Identification of ligand-target pairs from combined libraries of small molecules and unpurified protein targets in cell lysates

Article abstract of DOI:10.1021/ja412934t

We describe the development and validation of interaction determination using unpurified proteins (IDUP), a method that selectively amplifies DNA sequences identifying ligand+target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. By operating in cell lysates, IDUP preserves native post-translational modifications and interactions with endogenous binding partners, thereby enabling the study of difficult-to-purify targets and increasing the potential biological relevance of detected interactions compared with methods that require purified proteins. In IDUP, target proteins are associated with DNA oligonucleotide tags either non-covalently using a DNA-linked antibody or covalently using a SNAP-tag. Ligand-target binding promotes hybridization of a self-priming hairpin that is extended by a DNA polymerase to create a DNA strand that contains sequences identifying both the target and its ligand. These sequences encoding ligand+target pairs are selectively amplified by PCR and revealed by high-throughput DNA sequencing. IDUP can respond to the effect of affinity-modulating adaptor proteins in cell lysates that would be absent in ligand screening or selection methods using a purified protein target. This capability was exemplified by the 100-fold amplification of DNA sequences encoding FRB+rapamycin or FKBP+rapamycin in samples overexpressing both FRB and FKBP (FRB·rapamycin+FKBP, K_d ≈ 100 fM; FKBP· rapamycin+FRB, K_d = 12 nM). In contrast, these sequences were amplified 10-fold less efficiently in samples overexpressing either FRB or FKBP alone (rapamycin+FKBP, K_d ≈ 0.2 nM; rapamcyin+FRB, K_d = 26 μM). Finally, IDUP was used to process a model library of DNA-linked small molecules and a model library of cell lysates expressing SNAP-target fusions combined in a single sample. In this library×library experiment, IDUP resulted in enrichment of sequences corresponding to five known ligand+target pairs ranging in binding affinity from K_d = 0.2 nM to 3.2 μM out of 67,858 possible combinations, with no false positive signals enriched to the same extent as that of any of the bona fide ligand+target pairs.

Full text of DOI:10.1021/ja412934t

Article
pubs.acs.org/JACS
Open Access on 02/04/2015
Identification of Ligand−Target Pairs from Combined Libraries of
Small Molecules and Unpurified Protein Targets in Cell Lysates
Lynn M. McGregor, Tara Jain, and David R. Liu*
Department of Chemistry and Chemical Biology and Howard Hughes Medical Institute, Harvard University, 12 Oxford Street,
Cambridge, Massachusetts 02138, United States
S
* Supporting Information
ABSTRACT: We describe the development and validation of
interaction determination using unpurified proteins (IDUP), a
method that selectively amplifies DNA sequences identifying
ligand+target pairs from a mixture of DNA-linked small
molecules and unpurified protein targets in cell lysates. By
operating in cell lysates, IDUP preserves native post-
translational modifications and interactions with endogenous
binding partners, thereby enabling the study of difficult-to-
purify targets and increasing the potential biological relevance of detected interactions compared with methods that require
purified proteins. In IDUP, target proteins are associated with DNA oligonucleotide tags either non-covalently using a DNA-
linked antibody or covalently using a SNAP-tag. Ligand−target binding promotes hybridization of a self-priming hairpin that is
extended by a DNA polymerase to create a DNA strand that contains sequences identifying both the target and its ligand. These
sequences encoding ligand+target pairs are selectively amplified by PCR and revealed by high-throughput DNA sequencing.
IDUP can respond to the effect of affinity-modulating adaptor proteins in cell lysates that would be absent in ligand screening or
selection methods using a purified protein target. This capability was exemplified by the 100-fold amplification of DNA
sequences encoding FRB+rapamycin or FKBP+rapamycin in samples overexpressing both FRB and FKBP (FRB·rapamycin+
FKBP, Kd ≈ 100 fM; FKBP·rapamycin+FRB, Kd = 12 nM). In contrast, these sequences were amplified 10-fold less efficiently in
samples overexpressing either FRB or FKBP alone (rapamycin+FKBP, Kd ≈ 0.2 nM; rapamcyin+FRB, Kd = 26 μM). Finally,
IDUP was used to process a model library of DNA-linked small molecules and a model library of cell lysates expressing SNAP-
target fusions combined in a single sample. In this library×library experiment, IDUP resulted in enrichment of sequences
corresponding to five known ligand+target pairs ranging in binding affinity from Kd = 0.2 nM to 3.2 μM out of 67,858 possible
combinations, with no false positive signals enriched to the same extent as that of any of the bona fide ligand+target pairs.
relevance of selection methods will significantly increase their
effectiveness. Here we report the development and validation of
interaction determination using unpurified proteins (IDUP), a
method to rapidly identify ligand+target pairs from one-pot
mixtures of DNA-linked ligands and unpurified protein targets
in cell lysates (Figure 1A).
IDUP is triggered by the formation of a ternary complex
involving a DNA-linked ligand, a target protein, and a DNA
oligonucleotide that identifies the target protein. The
association of the target protein with its corresponding DNA
oligonucleotide can be established either non-covalently using a
DNA-linked antibody (Figure 1A) or covalently using a self-
labeling protein that reacts with a DNA-linked small molecule
(Figure 1B). Formation of this ternary complex is dependent
on ligand−target binding and promotes hybridization of short
complementary regions on the target- and ligand-linked
oligonucleotides. A DNA polymerase can then extend this
hybridized region to generate a double-stranded product that
contains DNA sequences identifying both the target and its
INTRODUCTION
■
Advances in genomic and proteomic studies continue to reveal
new targets for therapeutic intervention. The identification of
ligands for such targets remains a major opportunity and
challenge. To this end, a variety of target-oriented ligand-
binding assays have been developed, including affinity
selections on DNA-encoded chemical libraries,1,2 selection-
like methods such as interaction-dependent PCR,3 and a wide
variety of screening platforms.4 Selections offer substantially
improved throughput and decreased time, cost, and material
consumption compared to screens, but they generally rely on
purified, heterologously expressed proteins in an artificial
context that includes an immobilized1 or DNA-linked3 protein,
the compound library, and buffer. Selections conducted in this
manner can be incompatible with poorly soluble, aggregation-
prone, difficult-to-purify, intrinsically disordered, or membrane-
bound targets. Moreover, the results of selections on
immobilized targets may lack biological relevance for proteins
that adopt non-native conformations or lack binding partners
or cofactors essential for their function when taken out of the
cellular context.5 Although successful selections have been
conducted using purified proteins,1 increasing the biological
Received: December 19, 2013
Published: February 4, 2014
© 2014 American Chemical Society
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SA, and either DNA-biotin or DNA-desthiobiotin were
amplified much more quickly than those containing DNA-
amine, containing DNA-GLCBS, or lacking SA, resulting in a
qPCR cycle threshold (CT) difference of five cycles (ΔCT = 5,
corresponding to a 32-fold difference in effective template
availability; Supporting Information, Figure S1). Together,
these results demonstrate the ability of an antibody+protein+
ligand ternary complex to trigger the selective amplification of a
DNA sequence identifying the protein and ligand.
Because potential applications of IDUP include selections on
DNA-encoded chemical libraries, we next asked whether
formation of an αSA+SA+biotin complex would result in
selective amplification of the DNA sequence encoding SA+
biotin when DNA-biotin was present in a mock library
containing an excess of DNA-GLCBS. After primer extension,
PCR, restriction digestion, and polyacrylamide gel electro-
phoresis (PAGE) of samples containing DNA-αSA, SA, and
mixtures containing a 1:10, 1:100, or 1:1000 ratio of DNA-
biotin/DNA-GLCBS, we found that the sequence correspond-
ing to SA+biotin was enriched ∼10-fold (Figure S1). This
relatively modest enrichment of the SA+biotin DNA sequence
suggested that an improvement in the signal-to-noise ratio of
IDUP would be required to enable enrichment of sequences
corresponding to interactions with affinities weaker than that of
SA+biotin.
To optimize the effectiveness of IDUP, we investigated key
aspects of the primer extension step using the related and
previously validated IDPCR system, in which the purified target
protein is covalently preconjugated to an identifiable DNA
oligonucleotide.3 In a series of model library×library experi-
ments with 258 DNA-linked proteins and 260 DNA-linked
ligands, we systematically varied parameters of the DNA
extension step using the DNA polymerase Klenow exo− and
found that varying primer extension conditions did not
substantially improve enrichment factors for sequences
corresponding to weaker interactions (Figures S2 and S3).
We next performed model library×library experiments using
other mesophilic polymerases, and consistent with a previous
report describing the ability of polymerases with 3′-exonuclease
activity to increase the signal-to-noise ratio of the proximity
extension assay,7 we found that IDPCR with T4 DNA
polymerase and a complementary region of 8-nt or 9-nt
resulted in 4.5- to 140-fold improvements in the enrichment of
DNA sequences corresponding to ligand+target pairs with
binding affinities ranging from 40 nM to 13 μM (Figures S4−
S6).
Next we tested whether these improvements would apply to
IDUP and enable the enrichment of sequences corresponding
to known binding interactions from a large excess of
nonbinding entities (Figure 2A). We incubated mixtures
containing 1:10, 1:100, or 1:1000 ratios of DNA-biotin/
DNA-GLCBS with DNA-αSA and SA. Because we are
interested in the ability of IDUP to detect protein+ligand
binding in a complex mixture, such as a cell lysate, we also
added HeLa cell lysate to the solution so that SA was present at
0.01 wt% relative to the total protein content of the HeLa cell
lysate, an amount that is representative of the endogenous
expression level of members of protein classes of interest such
as MAP kinases, histone deacetylases, Ras-related proteins, and
isocitrate dehydrogenases.10,11 After IDUP, restriction diges-
tion, and PAGE analysis, we observed ∼1000-fold enrichment
of the sequence corresponding to SA+biotin. Replacing Klenow
exo− with T4 DNA polymerase and replacing a 6-nt
Figure 1. (A) Antibody-mediated interaction determination using
unpurified proteins (IDUP) uses DNA-linked antibodies to recognize
a target protein or epitope tag. (B) Alternatively, a covalent bond can
be formed between the target and identifying DNA strand in IDUP by
fusing the target to a self-labeling protein tag such as SNAP-tag, CLIP-
tag, or HaloTag. After primer extension and PCR, the resulting DNA
encodes all ligand+target combinations.
bound ligand. This extension product contains two primer-
binding sites and therefore can be amplified by PCR (Figure
1).3 By removing the requirement for purified protein targets,
the IDUP approach enables ligand-binding “selections” to be
performed on proteins that are free to undergo post-
translational modification, interact with endogenous accessory
proteins and metabolites, and access physiologically relevant
conformational states.5
RESULTS AND DISCUSSION
■
Pairs of DNA-linked antibodies have been used to measure the
presence of proteins and protein−protein complexes by the
proximity ligation assay6 and the proximity extension assay.7
On the basis of these concepts and our previous development
of interaction-dependent PCR (IDPCR),3 we speculated that
formation of a ternary complex of a DNA-linked antibody, a
protein target, and a DNA-linked small molecule could
promote hybridization of the linked oligonucleotides and
enable primer extension by a DNA polymerase in a manner
dependent on binding of the ligand to the target (Figure 1A).
Such a system would offer the benefits of IDPCR but without
the significant limitation of requiring a purified target protein
conjugated to a DNA oligonucleotide.
To test this hypothesis, quantitative PCR (qPCR) was used
to compare the amount of primer extension product from
reactions containing a DNA-linked anti-streptavidin antibody
(DNA-αSA), streptavidin protein (SA), and a DNA-linked
ligand. The DNA-linked ligand was varied among four small
molecules: DNA-linked hexylamine (DNA-amine, no signifi-
cant affinity for SA), DNA-linked Gly-Leu-carboxybenzene
sulfonamide (DNA-GLCBS, no significant affinity for SA), and
either one of two ligands of SA, DNA-desthiobiotin (Kd = 2
nM)8 or DNA-biotin (Kd = 40 pM).9 Consistent with
formation of an antibody+SA+biotin complex or an antibody
+SA+desthiobiotin complex, samples containing DNA-αSA,
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Figure 2. (A) The ability of IDUP to enrich the sequence
corresponding to a particular interaction is evaluated by incubating
mixtures of binding and nonbinding DNA-linked ligands with a target
protein and a DNA-linked antibody. After primer extension and PCR,
a restriction digest is used to determine the fraction of the amplified
sequences corresponding to the target+ligand interaction. (B) IDUP
with DNA-αSA and 0.01% SA in HeLa lysate shows selective
amplification of a sequence corresponding to SA+desthiobiotin in
qPCR (ΔCT = 4.7). (C) IDUP on a mock library containing mixtures
of DNA-desthiobiotin and DNA-GLCBS shows ∼1000-fold enrich-
ment of a sequence corresponding to SA+desthiobiotin. (D) When
analyzed by qPCR, IDUP with DNA-αCAII and 0.01% CAII in HeLa
lysate shows rapid amplification of sequences corresponding to CA+
GLCBS and CA+CBS, but not CA+desthiobiotin or CA+amine (ΔCT
= 4−5). (E) IDUP on a mock library containing mixtures of DNA-
GLCBS and DNA-desthiobiotin shows ∼10-fold enrichment of a
sequence corresponding to CAII+GLCBS.
Figure 3. (A) IDUP using DNA-αHis and genetically encoded His6-
tagged target proteins. (B) IDUP using DNA-αHis with 293T cell
lysate expressing CAII-His6 shows rapid amplification of sequences
corresponding to CA+GLCBS and CA+CBS but not CA+
desthiobiotin (ΔCT = 5−6). (D) IDUP using a mock library shows
∼10-fold enrichment of the sequence corresponding to CAII+GLCBS
and (F) ∼100-fold enrichment of a sequence corresponding to CAII+
CBS. (E) When the transfected lysate was diluted 1:10 into
untransfected lysate, the enrichment of the CAII+GLCBS sequence
increased to ∼100-fold. (C) IDUP using DNA-αHis with 293T cell
lysate expressing His6-BclxL shows rapid amplification of DNA-Bad
but not DNA-GLCBS or DNA-biotin (ΔCT = 8) and ∼100-fold
enrichment of a sequence corresponding to Bcl-xL+Bad (G).
complementary region with an 8-nt complementary region
resulted in a 100-fold improvement in enrichment of the
sequence encoding SA+biotin (Figures 2B,C and S1).
performed IDUP with DNA-αHis and purified, C-terminally
His6-tagged CAII (CAII-His6, 0.01% in HeLa cell lysate) and
observed that using DNA-αHis resulted in 100-fold enrichment
of DNA encoding CAII+GLCBS, representing a 10-fold
improvement over the enrichment factor using DNA-αCAII
(Figure S8). These results demonstrate the feasibility of IDUP
mediated by an epitope tag-binding antibody instead of an
antibody that directly binds the target protein’s coding
sequence.
To assess the compatibility of IDUP with unpurified,
genetically encoded targets, we transiently transfected HEK-
293T cells with a plasmid expressing CAII-His6. We performed
an IDUP enrichment experiment in the resulting cell lysate
using DNA-αHis and observed ∼10-fold enrichment of the
sequence corresponding to CAII+GLCBS (Figure 3B,D).
When we similarly performed IDUP using mixtures of DNA-
CBS/DNA-biotin, we observed ∼100-fold enrichment of the
sequence corresponding to CAII+CBS (Figure 3F). Enrich-
ment by antibody-mediated IDUP depends on formation of a
ternary complex of DNA−antibody+target+ligand−DNA.
According to a recent model of three-body binding,15 for
αHis-mediated IDUP (His+αHis, Kd = 10 nM), a target protein
concentration of 30 nM is optimal for CAII+GLCBS (Kd = 40
nM),12 but the optimal target concentration is 190 nM for CAII
+CBS (Kd = 3.2 μM).16,15 By Western blot, we determined that
the concentration of CAII-His6 in the 293T cell lysate was
To determine whether IDUP using T4 DNA polymerase
could enrich DNA sequences corresponding to weaker ligand−
target interactions, we similarly studied the interaction between
carbonic anhydrase II (CAII) and its ligand GLCBS (Kd = 40
nM)12 and observed 10-fold enrichment of the sequence
corresponding to CAII+GLCBS (Figure 2D,E), despite the
observation that the polyclonal antibody used to generate
DNA-αCAII appears to partially compete for ligand binding,
likely reducing the enrichment obtained by IDUP using DNA-
αCAII (Figure S7). Together, these results demonstrate that,
for targets for which suitable antibodies exist, antibody-
mediated IDUP provides a selection-like method for the
detection and reporting of small molecule−protein interactions
from cell lysates.
For some targets of interest, an antibody capable of
selectively binding the target in solution without obscuring
the ligand-binding site may be difficult to obtain. Because
oligohistidine is a rare sequence among naturally occurring
proteins,13 we expected that an antibody against the His6
epitope tag would be less likely to interfere with target protein
function, including ligand binding. We therefore investigated
the ability of an anti-His5 antibody (Qiagen, His6+αHis Kd =
1−50 nM)14 linked to DNA (DNA-αHis) to participate in
IDUP with His6-tagged target proteins (Figure 3A). We
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∼300 nM, corresponding to ∼130 nM in the IDUP assay
(Figure S9). Consistent with the model of three-body
binding,15 we observed optimal IDUP enrichment of the
sequence corresponding to CAII+GLCBS (∼100-fold) when
the 293T cell lysate transfected with a CAII-His6 expression
plasmid was diluted 1:10 into untreated 293T cell lysate
(Figures 3E and S9). The enrichment of the sequence
corresponding to CAII+CBS decreased to ∼10-fold in this
diluted lysate sample (Figure S9).
IDUP using DNA-αHis also resulted in ∼100-fold enrich-
ment of a sequence corresponding to Bcl-xL+Bad (Kd = 0.6
nM)17 when performed in lysates of 293T cells transfected with
a plasmid expressing His6-Bcl-xL (amino acids 1−212) (Figure
3G). Taken together, these results suggest that IDUP using the
DNA-αHis antibody can enrich sequences corresponding to
ligand+target combinations for unpurified targets in cell lysates.
Covalent protein−DNA linkages offer several potential
advantages during IDUP compared to non-covalent anti-
body−target or antibody−tag associations. We anticipated
that formation of a covalent bond between a target protein and
its identifying DNA might increase the stability of the DNA−
target entity and the sensitivity of IDUP for weaker small
molecule−target binding interactions. In principle, replacing
non-covalent antibody−target binding with a covalent linkage
can be accomplished by expressing the target protein as a fusion
to a self-labeling protein domain such as SNAP-tag,18,19 CLIP-
tag,20 or HaloTag.21 Moreover, the use of a small-molecule-
reactive tag removes the requirement for a non-covalent ternary
complex to form, and therefore reduces the assay’s dependence
on target protein concentration.15 Finally, the small size of self-
labeling proteins compared to antibodies (∼30 kDa vs ∼150
kDa) suggests that the former are less likely to obscure ligand-
binding sites or disrupt native protein−protein interactions.
Self-labeling proteins have been used successfully in protein−
ligand binding assays19,20 and in linking target proteins to
DNA.22 We therefore speculated that self-labeling proteins
expressed as fusions to target proteins might serve as effective
reagents for linking targets to DNA during IDUP.
We transiently transfected 293T cells with vectors expressing
N- or C-terminally SNAP-tagged CAII (SNAP-CAII or CAII-
SNAP). The resulting lysates were individually incubated with a
SNAP substrate, O6-benzylguanine (BG), linked to DNA
(DNA-BG) for 15 min before incubation with mixtures of
DNA-GLCBS/DNA-desthiobiotin or DNA-CBS/DNA-des-
thiobiotin. After IDUP and restriction digestion, we observed
∼100-fold enrichment of the sequences encoding CAII+
GLCBS and CAII+CBS in samples expressing SNAP-CAII or
CAII-SNAP, but not in untransfected samples or samples
expressing SNAP-tag alone (Figures 4B,D and S10).
Similarly, IDUP performed on 293T cell lysate expressing
SNAP-Bcl-xL or Bcl-xL-SNAP with DNA-BG and mixtures of
DNA-Bak/DNA-BakL78A/DNA-biotin resulted in ∼100-fold
enrichment of a sequence corresponding to Bcl-xL+Bak (Kd =
340 nM)23 but no enrichment of the DNA sequence encoding
an interaction between Bcl-xL and the closely related negative
control peptide BakL78A (Kd = 270 μM).23 (Figures 4C,E and
S10). Collectively, these results demonstrate the ability of
protein targets fused to self-labeling domains to participate in
IDUP. In contrast to our results with αHis-mediated IDUP, we
noticed that both N- and C-terminally SNAP-tagged proteins
resulted in roughly equivalent enrichment levels of DNA
encoding known ligand−target pairs, suggesting that the SNAP-
Figure 4. (A) IDUP in cell lysates expressing a SNAP-tagged target
protein. DNA was rapidly amplified in samples corresponding to
known interactions: (B) SNAP/CAII+(GL)CBS (ΔCT = 5−6), (C)
SNAP/Bcl-xL+Bad or Bak (ΔCT = 8−9), (F) SNAP-FRB+rapamycin
(ΔCT = 6), and (G) FKBP-SNAP+rapamycin (ΔCT = 6). (D) A
sequence corresponding to CAII+CBS was enriched ∼100-fold in a
sample expressing either SNAP-CAII (lane 7) or CAII-SNAP (lane 8).
(E) In samples expressing SNAP-Bcl-xL, a sequence corresponding to
the interaction between Bcl-xL+Bak was enriched ∼100-fold (lane 11),
but no enrichment was observed for a sequence corresponding to
BclxL+BakL78A, a weakly binding mutant of the Bak peptide (lane 9).
(H) Overexpression of FKBP with SNAP-FRB increased the
enrichment of a sequence encoding FRB+rapamycin by 10-fold
compared to a sample transfected with SNAP-FRB alone. (I)
Overexpression of FRB with FKBP-SNAP also increased the
enrichment of a sequence corresponding to FKBP+rapamycin by 10-
fold compared to a sample transfected with FKBP-SNAP alone.
tag also offers increased generality compared to the His6-tag+
αHis approach.
The ability of IDUP to evaluate ligand−protein binding in
complex mixtures enables the detection of interactions that
require exogenous factors. For example, the interaction of
rapamycin with FRB, the rapamycin-binding domain of mTOR,
is substantially increased in the presence of another rapamycin-
binding protein, FKBP; the Kd of rapamcyin+FRB is 26 μM,
while the Kd of FKBP·rapamycin+FRB is 12 nM.24 We
wondered whether the FKBP-dependent modulation in the
strength of the FRB+rapamycin interaction could be detected
by IDUP. We conjugated azide-linked rapamycin24 to DNA
using the Cu(I)-catalyzed azide−alkyne cycloaddition reac-
tion.25 When we performed IDUP using 293T cell lysates
overexpressing SNAP, SNAP-FRB, or SNAP-FRB and FKBP,
we observed ∼10-fold enrichment of the sequence correspond-
ing to FRB+rapamycin in the sample overexpressing SNAP-
FRB and ∼100-fold enrichment in the sample overexpressing
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SNAP-FRB and FKBP (Figure 4F,H). Similar results were
obtained with recombinant, preconjugated DNA-FRB (Figure
S11). The rapamycin·FRB complex also has a higher affinity for
FKBP (Kd ≈ 100 fM) than rapamycin alone (Kd ≈ 0.2 nM).24
293T cell lysate overexpressing FRB and FKBP-SNAP also
showed ∼10-fold greater enrichment for a sequence encoding
FKBP+rapamycin than a sample overexpressing FKBP-SNAP
alone (Figure 4G,I). Together these results demonstrate that
IDUP results can reflect the influence of accessory proteins on
the target affinity of small-molecule ligands.
Because a key advantage of IDUP is the ability to
simultaneously assay all interactions between combined
libraries of targets and ligands in a single solution, we next
tested the ability of IDUP to selectively enrich known target+
ligand interactions from a model library containing 262 DNA-
linked ligands (comprising DNA-linked GLCBS, CBS,
rapamycin, Bad, Bak, BakL78A, and hexylamine linked to a
set of 256 DNA sequences) and 259 DNA-linked targets. To
generate a library of 259 DNA-linked targets, lysates from 293T
cells previously transfected with vectors encoding SNAP-FKBP,
SNAP-CA, SNAP-Bcl-xL, or SNAP were individually incubated
with BG linked to unique DNA sequences (in the case of
SNAP-FKBP, SNAP-CA, or SNAP-Bcl-xL) or to a library of
256 sequences (in the case of SNAP alone), quenched with a
free BG derivative, and pooled to obtain an equimolar ratio of
the 259 DNA-linked targets. As a control, aliquots of the same
lysates were separately incubated with DNA sequences lacking
conjugated BG. Both samples were incubated with the library of
DNA-linked ligands and processed by primer extension, PCR,
and high-throughput DNA sequencing using conditions
identified in previous experiments (Figures S2 and S6). We
divided the number of sequence counts for each protein+ligand
sequence from the sample treated with DNA-BG by the
corresponding number of counts from the sample treated with
DNA alone and observed enrichment factors from 68.9 to
328.7 for sequences corresponding to all five known ligand−
target binding interactions, including FKBP+rapamycin, CAII+
GLCBS, CAII+CBS, Bcl-xL+Bad, and Bcl-xL+Bak (Figure 5).
The mean enrichment for all 67,858 possible ligand+target
sequences was 1.5. We observed strong enrichment of the
sequences corresponding to all of the known target+ligand
interactions, despite the fact that the corresponding dissociation
constants vary over 4 orders of magnitude (Kd ≈ 0.2 nM−3.2
μM). No sequences corresponding to any presumed non-
binding interactions were enriched greater than 31-fold, and
only 21 presumed false-positive sequences had enrichment
factors greater than 20, a signal level less than one-third that of
the weakest bona fide positive (Tables S1 and S2).
Figure 5. (A) Cell lysates expressing SNAP-CA, SNAP-Bcl-xL, and
SNAP-FKBP were individually labeled with one of three DNA
sequences and combined with a cell lysate expressing SNAP and
labeled with 256 DNA sequences. The pooled lysates were combined
with a library of 262 DNA-linked small molecules, including DNA-
linked GLCBS, CBS, Bad, Bak, BakL78A, and rapamycin, for a model
library×library IDUP “selection”. (B) IDUP using a library of cell
lysates expressing SNAP-target fusions identified all five known target+
ligand pairs, including A - FKBP+rapamycin, B - Bcl-xL+Bad, C - Bcl-
xL+Bak, D - CAII+GLCBS, and E - CAII+CBS, despite having
affinities from 0.2 nM to 3.2 μM. (C) For interactions with Kd = 40
nM−26 μM, we observed a strong relationship between the log of
target−ligand Kd and the number of sequence counts after selection.
including concentrations of individual library members, differ-
ences in the expression levels of SNAP-tagged targets, and
differences in the extent to which both DNA-linked ligands and
SNAP-tagged targets are obscured by factors present in the cell
lysate. For example, 293T cells have been shown to natively
express FK506-binding proteins,28 mTor,29 CAII,26 and
BclxL,27 and these untagged targets may compete for ligand
binding. Despite these potential complications, for interactions
with dissociation constants from 40 nM to 26 μM, we observed
a surprisingly strong relationship between log(Kd) and the
number of counts observed after selection (Figures 5C and
S13). In principle, this relationship can be used to estimate the
detection limit of the IDUP assay (here 30−60 μM; see Figure
S13B) and to infer the affinities of newly detected interactions.
The relationship between affinity and enrichment factor was
less strong than that between affinity and sequence counts.
Enrichment factor values also depend on the number of
sequence counts in the negative control sample, which are
generally smaller (despite 12- to 18-fold sequence coverage)
and more susceptible to variation caused by DNA sequence
bias and sampling stochasticity during PCR or high-throughput
sequencing. Plotting enrichment factors is, however, an effective
way to distinguish true binding events from presumed false
positives by eliminating sequences likely amplified due to PCR
bias (Figure S13C,D). Interactions with Kd ≈ 0.2−0.6 nM did
not follow the linear trend (Figure S13A). A plausible
explanation for this observation is that the concentration of
each library member during IDUP is 0.4 nM, and thus
When we performed a similar IDUP experiment containing
260 DNA-linked targets, including both SNAP-FKBP and FRB-
SNAP, we observed all of the known interactions except for
that of FRB+rapamycin, likely due to both the relatively weak
affinity of rapamycin for FRB (Kd = 26 μM)24 and the relatively
low expression level of FRB-SNAP (Figure S12). In an IDUP
experiment containing FRB-SNAP but lacking SNAP-FKBP, we
observed 23.4-fold enrichment of the sequence corresponding
to rapamycin+FRB, with only three presumed false positive
sequences enriched as strongly (Figure S12). Together, these
results validate the ability of IDUP to identify interactions
between combined libraries of small molecules and SNAP-
tagged target proteins in crude cell lysates.
The relationship between target−ligand affinity and sequence
counts following IDUP should be governed by several factors,
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interactions with affinities in this range (here, FKBP+rapamycin
and Bcl-xL+Bad) could approach binding saturation.
REFERENCES
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CONCLUSIONS
■
IDUP is a method for rapidly evaluating potential small-
molecule−target interactions from mixtures in a single solution
that is compatible with unpurified targets in biological samples.
The ability to identify ligand+target pairs from complex
samples including cell lysates offers significant advantages
compared to other methods for evaluating DNA-encoded
chemical libraries. Samples in cell lysates are able to undergo
native post-translational modification and interact with
accessory proteins and metabolites in ways that better reflect
their relevant biological environment. Because IDUP is
compatible with crude cell lysates, difficult-to-purify, poorly
soluble, intrinsically unstable, and aggregation-prone targets
may also be compatible with this method, without requiring
truncation or other strategies used to promote heterologous
expression. We demonstrated the ability of IDUP to selectively
amplify DNA sequences corresponding to interactions between
His6-tagged or SNAP-tagged target proteins and their ligands in
cell lysates, and we have demonstrated that IDUP results reflect
the ability of accessory proteins to modulate ligand−target
affinity. We also demonstrated the ability of IDUP to selectively
enrich DNA sequences corresponding to known protein+ligand
interactions with affinities from 0.2 nM to 26 μM from a library
of SNAP-target-expressing cell lysates and a library of DNA-
linked small molecules. Moreover, we observed a relationship
between sequence counts and ligand−target identity, suggest-
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identify new interactions but also estimate their affinities. We
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strategy to evaluate DNA-encoded libraries under conditions in
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ASSOCIATED CONTENT
■
S
* Supporting Information
Additional information and experimental methods. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
drliu@fas.harvard.edu
(15) Douglass, E. F., Jr.; Miller, C. J.; Sparer, G.; Shapiro, H.; Spiegel,
D. A. J. Am. Chem. Soc. 2013, 135, 6092.
Notes
The authors declare the following competing financial
interest(s): D.R.L. is a consultant for Ensemble Therapeutics,
a company that uses DNA-templated synthesis and in vitro
selection for drug discovery.
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ACKNOWLEDGMENTS
■
This work was supported by the Howard Hughes Medical
Institute and the NIH/NIGMS (R01GM065865). L.M.M.
gratefully acknowledges fellowships from the NSF GRFP and
the Harvard Graduate Society. Plasmids expressing CAII and
Bcl-xL were the kind gifts from Carol Fierke and Loren
Walensky, respectively. We thank Rick McDonald and Ryan
Hili for many helpful discussions.
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