Selection of DNA-Encoded Libraries to Protein Targets within and on Living Cells

Article abstract of DOI:10.1021/jacs.9b08085

We report the selection of DNA-encoded small molecule libraries against protein targets within the cytosol and on the surface of live cells. The approach relies on generation of a covalent linkage of the DNA to protein targets by affinity labeling. This c

Full text of DOI:10.1021/jacs.9b08085

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Communication
Selection of DNA-encoded Libraries to
Protein Targets Within and On Living Cells
Bo Cai, Dongwook Kim, Saeed Akhand, Yixing Sun, Robert Cassell, Aktan Alpsoy,
Emily C. Dykhuizen, Richard M. van Rijn, Michael K. Wendt, and Casey Krusemark
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b08085 • Publication Date (Web): 15 Oct 2019
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Selection of DNA-encoded Libraries to Protein Targets Within and
On Living Cells
Bo Cai, Dongwook Kim†, Saeed Akhand, Yixing Sun, Robert J. Cassell, Aktan Alpsoy, Emily C.
Dykhuizen, Richard M. Van Rijn, Michael K. Wendt and Casey J. Krusemark*
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Department of Medicinal Chemistry and Molecular Pharmacology, Purdue Center for Cancer Research, Purdue University,
West Lafayette, IN 47907, USA
Supporting Information Placeholder
over non-ligands, particularly for low affinity binders or
with protein targets at low concentration. Crosslinking
also allows assessment of binding interactions in
solution and can be conducted in complex environments
such as cell lysates. Extension of this approach into live
cells would have further advantages in limiting protein
dilution, which is typically 10-fold or greater in cell
lysates.6
ABSTRACT: We report the selection of DNA-encoded
small molecule libraries against protein targets within
the cytosol and on the surface of live cells. The
approach relies on generation of a covalent linkage of
the DNA to protein targets by affinity labeling. This
crosslinking event enables subsequent co-purification
by a tag on the recombinant protein. To access targets
within cells, a cyclic cell-penetrating peptide is
appended to DNA-encoded libraries for delivery
across the cell membrane. As this approach assesses
binding of DELs to targets in live cells, it provides a
strategy for selection of DELs against challenging
targets that cannot be expressed and purified as
active.
non-ligand
Control
cCPP
cCPP
cCPP
Reactive group
ssDNA
Ligand
cCPP
DNA-encoded
Chemical LIbrary
1. Live Cells
expressing
Halo-Tag protein
1. Lyse cells
2. Protein capture
The discovery of small molecule ligands to protein
targets is of fundamental importance for biological
research and therapeutic development. DNA-encoded
chemical libraries (DELs) have emerged as an important
tool in molecular discovery.1 The high sensitivity and low
cost of DELs allow exploration of large portions of
chemical space. These benefits arise because DNA-
encoding allows collective, rather than individual,
assessment of molecular function via an in vitro selection
assay.2 Unlike high throughput screening, the cost and
difficulty of selection assays do not scale appreciably
with the number of compounds. The traditional DEL
selection using purified, immobilized protein targets
cannot be applied to many important drug target
classes.3 This includes proteins that cannot be expressed
recombinantly and purified in their fully active form. An
approach to enable selection of DELs to targets within
live cells would not only remove the onerous
requirement for a pure, active protein but also assess
proteins in a more functionally relevant state, where
critical binding partners or posttranslational
modifications can be maintained.
cCPP
2. Incubation with
Reactive DEL
3. Denaturing
washes
Enriched Ligand
Scheme 1. Crosslinking approach to live cell DEL selections.
While there are reports of cell-based selections for
aptamers,7 this has been relatively unexplored with
DELs. Israel and coworkers applied a cell-based selection
with DELs to a cell surface protein, the NK3 Tachykinin
GPCR.8 This approach required high expression of the
protein target. Thus, limitations arise for proteins that
are toxic when expressed at high levels (ion channels, e.g.)
or for discovery of ligands with low or modest affinity.
Here, we apply covalent crosslinking to enable DEL
selections against targets both within and on living cells.
For selection of targets within live cells, delivery of
DELs into the cytosol presents a challenge. To achieve
this, we conjugated DNA-linked molecules to a cyclic,
cell-penetrating peptide developed by Pei and
coworkers,9 which has shown high cell entry and
endosomal escape efficiency. Our approach is outlined in
Scheme 1. A protein target is expressed in cells
containing a fusion tag to enable later covalent capture.
Upon cytosolic delivery of a DEL, the binding event
between a protein and a DNA-linked ligand enables a
covalent bond to be formed between the protein and
DNA by affinity labeling. Cells are then lysed, and all
target proteins are captured on beads. After stringent,
Prior work by our lab and others has applied covalent
crosslinking by affinity labeling to expand the
capabilities of DEL selections.4,5 By trapping the
transient interaction of DEL ligands to the target,
crosslinking can give improved enrichment of ligands
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protein-denaturing washes, enriched ligands are
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~11% cCPP12–DNA was recovered, 170-fold higher
than the recovery of DNA lacking the cCPP12. In contrast,
the recovery of a 140 bp DNA by cCPP12 was ~1%
(Figure 1B). These qPCR tests indicated that significant
amount of DNA was amplifiable after incubation and that
cell entry is length-dependent. Treatment of cells with
cationic lipid transfection reagents (Lipofectamine, e.g.)
showed slightly improved DNA uptake compared to
cCPP12 (Table S3); however, subsequent affinity
labeling and selection experiments were unsuccessful,
suggesting that DNA in these cases may be trapped
within endosomes.
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identified by DNA sequencing.
To evaluate the cellular uptake, we appended cCPP12
to a 20-base oligonucleotide via a disulfide bond
(Schemes S1 and S2), such that constructs might
accumulate in the cytosol under reducing conditions.
This cCPP oligo was hybridized to a 6-carboxyfluorescein
amide (FAM)-linked oligo to yield a 20-base pair (bp),
double-stranded DNA (cCPP12–DNA–FAM). Cell entry
was assessed by fluorescence microscopy (Figure 1A).
We observed comparable cell entry of cCPP12–DNA–
FAM to cCPP12-fluorescein, while no fluorescence was
observed with DNA–FAM.
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In addition to overall uptake, the actual cytosolic
delivery of DNA to protein targets is critical. To evaluate
the cytosolic penetration of cCPP12-DNA, we performed
the HaloTag-based12 chloroalkane penetration assay
(CAPA).13 When cells were treated with 5 M cCPP12-
linked chloroalkane (CA)-DNA (20-base dsDNA), the
fluorescence intensity was reduced by ~30% (Figure
1C), indicating cCPP12 allows cytosolic delivery of DNA.
Results were similar, but less pronounced (~20%
reduction), using 2.5 M CA-DNA (Figure S1).
A
Overlay
Transmitted
FITC
DNA-FAM
cCPP12-
DNA-FAM
The cGAS–STING pathway detects the presence
of cytosolic DNA and triggers an immune response.14
While prior experiments showed no concerns with cell
viability, we sought to avoid this and investigated cell
lines that lack cGAS or STING expression: HEK293T,
FreeStyle™ 293-F, U937 cGAS-/- and U937 STING-/-.14 We
found the FreeStyle™ 293-F cells, which are adapted to
suspension culture, gave the best results. These cells
showed high transfection efficiency and were easy to
handle at high cell density, which gave improved cell
entry of DNA by qPCR (Table S4).
cCPP12-
FAM
We evaluated interactions between DNA-linked ligands
and cytosolic proteins by affinity labeling and gel
analysis. Within 293-F cells, we expressed two HaloTag
fusion proteins: the Chromobox Protein Homolog 7
Chromodomain (CBX7-ChD) and E. coli-dihydrofolate
reductase (eDHFR). Affinity labeling within live cells
used DNA constructs containing a ligand, FAM, and a
sulfonyl fluoride as a reactive crosslinker (Figure S2).
For CBX7-ChD, we used a previously reported modified
peptide ligand (4-BrBA) (Figure 2A, Kd ~ 270 nM off-
Figure 1. Cell penetration of DNA is enabled by cCPP12. (A)
Live cell entry of a dsDNA-cCPP12 conjugate. 20-base
dsDNAs containing FAM labels and
a cCPP12-FAM
conjugate were incubated with HMLE cells at 2 M for 4.5
hours and imaged. (B) qPCR analysis of DNA cell entry. (C)
CAPA performed with 5 M 20-base dsDNA. P-values were
calculated using unpaired Student’s two-tailed t-test: ***P <
0.001.
DNA, 26 nM on-DNA).15 For eDHFR,
a modified
trimethoprim (TMP) was used (Figure 2B, Kd ~1 nM off-
DNA).16 After incubation, cells were washed and lysed
directly with SDS-PAGE buffer and excess free ligand to
ensure that labeling occurred within the cell rather than
the lysate. Labeling was observed and was dependent on
the presence of the cCPP12, the ligand, and the sulfonyl
fluoride on the DNA. With Halotag-eDHFR tests, addition
of excess TMP ligand eliminated labeling (Figure 2B, lane
5), additionally verifying that labeling occurred within
the cell. Bands occurred at expected molecular weights
for the proteins linked to 40 bp DNA. Although
Lipofectamine gave higher cellular DNA delivery as
While the cCPP12 allows uptake of DNA, the amount of
amplifiable DNA remaining in cells is another key
consideration. Nuclease degradation of DNA constructs
may be problematic. DNase II is the predominant
nuclease within endosomes and lysosomes that
degrades exogenous DNA.10,11 To determine the amount
of amplifiable DNA remaining in cells, we performed
qPCR analysis of cell lysates prepared after treatment of
cCPP12-DNA. We also investigated the effect of DNA
length on cellular uptake. When delivering a 60 bp DNA,
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assessed by qPCR, the absence of conjugated band in this
case (Figure 2A, lane 4) suggests poor cytosolic entry.
ligand displacement assay.15 Values roughly correlated
to observed enrichments (Figure 3D and Table S13).
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Figure 2. Assessing DNA-linked ligand-target interactions
inside live cells. Affinity labeling of CBX7-ChD (A) and
eDHFR (B) with DNA-linked ligands analyzed by SDS-PAGE.
Encouraged by successful labeling experiments, we
performed a test selection assay against Halotag-CBX7-
ChD within live cells using three model constructs
(Figure 3A and S3). An encoding DNA of 60 bp was used,
which is the approximate size within many DELs.1 In an
initial test, the recovery of the positive control ligand P1
(BrBA-60 bp DNA-cCPP12, Figure 3A) was 0.012%, 20-
fold higher than that of N1 (non-ligand control, Figure 3A)
and 22-fold higher than that of N2 (non-cCPP12 control,
Figure 3A). Enrichments were significantly greater
(~600-fold, Table S5) in analogous experiments in cell
lysates, suggesting that much of the DNA recovered at
cell lysis was unable to access the cytosol. Enrichment
and recovery values are critical parameters that govern
the amount and complexity of DELs used for a selection
to ensure against under sampling of the library.17
Figure 3. DEL selection against Halotag-CBX7-ChD
within living cells. (A) Recovery and enrichment of
tested DNA constructs in a mock selection at 1000-fold
dilution. (molar ratios P1:N1:N2 = 1:500:500; [P1] = 1
nM). (B) Correlation plot of live cell and cell lysate
selections. (C) The parental peptide for a DNA-encoded
PSL and a heat map of live cell enrichments. The color
scale represents the log enrichment relative to the non-
ligand control. See Figures S6 and S7 for structures. (D)
IC50 values of off-DNA hits in binding assay to Halotag-
CBX7-ChD. Line colors correspond to live cell
enrichment values from the selection, as indicated on the
color scale.
We constructed a 96-membered DEL by varying 24
building blocks at each of four monomer positions within
a known CBX7 ChD ligand (4-BrBA) to create a positional
scanning library (PSL). Constructs from the 3-member
test selection were included as controls. Selections were
additionally performed against Halotag-CBX7-ChD in
lysates and purified Halotag-CBX7-ChD. Consistent with
the qPCR selection, recovery of the positive control
ligand was ~20 fold higher than the non-ligand control
(Figure 3C) when assessed by DNA sequencing (Table
S5). An additional control, N2, which contained the BrBA
ligand but lacked the cCPP, showed no enrichment in the
live cell selection (Figure 3C), but showed comparable
enrichment to the positive control in cell lysate
selections (Table S5 and S11). Sequencing results
showed differential enrichment of molecules (Figure S6
and S7) consistent with the reported SAR to the CBX7
ChD.15 Enrichment of each library member in the live cell
selection correlated well with the cell lysate selection
(Figure 3B). To validate selection hits, we synthesized 10
molecules off-DNA and assessed their IC50 values in a
In addition, we sought to test this approach with an
integral membrane protein target, the  opioid receptor
(DOR), as an example of a target that is not amenable to
purification and immobilization. First, we prepared an
on-DNA control ligand for this target, Dmt-Tic-Lys (DTK,
2,6-dimethyl-L-tyrosine (Dmt), 1,2,3,4-tetrahydroiso-
quinolone-3-carboxylic acid (Tic)), which binds with
high affinity (Ki ~ 150 pM, Figure S4).18 Imaging of HEK-
293T cells expressing SNAPtag-DOR after treatment with
fluorescently-labeled DNA constructs showed that
retention of the fluorescent signal was dependent on
both the ligand and the crosslinker (Figure S5).
Application of the selection approach with 293F cells
expressing DOR (Figure 4A) gave a 180-fold enrichment
of the DTK ligand over a non-ligand control at 100-fold
dilution (Figure S4). Similar enrichment and recovery
were observed when reducing the DTK ligand
concentration 10-fold (from 1 nM to 0.1 nM).
We constructed a 96 compound DEL using Dmt-Pro-
Phe-Phe (Ki ~ 28 nM)19 and Tyr-Tic (Ki ~ 240 nM)20 as
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parental peptides and applied the selection. Compounds
from the higher affinity parental peptide were more
greatly enriched (Figures 4B, S8 and S9). The DTK ligand
doped into the DEL gave 300-fold enrichment. Similarly,
the Dmt-Tic pair was the highest enriching ligand among
the low affinity set (LA-P1-#1, 13-fold). We synthesized
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expression, further work may enable targeting
endogenous proteins by using immunopurification or
incorporation of affinity tags on targets using
CRISPR/Cas. In addition to the PSLs demonstrated here,
we expect this approach to find application in de novo
ligand discovery with combinatorial DELs, on-DNA hit
validation, and DNA-based protein activity assays.22
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off-DNA ligands and evaluated their Ki by a
radiolabeled ligand displacement assay.21 Ki values
roughly correlated to the enrichments with the
exception of (HA-P1-#6), which did not show binding
(Figure 4C and Table S14). Compounds HA-P(2)-#10 and
HA-P(2)-#11 (both containing non-alpha amino acids)
are novel DOR ligands of high affinity.
ASSOCIATED CONTENT
9
Supporting Information. Synthetic procedures,
evaluation of DNA cell entry, protein expression, cell
culture, selection details, off-DNA hit validation, library
information, and characterization data is available free
of charge on the ACS Publications website.
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AUTHOR INFORMATION
Corresponding Author
cjk@purdue.edu
Present Addresses
† Dencoda, LLC, West Lafayette, IN 47907, USA
Notes
DK is the founder and CEO of Dencoda, LLC, a company that
uses DNA-linked molecules as sensors for diagnostic and
drug discovery purposes.
ACKNOWLEDGMENT
The authors would like to thank Dr. Joshua A. Kritzer for
HeLa cells stably expressing the HaloTag-GFP and Dr.
Lingyin Li for U937 cell lines. This work was supported
by NIH (1R35GM128894-01 to CJK; CA207532 to ECD;
AA025368, AA026949, AA026675, and DA045897 to
RMvR), the American Cancer Society (RSG-16-172-01 to
MKW), and by fellowship support to BC from the Lilly
Endowment to the College of Pharmacy at Purdue
University. The Purdue University Mass Spectrometry
and Genome Sequencing Shared Resources are
supported by P30 CA023168 from the National Institutes
of Health.
Figure 4. DEL selection against SNAPtag-DOR on live
cell surfaces. (A) Crosslinking approach to DEL
selections on live cell surfaces. BG-biotin = O-(6)-
benzylguanine-biotin. (B) Parental peptides for DNA-
encoded PSLs and heat maps of live cell enrichments. The
color scale represents the log enrichment relative to the
non-ligand control. See Figures S8 and S9 for structures.
(C) Ki values of off-DNA hits binding to SNAPtag-DOR.
Line colors correspond to live cell enrichment values
from the selection, as indicated on the color scale. *Ki of
HA-P(2)-#5 was determined on Prolink-tagged DOR
(DiscoverX).
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