Photoaffinity labeling of small-molecule-binding proteins by dna-templated chemistry

Article abstract of DOI:10.1002/anie.201302161

DNA-templated affinity labeling: Accurate characterization of small-molecule (SM)-protein interactions is one of the most important but also very challenging tasks in chemical biology and drug discovery. A novel method named DNA-programmed photoaffinity labeling is reported. The introduction of DNA encoding and template effect enable multiplexed protein labeling by multiple probes. Copyright

Full text of DOI:10.1002/anie.201302161

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DOI: 10.1002/anie.201302161
DNA-Templated Chemistry
Photoaffinity Labeling of Small-Molecule-Binding Proteins by DNA-
Templated Chemistry**
Gang Li, Ying Liu, Yu Liu, Li Chen, Siyu Wu, Yang Liu, and Xiaoyu Li*
Dedicated to the Bayer company on the occasion of its 150th anniversary
Small molecules are important tools in
exploring biology.[1] Accurate character-
ization of small-molecule- (SM) binding
proteins is of great importance in appli-
cations such as target identification and
proteomic profiling,[2] which calls for
general, specific, and high-throughput
methods to characterize the multitude
of SM–protein interactions in the pro-
teome.
Classic “affinity pull-down” employs
immobilized or tagged SM probes to
isolate binding proteins (Figure 1a, left
and center); but they are usually limited
to high abundance/affinity SM–protein
interactions. Covalent affinity probes,
equipped with additional reactive
groups, provide a straightforward way
for protein capture and isolation (Fig-
ure 1a, right). Since first developed in
Figure 1. a) Common types of affinity probes. b) Scheme of DNA-programmed photoaffinity
labeling (DPAL).
1994,[3] these probes received extensive
use in numerous studies.[4] However, the
basic probe architecture with multiple
functions combined in one entity remains largely unchanged,
which lacks modularity and presents several issues. First, the
crosslinking group should be distal from the binding site in
order not to alter binding, but it also should be close to the
protein for efficient crosslinking. Recently, Cravatt and co-
workers incorporated benzophenone, a crosslinking group, as
part of the binding structure. This elegant strategy solves the
dilemma, but probes are then limited to benzophenone
derivatives.[4f] Second, for each SM, preparation of multiple
probes for different purposes are often required (e.g. with
a fluorophore for detection and with an affinity tag for
purification). Finally, probes often face proteins with varied
binding properties. A collection of probes with diverse small
molecules is highly desired for proteome-wide protein profil-
ing.[5] Previously, most affinity probes offer very limited
multiplexing capability.
Here we report a novel method named “DNA-pro-
grammed photoaffinity labeling (DPAL)”. DNA-templated
chemistry has emerged as a versatile approach to control
chemical reactions;[6] and DNAwas used for protein detection
in immuno-polymerase chain reaction (immuno-PCR)[7] and
proximity-ligation assay.[8] Recently nucleic acids (DNA and
PNA) have been used to program the assembly of SM ligands
with controlled combinations or distance to explore spatial
information of ligand binding to protein targets.[9] We
implemented DNA-templated chemistry to establish
a simple, modular, and multiplexed system for labeling the
binding proteins of small molecules.
[*] Prof. X. Li
Key Laboratory of Bioorganic Chemistry and
Molecular Engineering of the Ministry of Education
Beijing National Laboratory of Molecular Sciences (BNLMS)
College of Chemistry and Molecular Engineering, Peking University
202 Chengfu Road, Beijing, 100871 (China)
E-mail: xiaoyuli@pku.edu.cn
G. Li, Y. Liu, Y. Liu, L. Chen, S. Wu, Y. Liu, Prof. X. Li
College of Chemistry and Molecular Engineering, Peking University
202 Chengfu Road, Beijing, 100871 (China)
[**] This work was supported by the Ministry of Science and Technology
Basic Research Program (grant number 2011CB809100), National
Natural Science Foundation of China (grant numbers 21002003,
91013003, and 21272016), the Beijing Nova Program (grant number
2010B002), and the Project Sponsored by the Scientific Research
Foundation for the Returned Overseas Chinese Scholars, State
Education Ministry.
DPAL consists a dual-probe system (Figure 1b): the small
molecule is conjugated to a DNA as the “binding probe (BP,
shown in red)”; and a photocrosslinking group is conjugated
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201302161.
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to a complementary DNA as the “capture probe (CP, shown
in blue)”. After BP/CP hybridization, irradiation triggers
DNA-templated photocrosslinking of CP to covalently cap-
ture the protein. DPAL decouples SM binding from cross-
linking/tagging. The crosslinking group can be flexibly
adjusted by DNA hybridization. In DPAL, only a single
modification (for DNA conjugation) on the SM is needed and
only a single BP is necessary, as BP can be paired with
multiple CPs for different purposes. Importantly, the intro-
duction of DNA provides multiplexing capability.
We first addressed an important issue in affinity labeling:
nonspecific crosslinking, a particularly severe issue for label-
ing low-abundance/low-affinity proteins when a high probe
concentration is necessary.[10] It is well known that the salt
concentration can affect specific and nonspecific molecular
interactions, such as protein–DNA,[11] protein–protein,[12]
protein–ligand,[13] and protein–matrix interactions.[14] Inter-
estingly, this phenomenon has not been widely exploited to
reduce nonspecific labeling by affinity probes. To test this
possibility, we first investigated photocrosslinking reactions
between a diazirine/fluorescein-labeled DNA probe (DZ/
FAM-CP, Figure 2) and bovine serum albumin (BSA),
a protein well-known for its extensive nonspecific interactions
with small molecules.[15] Significant BSA photocrosslinking by
DZ/FAM-CP was observed at concentrations > 5 mm in H2O
(Figure 2a, top); in sharp contrast, with additional salt (1 ꢀ
PBS + 0.1m NaCl), crosslinking was nearly completely sup-
pressed even with 100 mm DZ/FAM-CP (Figure 2a, bottom).
In HeLa cell lysate (alone or with purified BSA), crosslinking
was also suppressed by the added salt (Figure 2b, lanes 2 and
4), indicating this effect is not limited to BSA but rather
general to other cellular proteins. We also performed studies
regarding protein and salt concentrations, salt type, irradi-
ation time, and other types of probes. The results collectively
suggest the salt effect is quite general. The ionic strength
appears to be the only major effecting factor (see the
Supporting Information for details). This salt effect has
been implemented throughout experiments in this study.
Next, we examined whether specific SM–target interac-
tions can be identified by DPAL under elevated salt concen-
trations. First, we conjugated a small molecule LPCBS (1) to
the 5’-NH2 of a 27 bp DNA as the binding probe (LPCBS-BP)
to label its well-known target carbonic anhydrase II (CA-II,
purified, 10 mm, Kd = 0.9 nm for free 1)[16] with BSA (10 mm) as
the background. DNA conjugation at the C-terminus of
LPCBS retains its binding property.[17] A 15 bp DZ/FAM-CP
fully complementary to LPCBS-BP was prepared as the CP.
After BP binding, CP hybridization, and irradiation, a 24 bp
displacement DNA complementary to BP was added to
eliminate the possibility of forming a noncovalent complex of
protein-bound BP/CP duplex. As shown in Figure 3a, SDS-
PAGE results show DZ/FAM-CP labeled CA-II only in the
presence of complementary BP/CP after irradiation (Fig-
ure 3a, left panel, lanes 1–2). Even with excess probes
(20 mm), nonspecific BSA labeling
was not detected. All negative controls
(no irradiation, no BP, mismatched
BP/CP, BP without LPCBS, no CA-II,
denatured CA-II; Figure 3a, lanes 3–
8) gave no detectable CA-II or BSA
labeling, indicating the labeling
requires specific SM–protein binding
and DNA hybridization. As expected,
without added salt, significant BSA
labeling was observed (see Figure S2
in the Supporting Information). Next,
we mixed purified CA-II with Jurkat
cell lysate and performed similar
DPAL experiments with same
probes, again specific labeling was
observed despite the presence of
numerous cellular proteins and
excess probes (Figure 3a, center and
right panels). Encouraged by this
result, we performed DPAL experi-
ments on a series of SM–protein pairs
covering a wide range of binding
affinities (structures shown in Fig-
ure 3b)[17] with the same DPAL
experiment setup (purified proteins
Figure 2. Suppression of nonspecific crosslinking by elevated salt concentrations. a) Crosslinking
between BSA and DZ/FAM-CP in H2O and in 1ꢀPBS/0.1m NaCl, monitored by FAM. Lanes 1–7:
with irradiation (concentrations are as marked). Lane 8: no irradiation. Lane 9: PBS added after
irradiation in H2O. b) Photocrosslinking between BSA and DZ/FAM-CP in lysate (HeLa,
1.0 mgmLꢀ1,10 mg per lane; CP: 20 mm). Left: fluorescence. Right: silver stain. M: marker. Lanes 1
and 2: lysate only. Lanes 3 and 4: lysate+20 mm BSA. Lanes 5 and 6: lysate without irradiation.
The arrow indicates the position of BSA-CP conjugate.
in a BSA background); again similar
labeling performances were observed
(Figure 3c). In addition, incubation
with DNase I diminished the FAM
signal (lane 9), further confirming the
covalent labeling by DPAL probes.
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Figure 3. a) DPAL of CA-II by LPCBS (1)-BP in BSA background (left) and in HeLa cell lysate (middle & right). CA-II, BSA: 10 mm. BP/CP: 20 mm.
Lysate: 10 mg/lane. Lane 1: BP/CP/hn; lane 2: pre-hybridized BP/CP/hn; lane 3: no hn; lane 4: no BP; lane 5: mismatched CP; lane 6: BP without
SM; lane 7: no CA-II or lysate; lane 8: CA-II pre-denatured; lane 9: with DNase I. Arrows indicate positions of CA-II and CA-II/CP conjugate.
Loading control: a 50 bp, FAM-labeled DNA. b) Structures of SMs used (1, LPCBS; 2, desthiobiotin; 3, GLCBS; 4, chymostatin; 5, CBS; 7,
AP1497). c) DPAL results of 2–5 with respective targets. Conditions and lane sequences are the same as in (a). d) DPAL of AP1497s endogenous
target FKBP12 in Jurkat cell lysate. Left panel: fluorescent image with DZ/FAM-CP. The lane sequence is the same as in (a). Jurkat lysate:
3.6 mgmLꢀ1, 54 mg per lane. Middle and right panels: after biotin-CP labeling, streptavidin beads enrichment, eluted proteins are detected by anti-
biotin and FKBP12 antibodies. M: marker. “!”: expected positions of FKBP12. M*: overlaid marker.
Next, we applied DPAL in labeling an endogenous
protein in cell lysate by using AP1497 (7), an immunophilin
inhibitor with a well-known target FKBP12.[18] In Jurkat cell
lysate, AP1497-BP (Figure 3b), along with DZ/FAM-CP,
specifically labeled a protein matching the molecular weight
of FKBP12 (Figure 3d, left). Furthermore, taking advantage
of the modularity of DPAL, without preparing additional BP,
we simply used a 5’-biotin-, 3’-diazirine-labeled DNA as CP
(biotin-CP) to pair with the existing BP. After DPAL and
filtration to remove free probes (ultracentrifugal tube,
MWCO: 30 kDa), streptavidin beads pull-down and elution
enriched a single protein band, which can be blotted by
biotin- and FKBP12-specific antibodies (Figure 3d, middle
and right panels), proving the identity of FKBP12 labeled by
biotin-CP.
We intentionally used high protein concentrations (10 mm)
and excess probes (20 mm) to challenge DPAL, for which
labeling is both SM-specific and DNA sequence-specific. This
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has been demonstrated by the absence of protein labeling,
either specific or nonspecific, in negative controls. For
example, experiments with the mismatched BP/CP or without
SM did not give detectable target or BSA labeling even with
excess probes.
It is common in affinity labeling that only a low-affinity
probe is available and target proteins are not abundant;
therefore we explored the performance of DPAL in labeling
low-abundance/low-affinity proteins. We spiked HeLa cell
lysate with purified CA-II at various dilutions to mimic the
low-abundance target scenario. Again, we simply used the
biotin-CP to pair with existing GLCBS-BP and CBS-BP
(Figure 3b, 3 and 5). Free CBS has a Kd of 3.2 mm against CA-
II,[19] which can be considered as a low-affinity ligand. After
DPAL and filtration to remove free probes, we performed
streptavidin pull-down to enrich any biotin-CP-labeled pro-
teins from the mixture. Figure 4 shows both probes specifi-
two-probe system of DPAL provides simple probe prepara-
tion and novel modularity in protein labeling and enrichment.
In DPAL, we can flexibly shift the hybridization position
of CP relative to the protein target. We selected four SM–
target pairs (Figure 3b, 2, 3, 4, 5) and prepared multiple CPs
with varied “n values”, able to hybridize at different locations
on these BPs (Figure 5). We performed DPAL experiments
Figure 5. Effect of the CP hybridization position (different n values) on
labeling yields with four SM–target pairs. SMs and their targets are
labeled above each plot. DPAL conditions are the same as in Fig-
ure 3a. Labeling yields were determined by an internal FAM-labeled
50 bp DNA and normalized to n=0. Error bars (standard deviation,
SD) are based on three replicates of each experiment.
Figure 4. Biotin Western blots of enriched CA-II from HeLa lysate with
a) GLCBS-BP (CA-II:lysate 1:1000), b) CBS-BP (CA-II:lysate 1:1000),
and c) CBS-BP (CA-II:lysate 1:5000). Lane 1: lysate only; lane 2: CA-II
only; lane 3: CA-II+CP in H2O with hn; lane 3*: CA-II+CP/BP in
PBS/NaCl with hn; lane 4: elution after enrichment; lane 5: same as 4
but with GLCBS competition; lane 6: same as 4 but with lysate only
(no CA-II added). BP, CP: 20 mm. Ratios: w:w. See the Supporting
Information for enrichment procedures. Dark smears in gels are due
to formamide used in elution buffer.
with these SM–target pairs (with purified proteins) and
compared their labeling yields. Results show that DNA-
templated photocrosslinking is “distance-dependent”:[21] in
general, CPs with negative n values have higher yields than
positive n values. Interestingly, n = ꢀ3 appears to be optimal,
instead of the typical “side-by-side” alignment in most DNA-
templated reactions. Overall, these data suggest the flexible
DNA hybridization can be used to tune for optimal cross-
linking in DPAL.
cally enriched a protein matching the expected molecular
weight of CA-II (lane 4; anti-biotin Western blot), even with
the low-affinity CBS probe at 1:5000 dilution (Figure 4c; see
Figure S10 for explanation of the two bands). With free
GLCBS, no enriched protein bands were observed (lane 5),
indicating the labeling is specific for small molecules. With
cell lysate only, no CA-II band was observed, proving the
enrichment was not from endogenous expression (lane 6).
Carefully optimized tri-functional probes can label less than
0.1 wt% of proteins in cell lysate.[20] Our data show DPAL is
comparable to conventional probes (Figure S11), although
both approaches benefited significantly from the salt effect to
suppress nonspecific labeling in this study. Nevertheless, the
Conventional affinity probes have very limited multi-
plexing capability. Previously, probes with two-color fluores-
cent tags[22] and chemically orthogonal handles have been
reported.[4e] Activity-based protein profiling (ABPP) has
been accomplished with PNA-based probes.[23] To demon-
strate the multiplexing capability of DPAL, we mixed avidin
(i), CA-II (ii), and trypsin (iii) at 1:1:1 molar ratio along with
HeLa cell lysate as the background. These proteins have
different molecular weights so that formed protein–DNA
conjugates can be easily distinguished by gel mobility (Fig-
ure 6a). The mixture was subjected to DPAL with different
combination of BPs (structures shown in Figure 6a) and
complementary DZ/FAM-CPs. We supplemented mis-
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however, only a single DNA conjugation and a single binding
probe are needed in DPAL. Capture probes are universal, and
can actually be pre-prepared and stored as a kit, ready to pair
with any BP regardless of a specific small molecule used.
The introduction of DNA in DPAL provides not only
simplicity and modularity, but also multiplexing capability
lacking in most conventional affinity probes. We used a three-
target mixture only as a model system to show that DNA
provides DPAL with virtual spatial separation and probe
encoding, two key features necessary for multiplexed affinity
labeling. We expect a combination with DNA microarray
would significantly improve the multiplexability and through-
put of DPAL. Moreover, DPAL may also be used as
a sensitive protein detection method benefiting from DNA
amplification. Our laboratory is actively applying DPAL in
research programs exploring these opportunities.
Received: March 14, 2013
Revised: May 14, 2013
Published online: June 17, 2013
Keywords: DNA · DNA-templated chemistry ·
.
photoaffinity labeling · protein labeling
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multiple small molecule probes in a single solution.
In summary, we have developed a novel affinity labeling
method based on DNA-templated photocrosslinking chemis-
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other functions that are usually combined in one probe,
therefore simplifying probe design and preparation. Struc-
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