Fluorophore targeting to cellular proteins via enzyme-mediated azide ligation and strain-promoted cycloaddition

Article abstract of DOI:10.1021/ja208090p

Methods for targeting of small molecules to cellular proteins can allow imaging with fluorophores that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the Escherichia coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 min. Second, we evaluated a panel of five different cyclooctyne structures and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655.

Full text of DOI:10.1021/ja208090p

Article
pubs.acs.org/JACS
Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated
Azide Ligation and Strain-Promoted Cycloaddition
Jennifer Z. Yao,† Chayasith Uttamapinant,† Andrei Poloukhtine,‡ Jeremy M. Baskin,§ Julian A. Codelli,§
Ellen M. Sletten,§ Carolyn R. Bertozzi,§,∥,⊥ Vladimir V. Popik,‡ and Alice Y. Ting*,†
†Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue. Cambridge, Massachusetts 02139,
United States
‡Department of Chemistry, University of Georgia, 212 Carlton Street. Athens, Georgia 30602, United States
∥
§Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical Institute, University of California,
Berkeley, California 94720, United States
⊥The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
S
* Supporting Information
ABSTRACT: Methods for targeting of small molecules to
cellular proteins can allow imaging with fluorophores that are
smaller, brighter, and more photostable than fluorescent
proteins. Previously, we reported targeting of the blue
fluorophore coumarin to cellular proteins fused to a 13-
amino acid recognition sequence (LAP), catalyzed by a mutant
of the Escherichia coli enzyme lipoic acid ligase (LplA). Here,
we extend LplA-based labeling to green- and red-emitting
fluorophores by employing a two-step targeting scheme. First,
we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly
quantitative yield after 30 min. Second, we evaluated a panel of five different cyclooctyne structures and found that fluorophore
conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in
cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental,
and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our
optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple
mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655.
one of its three natural acceptor proteins (Figure 1A).12 We
INTRODUCTION
■
used in vitro evolution to engineer a 13-amino acid replacement
for these acceptor proteins, called LAP13 (LplA Acceptor
Peptide), which can be genetically fused to any protein of
interest. We then showed that mutagenesis of the lipoic acid
binding pocket could allow LplA to ligate unnatural small
molecules instead of lipoic acid, including 7-hydroxycoumarin,7
7-aminocoumarin,14 Pacific Blue,15 and aryl azide.16 Coumarin
ligation has been demonstrated in living cells and used to image
various cytoskeletal proteins and neurexin.7,14,15
Though PRIME with coumarin ligase is versatile and specific,
coumarin is a blue fluorophore with excitation and emission
maxima of 387 and 448 nm,7 which are not optimal for live cell
imaging. Red-shifted fluorophores are more desirable as they
enable higher signal to background ratios due to their greater
separation from cellular autofluorescence. Furthermore,
coumarin does not exhibit the useful photoswitching properties
and higher photon count possessed by cyanine and ATTO
The use of small-molecule fluorophores in live cells to replace
fluorescent proteins has grown in recent years due to the need
for improved photophysical properties for advanced imaging
modalities such as single-molecule and super-resolution
imaging,1,2 and also because of the availability of new methods
for targeting such probes to cellular proteins.3 Our lab has been
working to develop better protein labeling methods, because
existing techniques still have significant shortcomings.
HaloTag4 and SNAP/CLIP5 methods offer high labeling
specificity but the tags are large, like fluorescent proteins, and
can interfere with the trafficking and function of the proteins to
which they are fused. FlAsH6 uses a small peptide tag for
labeling, but the specificity is imperfect, as we7 and other labs8
have shown. Several other methods, such as sortase,9 Sfp/
AcpS,10 and aldehyde tag11 are restricted to labeling of cell
surface proteins rather than intracellular proteins.
Our lab has developed a protein labeling method based on
enzyme-catalyzed probe ligation, called PRIME7 (PRobe
Incorporation Mediated by Enzymes). The platform for this
method is the Escherichia coli enzyme lipoic acid ligase (LplA),
which normally conjugates lipoic acid to a lysine side chain of
Received: September 1, 2011
Published: January 4, 2012
© 2012 American Chemical Society
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Figure 1. Fluorophore targeting via LplA-catalyzed azide ligation followed by strain-promoted azide-alkyne cycloaddition. (A) Top: natural ligation
of lipoic acid catalyzed by wild-type LplA.12 Bottom: two-step fluorophore targeting used in this work. First, the W37ILplA mutant ligates 10-
azidodecanoic acid (“azide 9”) onto the 13-amino acid LplA acceptor peptide (LAP).13 Second, the azido moiety is chemoselectively derivatized
using a cyclooctyne−fluorophore conjugate, via strain-promoted, copper-free [3 + 2] cycloaddition. The red circle represents any fluorophore or
probe. (B) Screening to identify the best LplA mutant/azide substrate pair. The table shows percent conversions of LAP to the LAP−azide product
conjugate. Wild-type LplA and six W37 point mutants were screened against four azidoalkanoic acid substrates of various lengths. N.D. indicates that
product was not detected. Screening was performed with 100 nM ligase, 600 μM LAP, and 20 μM azide substrate for 20 min at 30 °C. Conversions
were measured in duplicate. Note that W37SLplA was active with the natural substrate, lipoic acid (data not shown). The starred entries in the table
were further evaluated in Figure 2.
dyes, which are necessary for super-resolution imaging by
PALM and STORM techniques.1,2,17
scheme inside living cells (Figure 1A, bottom). First, the LplA
enzyme was expressed inside the cell instead of exogenously
added to the cell media. Second, the azide ligation step was
optimized to give maximum yield, while leaving minimal
residual azide probe, which would interfere with the subsequent
derivatization if it could not be completely removed from cells.
This optimization required the use of a different LplA mutant/
azide probe pair with improved kinetic properties. Third, we
developed a protocol to remove excess unconjugated alkyl azide
as completely as possible. Finally, we investigated a variety of
cyclooctyne structures to select the ones with the best reactivity
and specificity for LAP−azide.
Our experiments ultimately yielded a two-step targeting
protocol using W37ILplA to ligate a 10-azidodecanoic acid
(“azide 9”) substrate to LAP inside living cells with nearly
quantitative yield in 30 min. Excess unligated azide is removed
with two rounds of media changes over 1 h. Out of five
cyclooctyne structures tested, the aza-dibenzocyclooctyne,
ADIBO21,22 was generally the best, giving the highest signal
for a variety of LAP fusion proteins in multiple cell types.
However, because ADIBO is fairly hydrophobic, we found that
it gives background when conjugated to more hydrophobic
fluorophores such as ATTO 647N, in which case an alternative,
less hydrophobic cyclooctyne, MOFO,20 is preferred. Our two-
step targeting protocol was successfully used to label multiple
LAP fusion proteins in living mammalian cells with small-
molecule fluorophores, ranging from fluorescein to ATTO
647N.
The goal of this work is to generalize PRIME for labeling of
intracellular proteins with diverse fluorophore structures. We
wanted a general strategy for targeting to intracellular LAP-
fused proteins any fluorophore that can cross the cellular
membrane and exhibit minimal nonspecific binding or
reactivity to endogenous proteins.
To accomplish this, we needed an alternative approach to
lipoic acid binding pocket mutagenesis. Most green and red
fluorophores such as fluorescein, rhodamine, cyanine, and
ATTO dyes are much larger than coumarin, the largest
unnatural substrate so far to be recognized by an LplA mutant.7
The crystal structure of LplA in complex with lipoyl-AMP ester,
the intermediate of the natural ligation reaction, shows that the
lipoyl moiety is completely enclosed within a binding pocket in
the center of the enzyme.18 Engineering an “exit tunnel” from
this binding pocket to accommodate structures much larger
than lipoic acid or coumarin is not straightforward.
Our approach was instead to use a two-step targeting scheme
in which we first use LplA to ligate a “functional group handle”
to a LAP fusion protein and then chemoselectively derivatize
the functional group on LAP with a suitably derivatized
fluorophore. We have previously accomplished such two-step
labeling with LplA for cell surface proteins,19 using an alkyl
azide substrate for wild-type LplA and derivatizing the resulting
LAP−azide conjugate with a monofluorinated cyclooctyne20
(MOFO)−fluorophore conjugate. Here, we addressed several
challenges to successfully implement a similar two-step labeling
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Figure 2. Identification of the best LplA mutant/azide substrate pair for intracellular protein labeling. (A) Labeling protocol. HEK cells coexpressing
LplA and LAP-tagged BFP were labeled with azide 7 or azide 9 for 1 h, washed for 1 h, then labeled with monofluorinated cyclooctyne (MOFO)
conjugated to fluorescein diacetate for 10 min. Thereafter, cells were washed again for 2 h to remove excess unconjugated fluorophore. (B) Images of
HEK cells labeled as in (A), with different LplA mutant/azide probe pairs (starred combinations in Figure 1B). Scale bars, 10 μm. Quantitation of
this data is shown in Figure S2A. A repeat of this experiment, with immunofluorescence staining to compare LplA expression levels, is shown in
Figure S2B. Note that 10 times more W37ILplA plasmid was required to give similar expression levels to WTLplA and W37ILplA. (C) Gel-shift analysis
of azide ligation in cells. HEK cells were prepared and labeled as in (A), except LAP−BFP was replaced by LAP−YFP. After azide ligation and the
first wash, cells were lysed and run on a native 12% polyacrylamide gel. Labeled LAP-YFP runs faster than unlabeled LAP−YFP due to removal of a
positive charge. Percent conversions to azide-LAP-YFP product are given at the bottom of the YFP fluorescence gel image. Lane 4 shows a negative
control with azide 9 omitted. Additional controls are shown in Figure S4A.
adherence. In general, cells were transfected with 200 ng W37ILplA
METHODS
■
plasmid and 400 ng LAP fusion plasmid using Lipofectamine 2000
In vitro Azide Ligation. For the screen in Figure 1B, reactions
(Invitrogen) at 50−70% confluency. For Figures 2B and S2B, WTLplA
containing 100 nM LplA enzyme, 20 μM alkyl azide probe, 600 μM
and W37VLplA plasmids were introduced at 20 ng rather than 200 ng,
to give comparable expression levels to W37ILplA (at 200 ng), since the
LAP peptide (sequence: H2N-GFEIDKVWYDLDA-CO2H), 2 mM
ATP, and 2 mM magnesium acetate in 25 mM Na2HPO4 pH 7.2 were
former express much more strongly (Figure S2C).
incubated at 30 °C for 20 min. Reactions were quenched with 40 mM
General Protocol for Intracellular Protein Labeling. Sixteen to
EDTA (ethylenediaminetetraacetic acid, final concentration). Percent
twenty hours after transfection, mammalian cells were incubated in
conversion to LAP−azide adduct was determined by HPLC with a
C18 reverse phase column, recording absorbance at 210 nm. Elution
conditions were 30−60% acetonitrile in water with 0.1% trifluoroacetic
acid over 20 min at 1.0 mL/min flow rate. The percent conversion was
calculated from the ratio of LAP−azide to sum of (unmodified LAP +
LAP−azide). For Figure S3A in the Supporting Information (SI),
reactions containing 1 μM LplA enzyme, 500 μM azide 9, and 300 μM
LAP peptide were incubated at 30 °C for 2 h. For the kinetic
measurements in Figure S3C, reactions containing 100 nM W37ILplA,
25−700 μM azide 9, and 600 μM LAP peptide were incubated at 30
°C, before quenching at various time points with EDTA.
complete media (10% FBS in MEM) containing 200 μM azide 9 for
1−2 h at 37 °C. To wash out excess azide 9, cells were rinsed three
times with fresh, prewarmed complete media every 30 min for 1−1.5 h
in total. Cells were then incubated with FBS-free MEM containing 10
μM cyclooctyne−fluorophore conjugate for 10 min at 37 °C, followed
by rinsing three times with MEM over 5 min. Thereafter, cells were
switched to fresh, prewarmed complete media, and the media was
changed every 30 min to 1 h, for 1.5 to 8 h at 37 °C, prior to imaging.
We have not observed any morphological changes in the cells during
the washout period. ATTO 647N and ATTO 655 conjugates were
loaded at 1 μM instead of 10 μM.
Mammalian Cell Culture and Transfection. HEK, HeLa, and
COS-7 cells were cultured in Modified Eagle medium (MEM; Cellgro)
supplemented with 10% v/v fetal bovine serum (FBS; PAA
Laboratories). All cells were maintained at 37 °C under 5% CO2.
For imaging, cells were plated on 5 mm × 5 mm glass coverslips
placed within wells of a 48-well cell culture plate (0.95 cm2 per well)
12−16 h prior to transfection. HEK cells were plated on glass
precoated with 50 μg/mL fibronectin (Millipore) to increase
Cell Imaging. Cells were imaged in Dulbecco’s phosphate buffered
saline (DPBS) on glass coverslips at room temperature. For confocal
imaging, we used a ZeissAxioObserver inverted microscope with a 60×
oil-immersion objective, outfitted with a Yokogawa spinning disk
confocal head, a Quadband notch dichroic mirror (405/488/568/
647), and 405 (diode), 491 (DPSS), 561 (DPSS), and 640 nm (diode)
lasers (all 50 mW). BFP (excitation 405 nm; emission 445/40 nm),
YFP/fluorescein/Oregon Green 488 (excitation 491 nm; emission
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Figure 3. Evaluation of various cyclooctyne structures for site-specific intracellular protein labeling. (A) Top: labeling protocol for HEK cells
coexpressing W37ILplA and nuclear-localized LAP−BFP (LAP−BFP−NLS). After labeling with azide 9 for 1 h and washing for 1 h, cells were treated
with the indicated cyclooctyne-fluorescein conjugate for 10 min. Cells were washed again for 2.5 h to remove excess unconjugated fluorophore,
except for MOFO, which was washed for only 1.5 h. Bottom: images of labeled HEK cells. The LAP−BFP−NLS image is overlaid on the DIC image.
Fluorescein signal intensities can be compared in the first two columns, which show the fluorescein images at lower contrast (left) and higher
contrast (middle). Cyclooctyne structures are shown at right, and second-order rate constants (with references below) are shown at left. ADIBO,
aza-dibenzocyclooctyne; DIBO, 4-dibenzocyclooctynol; MOFO, monofluorinated cyclooctyne; DIMAC, 6,7-dimethoxyazacyclooct-4-yne; DIFO,
difluorinated cyclooctyne. All scale bars, 10 μm. (B) Quantitation of data in (A). For the top three cyclooctynes (ADIBO, DIBO, and MOFO), the
mean nuclear fluorescein intensity (representing specific labeling) was plotted against the mean cytosolic fluorescein intensity (representing
nonspecific labeling), for the same cell. More than 50 single cells were analyzed for each cyclooctyne.
528/38 nm), Alexa Fluor 568/TMR/X-rhodamine (excitation 561
nm; emission 617/73 nm), and Alexa Fluor 647/ATTO 647N/ATTO
655 (excitation 640 nm; emission 700/75 nm) images were acquired
using Slidebook 5.0 software (Intelligent Imaging Innovations).
Acquisition times ranged from 100 ms to 3 s. Fluorophore intensities
in each experiment were normalized to the same intensity ranges.
the LplA-catalyzed azide ligation reaction to improve its kinetic
properties.
Previous work has shown that Trp37 in the lipoic acid
binding pocket serves as a “gatekeeper” residue, and its
mutation to smaller side chains allows LplA to recognize a
variety of unnatural substrates.7,14−16 To identify an improved
LplA/azide pair, we prepared a panel of LplA Trp37 mutants
(W37G, A, V, I, L, and S) and screened them against a panel of
alkyl azide substrates of various lengths (Figure 1B). An HPLC
assay was used to determine the percent conversion of LAP
into LAP−azide conjugate, using 20 μM probe for 20 min
(Figure 1B). We found that wild-type LplA and W37VLplA were
the best ligases for the shortest azide 7 probe. For the longer
probes, wild-type LplA was no longer effective, and W37VLplA
and W37ILplA mutants were best. The four best ligase/probe
pairs are starred in Figure 1B.
To differentiate between these top four ligase/azide pairs, we
tested their performance in living cells. Human embryonic
kidney (HEK) cells were transfected with plasmids for each
LplA mutant and LAP−BFP (blue fluorescent protein). Azide 9
was added to the cells for 1 h. We empirically optimized the
washout time required to fully remove excess azide, using
cyclooctyne−fluorescein retention as a readout, and found that
1 h was adequate (Figure S1A). Therefore excess azide 7 and
azide 9 were each washed from cells for 1 h, before addition of
monofluorinated cyclooctyne (MOFO), conjugated to fluo-
RESULTS
■
Screening for the Best Alkyl Azide Ligase. To
generalize PRIME for targeting of diverse fluorophore
structures, our first challenge was to develop a method to
efficiently and specifically ligate a functional group handle to
LAP fusion proteins inside living cells. Previously we reported
that wild-type LplA can catalyze the conjugation of 8-
azidooctanoic acid (“azide 7”) to LAP with a kcat of 6.66
min−1 and Km of 127 μM.19 This works well for cell surface
labeling, where the azide probe can be added at high
concentrations and then excess unligated probe can be easily
washed away. For intracellular labeling, however, it is more
difficult to thoroughly wash away excess unused probe. It is
therefore preferable to deliver the azide probe at lower
concentrations so that less residual azide remains after the
ligation reaction, to minimize interference with the subsequent
[3+2] cycloaddition. To use lower azide concentrations,
without sacrificing azide ligation yield, we needed to engineer
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rescein diacetate (structure in Figure 3A), to derivatize the
azide−LAPs.
Numerous cyclooctyne structures have been developed by
our laboratories20,22,33−36 and other laboratories.21,37 These
structures vary in terms of ring strain and electron deficiency,
which in turn affect reactivity and specificity. In addition, more
hydrophilic cyclooctyne structures have been developed33 to
reduce the extent of nonspecific hydrophobic binding to cells.
Because it was not clear which cyclooctyne structure(s) would
be the best for our purpose, we selected a panel of five
structures, derivatized each with 5(6)-carboxyfluorescein
diacetate (Figure 3A), and compared the performance of
these conjugates for LAP−azide labeling inside living cells.
Figure 3A shows that, for labeling of LAP−BFP−NLS (NLS
is a nuclear localization signal) in HEK cells, ADIBO− and
DIBO−fluorescein diacetate conjugates give the highest signal,
consistent with their superior second-order rate constants (0.31
Following the labeling protocol shown in Figure 2A, after 10
min incubation and 2 h of washing to remove excess
fluorophore, cells were imaged. Figure 2B shows specific
labeling of LAP−BFP for all four combinations, but the highest
signal-to-background ratio was obtained for the W37ILplA/azide
9 pair. Note the substantial improvement in signal intensity
(∼4-fold greater on average) compared to the wild-type LplA/
azide 7 pair previously used for cell surface protein labeling.19
These differences are quantified in Figure S2A, in which
fluorescein intensity is plotted against LAP−BFP expression
level for >100 single cells for each condition. Anti-FLAG
immunofluorescence staining to detect FLAG-tagged LplA in
cells showed that ligase expression levels are all comparable
under our experimental conditions (Figure S2B).
M−1 s−1 and 5.9 × 10−2 M−1
s
−1, respectively21,34).
We also used a gel shift assay as a separate readout of azide
ligation yield inside cells (Figures 2C and S4A). HEK cells were
prepared expressing LAP−YFP (yellow fluorescent protein)
and either wild-type LplA or W37ILplA. Azide 7 or azide 9 was
added for 30 min or 1 h before washing and cell lysis. The yield
of azide ligation to LAP−YFP was determined by shift on a
native polyacrylamide gel. The unmodified fusion protein,
visualized by YFP fluorescence, runs at an apparent molecular
weight of ∼42 kD. Upon modification, the positively charged
lysine of LAP converts into a neutral amide, and the apparent
molecular weight of the fusion protein shifts down to ∼38 kD.
Using densitometry, we found that the WTLplA/azide 7 pair
gave 73% ligation yield after 1 h labeling in cells, whereas the
W37ILplA/azide 9 pair gave nearly quantitative ligation after only
30 min of azide 9 incubation. On the basis of these data and the
cell imaging results, we selected W37ILplA/azide 9 as our best
ligase/azide pair.
Surprisingly, significant nonspecific labeling is seen with
DIMAC, even in untransfected cells, despite its more
hydrophilic structure.33 Most of this nonspecific signal can be
washed away after cells are fixed, suggesting that it arises from
nonspecific binding rather than covalent labeling (Figure S5A).
DIFO35 also gave background, which unlike DIMAC, persisted
to some extent after cell fixation (Figure S5A); this may reflect
covalent addition to endogenous cellular nucleophiles such as
glutathione, which has previously been observed.27,32 Lowering
the DIFO−fluorescein diacetate concentration by 10-fold to 1
μM, and shortening the labeling time to 40 s reduced the
background somewhat, but it was still higher than the
background seen with ADIBO and DIBO (data not shown).
Labeling with MOFO-fluorescein diacetate was specific, like
with ADIBO and DIBO, although the signal was lower,
consistent with MOFO's slower rate constant (4.3 × 10−3 M−1
s−1).20 We quantitatively analyzed the signal-to-background
ratios resulting from cellular labeling with ADIBO, DIBO, and
MOFO, by calculating the cytosolic to nuclear signal intensity
ratios for >50 single cells from each condition. Because the LAP
fusion is nuclear-localized, a nuclear fluorescein signal
represents specific labeling, whereas cytosolic fluorescein signal
represents nonspecific background. Figure 3B shows that while
absolute signals are ∼4-fold higher with ADIBO and DIBO
compared to MOFO, the signal-to-background ratios are
comparable for all three cyclooctynes. We hypothesize that
MOFO gives lower background because it is not as
hydrophobic as ADIBO and DIBO. This is supported by the
fact that shorter dye washout time is required for MOFO (1.5
h) compared to ADIBO and DIBO (2.5 h).
On the basis of these results, we selected ADIBO and DIBO
for most of our cellular protein labeling experiments. However,
as shown later, due to ADIBO’s hydrophobicity, we find that
MOFO is a better option when working with very hydrophobic
fluorophores such as ATTO 647N.
Intracellular Protein Labeling with Azide 9 Ligase and
ADIBO-Fluorescein. Having optimized both the azide ligase
and the cyclooctyne, we proceeded to characterize two-step
labeling inside cells and explore its generality. HEK cells
expressing W37ILplA and LAP−BFP were labeled with azide 9
for 1 h followed by ADIBO−fluorescein diacetate. We
empirically optimized the ADIBO−fluorophore loading con-
centration and washout time (Figure S1B). Since cycloaddition
yield in cells increases with cyclooctyne concentration, we
determined the highest concentration that we could load and
yet obtain a clean washout in a reasonable period of time. We
Characterization of the Azide 9 Ligase, W37ILplA. We
proceeded to fully characterize our best azide ligation reaction.
Figure S3A shows an HPLC analysis of W37ILplA-catalyzed
ligation of azide 9 onto purified LAP peptide. The identity of
the LAP−azide 9 product peak was confirmed by mass
spectrometry (Figure S3B). Negative control reactions with
ATP omitted or wild-type LplA in place of W37ILplA were also
ran and showed no product formation. We also used HPLC to
quantify product amounts in order to measure kcat and Km
values. Figure S3C shows the Michaelis−Menten plot giving a
k
cat of 3.62 min−1 and a Km of 35 μM for azide 9 ligation to LAP
catalyzed by W37ILplA. Compared to our previously reported
azide 7 ligation, catalyzed by wild-type LplA,19 this Km is 4-fold
lower. The kcat is 1.8-fold reduced, giving an overall 2-fold
improvement in kcat/Km.
Comparison of Cyclooctyne Structures. Next, we
focused on the optimization of the azide derivatization
chemistry in cells. Numerous bioorthogonal ligation reactions
have been reported to derivatize alkyl azides, including the
Staudinger ligation,23 and copper-catalyzed24 as well as strain-
promoted25 [3+2] azide−alkyne cycloadditions. Of these,
copper-catalyzed [3+2] cycloaddition is the fastest, but
copper(I) is toxic to cells26 and not easily delivered into the
cytosol, where it also could become sequestered by endogenous
thiols. On the other hand, copper-free, strain-promoted
cycloaddition has been successfully demonstrated inside living
cells,27−29 and on the surface of cells within living animals.30−32
For this reason, we selected cyclooctyne−fluorophore con-
jugates to derivatize LAP−azide.
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Figure 4. Intracellular protein labeling with azide 9 ligase and ADIBO−fluorescein. (A) HEK cells coexpressing W37ILplA and LAP−BFP were
labeled with azide 9 and ADIBO−fluorescein as in Figure 3A, then imaged live. Negative controls are shown with an alanine mutation in LAP, azide
9 omitted, and a catalytically inactive mutant of LplA (last column). (B) ADIBO−fluorescein labeling of three localized LAP−BFP fusions, LAP−β-
actin, and LAP−MAP2 (microtubule-associated protein 2). Labeling in the cell type indicated beneath each image was performed as in Figure 3A,
except that for LAP−β-actin and LAP−MAP2, azide 9 was incubated for 2 h, and washed for 1.5 h before fluorophore addition. NES = nuclear
export sequence; CAAX = prenylation tag; NLS = nuclear localization sequence. All scale bars, 10 μm.
found that 10 μM of ADIBO−fluorescein diacetate, followed by
2.5 h of washout, was optimal.
647N gives a high level of nonspecific cell staining, primarily in
the mitochondria, which is known to concentrate positively
charged hydrophobic dyes (data not shown). We wondered if
replacing ADIBO with the less hydrophobic cyclooctyne
MOFO might reduce the background. Figure 5B shows a
comparison of LAP−BFP−NLS labeling with ADIBO- and
MOFO-conjugates to ATTO 647N. The graph on the right
plots the specific labeling (nuclear ATTO signal) against the
nonspecific labeling (cytosolic ATTO signal) for >50 single
cells for each probe. It can be seen that MOFO−ATTO 647N
gives more specific labeling than ADIBO−ATTO 647N, likely
because the total hydrophobicity of the conjugate is reduced.
This permitted us to perform MOFO−ATTO 647N labeling of
LAP−β-actin in live COS-7 cells (Figure 5A).
Figure 4A shows that HEK cells expressing LAP−BFP were
labeled with ADIBO-fluorescein, whereas neighboring un-
transfected cells were not labeled. Negative controls with
azide 9 omitted, LAP mutated, or a catalytically inactive LplA
mutant, W37I/K133RLplA,18 did not show fluorescein labeling.
We also tested labeling of different LAP fusion proteins
(Figure 4B). Using the two-step protocol shown in Figure 3A,
we successfully labeled LAP in the nucleus, cytosol, and plasma
membrane, as well as LAP fusions to β-actin and MAP2
(microtubule-associated protein 2). These experiments were
performed in multiple mammalian cell lines (HEK, HeLa, and
COS-7), demonstrating the versatility of the method.
Extension to Diverse Fluorophore Structures. To test
our method with other fluorophores, we prepared ADIBO
conjugates to tetramethylrhodamine (TMR), ATTO 647N, and
ATTO 655. ADIBO−TMR and ADIBO−ATTO 655 both gave
specific labeling (Figure 5A), but ADIBO−ATTO 647N
produced a high level of nonspecific background. We
hypothesized that this is due to the hydrophobicity of ATTO
647N (structure in Supporting Information). Even by itself,
without a conjugated cyclooctyne, we have found that ATTO
We also tested the effect of varying the linker structure
between MOFO and ATTO 647N in an attempt to further
reduce the labeling background. The N,N′-dimethyl-1,6-
hexanediamine (HDDA) linker that we used for most
fluorophore conjugates in this work was replaced by a more
hydrophilic polyethylene glycol (PEG) linker. Figure S5B
shows that for labeling of LAP−BFP−NLS, no significant
reduction in staining background was observed with MOFO−
PEG−ATTO 647N, suggesting that the cyclooctyne and
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Figure 5. Intracellular protein labeling with diverse fluorophore structures. (A) COS-7 cells coexpressing W37ILplA and the indicated LAP fusion
protein (labeled across bottom) were labeled with azide 9, followed by the indicated cyclooctyne−fluorophore conjugate (labeled across top).
MOFO was used for the more hydrophobic fluorophores (X-rhodamine and ATTO 647N); ADIBO and DIBO were used for the others. Chemical
structures are shown in the Supporting Information. TMR = tetramethylrhodamine. (B) Comparison of labeling specificity with ATTO 647N
conjugates to MOFO and ADIBO. After labeling of COS-7 cells expressing nuclear-localized LAP−BFP−NLS with azide 9 and the indicated
cyclooctyne−ATTO 647N conjugate, the mean nuclear ATTO 647N intensity (representing specific signal) was plotted against the mean cytosolic
ATTO 647N intensity (representing nonspecific signal), for the same cell, for >50 single cells for each condition. Images are shown after 8 h of
ATTO 647N conjugates washout. All scale bars, 10 μm.
fluorophore moieties dominate the hydrophobic properties of
the probe.
NLS and W37ILplA. After azide ligation, DIBO−biotin was
added for 10 min; then cells were washed and fixed, and
biotinylated LAP was detected with streptavidin-Alexa Fluor
568.
Figure 5A shows live cell labeling of multiple LAP fusion
proteins with a diverse palette of fluorophores. ADIBO is used
for the more hydrophilic dyes such as fluorescein, TMR, and
ATTO 655. DIBO is used for Oregon Green 488. MOFO is
used for the more hydrophobic dyes, X-rhodamine and ATTO
647N.
We used this two-step intracellular azide 9/DIBO−biotin
labeling to measure our overall two-step labeling yield in cells.
After performing labeling using the protocol in Figure 3A, HEK
cells were lysed, incubated with excess streptavidin protein to
bind biotinylated LAP−mCherry fusion protein, and the lysate
was analyzed by gel. In-gel mCherry fluorescence imaging in
Figure S4B shows that LAP−mCherry runs at the expected
molecular weight (27 kD) in negative control samples in which
azide 9 or streptavidin are omitted. In lane 1, however, 21% of
LAP−mCherry is shifted up to ∼80 kD, reflecting binding by
streptavidin. We conclude that using the labeling protocol
shown in Figure 3A, the two-step labeling yield in cells is
approximately 20%.
Cell Surface Labeling and Measurement of Two-Step
Ligation Yield in Cells. In addition to intracellular labeling,
we performed cell surface labeling using commercially available
cyclooctyne-probe conjugates (Figure S6A). LAP-tagged LDL
receptor and neurexin-1β were labeled on the surface of HEK
cells, by adding purified W37ILplA, azide 9, and ATP to the cell
medium for 20 min. Thereafter, LAP−azide was derivatized
using either membrane-impermeant DIBO−Alexa Fluor 647, or
DIBO−biotin. The DIBO−biotin was visualized by staining
with streptavidin−Alexa Fluor conjugates. Specific, azide-
dependent cell surface labeling was seen in all cases.
Because DIBO−biotin is membrane-permeant, it is also
possible to perform this labeling inside cells, although
biotinylated LAP proteins can only be detected after membrane
permeabilization and streptavidin staining. Figure S6B shows
intracellular labeling in HEK cells coexpressing LAP−BFP−
DISCUSSION
■
We have developed a methodology for targeting diverse
fluorophores to recombinant cellular proteins modified by a
13-amino acid peptide tag (LAP). The targeting is accom-
plished first by enzyme-mediated alkyl azide ligation and then
by strain-promoted cycloaddition with a fluorophore-con-
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jugated cyclooctyne. To develop the method, we systematically
optimized the azide ligation reaction through screening of lipoic
acid ligase mutants and alkyl azide variants. We then evaluated
five different cyclooctyne structures differing in reactivity,
selectivity, and extent of nonspecific binding to cells, using a
live-cell fluorescein targeting assay. Our final, optimized two-
step labeling scheme was used to target a diverse panel of
fluorophores ranging from fluorescein to ATTO 647N, to a
variety of LAP fusion proteins in multiple mammalian cell lines.
Our comparison of cyclooctynes in cells yielded observations
that should prove useful even beyond the context of PRIME,
due to the numerous and diverse applications to which
cyclooctynes are being applied.27−32,38,39 One of the earliest
cyclooctynes, MOFO (monofluorinated),20 performed well
inside cells, giving signal to background ratios consistently >5:1
in the context of fluorescein targeting to nuclear LAP. This
same cyclooctyne was used for cell surface LplA-mediated
labeling in our previous study.19 In next-generation cyclo-
octynes, fusion to benzene rings increased ring strain and hence
the second-order rate constants. Not surprisingly, we found
that these cyclooctynes, ADIBO and DIBO, gave ∼4-fold
higher absolute signal in cells, compared to MOFO, probably
due to increased yield of the cycloaddition product. However,
the increase in signal was accompanied by an increase in
background, likely due to the greater hydrophobicity and hence
nonspecific binding of these probes. Consequently, the signal to
background ratios were comparable for ADIBO−, DIBO−, and
MOFO−fluorescein conjugates.
we demonstrate two-step fluorophore targeting using LplA in
combination with Diels−Alder cycloaddition between a trans-
cyclooctene and tetrazine.46 The very fast cycloaddition kinetics
(k ∼ 104 M−1 s−1) yields substantial improvements in signal to
background ratio for intracellular protein labeling. Another
interesting advance is in copper-catalyzed click chemistry.
Previously discounted for cellular applications due to copper
toxicity, new improvements in copper ligand design and
reactive oxygen species scavenging have made it possible to
perform click chemistry on live cell surfaces and even animals.24
If the toxicity can be further reduced, while preserving the fast
kinetics of ligation (currently 104−107 fold greater than strain-
promoted cycloaddition25), then copper-catalyzed click chem-
istry will be competitive with other methods for bio-orthogonal
derivatization on the cell surface.
Considered in the context of other protein labeling
methods,3 the disadvantages of the approach presented here
are the requirement for coexpression of the LplA labeling
enzyme, the unavoidable background caused by nonspecific
binding of cyclooctyne−fluorophore conjugates (albeit low in
the case of hydrophilic fluorophores such as fluorescein and
Oregon Green), and the signal which is fundamentally limited
by the kinetics of strain-promoted cycloaddition chemistry.
Given these factors, the methodology will be most useful as a
nontoxic (in contrast to FlAsH6) labeling method for abundant
proteins, whose fusion to large tags (such as fluorescent
proteins, HaloTag,4, or SNAP tag5) perturb function, such as
actin.
When we extended the cyclooctyne comparison to other
fluorophores, we found that ADIBO and DIBO conjugates to
well-behaved hydrophilic fluorophores such as fluorescein and
Oregon Green gave satisfactory labeling, but when we tried to
target very hydrophobic fluorophores such as ATTO 647N, the
combined hydrophobicity of the dye and the cyclooctyne
(ADIBO) precluded successful labeling, due to high nonspecific
binding. This was alleviated by using the less hydrophobic
MOFO instead. Thus MOFO−ATTO 647N but not ADIBO−
ATTO 647N was used to label and image actin in living COS-7
cells. Our study illustrates the need for new cyclooctyne probes
that combine high reactivity (as displayed by ADIBO) with low
hydrophobicity/nonspecific binding (as displayed by MOFO).
In this regard, bicyclononynes (BCNs) show promise.40
Alternatively, fluorogenic cyclooctynes41 could be extremely
helpful, hiding nonspecific binding, and producing fluorescence
only upon specific reaction with azide-conjugated LAP.
ASSOCIATED CONTENT
■
S
* Supporting Information
Figures S1−S6, experimental procedures for the synthesis of
alkyl azides and cyclooctyne−fluorophore conjugates, plasmid
construction, immunofluorescence detection of LplA, kinetic
analysis of azide 9 ligation by W37ILplA, measurement of ligation
yields, cell surface labeling, and quantitative analysis of cell
images. Complete reference 4 with all authors’ names listed.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
ating@mit.edu
Several of the fluorophores targeted using LplA and strain-
promoted cycloaddition in this study have exemplary properties
that make them attractive alternatives to fluorescent proteins.
For instance, X-rhodamine is a bright and photostable
fluorophore commonly used for speckle imaging of actin.42
ATTO 647N is one of the best fluorophores of any kind for
both STED (stimulated emission depletion)43,44 and STORM-
type17 super-resolution microscopies. On the cell surface, we
targeted Alexa Fluor 647, an excellent fluorophore that has
been used for countless ensemble and single-molecule imaging
experiments. If methods can be developed to deliver sulfonated
fluorophoreswhich include the cyanine dyes and Alexa
Fluorsacross cell membranes,45 then these too should be
targetable to specific intracellular proteins using PRIME.
In this work, we focus on the use of strain-promoted
cycloaddition to accomplish two-step fluorophore targeting, but
the availability of new and improved bio-orthogonal ligation
chemistries opens up alternative possibilities. In separate work,
ACKNOWLEDGMENTS
■
Hemanta Baruah and Marta Fernan
́
dez-Suar
́
ez performed
preliminary experiments. We thank Daniel Liu, Justin Cohen,
and Katie White for plasmids and helpful discussions. DIBO−
Oregon Green 488 diacetate was a gift from Kyle Gee (Life
Technologies). This work was supported by the National
Institutes of Health (R01 GM086214), the Dreyfus Founda-
tion, the American Chemical Society, and MIT.
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