motif of claudin-1 (fused with GFP) using time resolved luminescence resonance energy transfer (LRET) technique (Fig. 5).198 Conventional FRET imaging suffers from fluorescence breed-through, leading to high background. In the LRET approach, background signals from cellular auto-fluorescence and direct excitation of GFP were effectively eliminated by imposing a time delay of 10 μs between excitation and detection.
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Fig. 5 Time resolved LRET for live-cell imaging of protein–protein interactions using the eDHFR tag. |
Photoactive yellow protein (PYP) is a small (14 kDa) soluble protein found in several purple bacteria. It binds to a natural cofactor, the CoA thioester of 4-hydroxycinnamic acid through transthioesterification with Cys69. It also binds to the thioester derivative of coumarin-3-carboxylic acid. Since PYP and its ligands do not exist in animal cells, they can therefore be employed for bioorthogonal labeling of proteins (k = 1.1–124 M−1 s−1).167,168
O 6-Alkylguanine transferase (AGT), a human DNA repair protein, has been used as a self-labeling tag.169,199 The reaction involves the irreversible transfer of the alkyl group of O6-benzylguanine (BG) derivatives to the reactive cysteine residue within the enzyme to generate a covalently modified protein. More efficient AGT mutants, termed SNAP-tags (19 kDa), have been developed.169 An orthogonal AGT-based tag, termed a CLIP-tag, reacts specifically with O2-benzylcytosine derivatives.157
Single-molecule imaging often requires photo-stable and bright organic dyes, which are made possible using chemical protein labeling approaches. The spliceosome is a complex machine responsible for removing introns from the precursors of messenger RNAs (pre-mRNAs). The SNAP-tag has been exploited in combination with the eDHFR-tag to enable single-molecule imaging of the spliceosome in yeast cell extracts (Fig. 6A). The SNAP- and eDHFR-tags facilitate labeling pairs of the small nuclear ribonucleoprotein (snRNP) components of the spliceosome in cell extracts with bright organic dyes (TMP-Cy5 and BG-DY549), thereby enabling imaging of their assembly on individual pre-mRNAs. The measurements revealed that individual spliceosomal subcomplexes associate with pre-mRNA sequentially through an ordered pathway and that subcomplex binding is reversible.200
Halo-tag is a modified haloalkane dehalogenase that covalently binds to synthetic chloroalkane derivatives.171,201 Halo-tag is commercially available and has been widely used to label proteins in cells. This approach has been applied to small molecule-induced protein degradation, namely, HaloPROTACs. PROTACs are a class of heterobifunctional molecules that link a ligand of E3 ligase to a ligand for a protein of interest (POI).202 PROTACs recruit the E3 ligase to the POI, resulting in its ubiquitination and subsequent degradation by the proteasome. A bifunctional HaloPROTAC contains a chloroalkane and a hydroxyproline derivative which binds an E3 ligase VHL. The compound induces binding between the HaloTag7 fusion protein and the E3 ligase, leading to the degradation of the HaloTag7 fusion proteins by the proteasome (Fig. 6B).203
β-Lactamase is a small bacterial enzyme (29 kDa) that hydrolyzes β-lactam antibiotics. The E166N mutation of β-lactamase leads to the accumulation of acyl-enzyme intermediates due to the dramatic suppression of deacylation. The mutant β-lactamase, termed BL-tag, and β-lactam probes have been used for the covalent labeling of proteins in cells.170 Enormous efforts that have been made on β-lactam antibiotics render it possible to design various β-lactam probes.204 Cephalosporin-based probes featuring substituent elimination facilitate the development of fluorogenic probes.205 Other examples in this category include cutinase172 and catalytic antibodies (Abs)206 (Table 4D).
Many other enzymes have also been used for the selective modification of proteins, including phosphopantetheine transferase (AcpS or Sfp),173–175,208 transglutaminases (TGases),176,177 sortase (SrtA),179 protein farnesyl transferase (PFTase),180 glycosyltransferase,209N-myristoyl transferase (NMT)210 and tubulin tyrosine ligase (TTL)184 (Table 4E). The advantages of enzymatic modifications lie in the small size of the tag and the highly efficient reactions. However, many of the substrates are not cell permeable and therefore are not suited for intracellular labeling.
The enzymatic modification approach has been elegantly applied to spatially-resolved proteomic mapping in living cells.211 An ascorbate peroxidase (APEX) fused with a “mito” sequence was targeted to the mitochondrial matrix. Labeling was initiated by the addition of biotin–phenol and H2O2 to live cells. The resulting phenoxyl radicals are short-lived and membrane-impermeant and therefore only label neighboring endogenous proteins. The biotinylated proteins were recovered with streptavidin-coated beads and identified using mass spectrometry (Fig. 7). This approach led to the identification of 495 proteins within the human mitochondrial matrix, including 31 proteins that were not previously linked to mitochondria.
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Fig. 7 Labeling the mitochondrial matrix proteome in living cells using mito-APEX and biotin-phenol. |
Despite successful applications in selective protein labeling, numerous challenges remain in bioorthogonal chemistry. Firstly, many bioorthogonal functional groups are not truly “bioorthogonal”. For instance, strained alkynes may react with free thiols in live systems. Aldehyde and ketone functionalities are also present in many metabolites in living systems. Secondly, some of the bioorthogonal groups are too large and lipophilic, such as cyclooctynes and cyclooctenes, causing non-specific staining and reduction of effective reactants in live cells. Thirdly, the instability of tetrazine, phosphine and other groups in live cells during a prolonged incubation time leads to the deactivation of the bioorthogonal moieties. Fourthly, some metal-catalyzed reactions are incompatible with living conditions, as exemplified by the cytotoxicity of the copper ion used for CuAAC. To address these issues, the development of new bioorthogonal chemistry is definitely required. Since there are few “perfect” bioorthogonal reactions available, one has to carefully consider both the pros and cons of each reaction in order to identify the most suited ones for a particular application.
Chemical tagging approaches allow for the incorporation of probes or bioorthogonal handles into proteins in cells and organisms. However, most of these approaches rely on exogenous expression of the POI with tags or UAAs. Although the investigation of exogenous proteins is useful for unraveling biological processes, the advance in selective labeling of endogenous proteins should facilitate the proteomic analysis of cellular organelles and protein complexes, target identification and diagnosis. An advantage of chemical probes over FPs lies in the flexibility of modifications on organic dyes. Therefore, the development of new organic dyes with special properties, e.g. photo-switchable, activatable, highly fluorogenic, bright, far-red emissive, etc., should substantially help to understand the biological mechanisms of proteins in the context of living systems.
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