Selecting a DNA-Encoded Chemical Library against Non-immobilized Proteins Using a "ligate-Cross-Link-Purify" Strategy

Article abstract of DOI:10.1021/acs.bioconjchem.7b00343

DNA-encoded chemical libraries (DELs) have recently emerged and become an important technology platform in biomedical research and drug discovery. DELs containing large numbers of compounds can be prepared and selected against biological targets to discov

Full text of DOI:10.1021/acs.bioconjchem.7b00343

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Article
Selecting DNA-encoded Chemical Library Against Non-
immobilized Proteins Using a “Ligate-Crosslink-Purify” Strategy
Bingbing Shi, Yuqing Deng, Peng Zhao, and Xiaoyu Li
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00343 • Publication Date (Web): 25 Jul 2017
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Selecting DNA-encoded Chemical Library Against Non-immobilized
Proteins Using a “Ligate-Crosslink-Purify” Strategy
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Bingbing Shi†,‡, Yuqing Deng‡, Peng Zhao‡,§, and Xiaoyu Li*,‡
†Key Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Peking University Shen-
zhen Graduate School, 2199 Lishui Road West, Shenzhen, China 518055
‡Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.
§Institute of Nuclear Physics and Chemistry, China Academy of Engineering Physics, 64 Mianshan Road, Mianyang,
Sichuan, China 621900
Email: xiaoyuli@hku.hk
ABSTRACT: DNA-encoded chemical library (DEL) has recently emerged and become an important technology platform in
biomedical research and drug discovery. DELs containing large numbers of compounds can be prepared and selected
against biological targets to discover novel ligands and inhibitors. In practice, DELs are usually selected against purified
and immobilized proteins using the typical “bind-wash-elute” protocol; however, selection methods compatible with non-
immobilized proteins would be able to greatly expand the target scope of DELs beyond purified proteins to more complex
and biologically relevant targets. Using a novel “ligate-crosslink-purify” strategy, here we report a method capable of select-
ing DELs against unmodified and non-immobilized protein targets. In addition, this method has shown excellent capability
in identify binders with moderate and weak affinities.
typical “bind-wash-elute” protocol: 1) the library is incu-
bated with a matrix-immobilized target, usually a purified
INTRODUCTION
Encoded combinational libraries are powerful
discovery tools in biomedical research. Large scale peptide,
antibody, protein, and nucleic acid libraries can be
protein; 2) non-binding library compounds are removed by
multiple washing steps; 3) bound compounds are eluted
under strong denaturing conditions and then subjected to
PCR amplification and DNA sequencing for hit deconvolu-
tion.65 However, not all proteins are suitable for purifica-
tion and immobilization, and many immobilized proteins
often display altered properties from their soluble form or
even a complete loss of activities.66-70 Therefore, selection
methods compatible with non-immobilized proteins
would be able to extend the target scope of DEL to more
complex biological targets such as protein complexes,71
membrane proteins,52, 72-74 and even live cells.75-77 In addi-
tion, selection with unpurified endogenous proteins can
also preserve some desirable features such as post-transla-
tion modifications and endogenous binding partners.78
routinely built with platforms such as phage display,1-4
6
mRNA display,5,
yeast surface display,7-9 ribosome
display,10-12 SELEX,13-17 etc. Recently, DNA-encoded
chemical library (DEL) has emerged and become an
important technology platform for ligand and inhibitor
discovery in biomedical research.18-48 In a DEL, each
compound is covalently conjugated with a unique DNA tag,
serving as the identifier for the chemical structure of the
compound. Therefore, the entire library can be
synthesized and processed simultaneously at minute scale.
The original concept was proposed and explored by
Brenner and Lerner18-19 and also Gallop20. During the next
two decades, this field has seen significant developments
on library synthesis, selection and deconvolution. Today,
DELs containing hundreds of millions to many billions of
compounds are routinely prepared and screened against
various biological targets.21, 32, 34-36, 39 Moreover, DEL has
also been widely adopted by research institutions and
pharmaceutical companies in numerous drug discovery
programs;22-29, 34, 39-43, 49-63 it is not unreasonable to foresee
In
a pioneering work, Gallop and co-workers
synthesized a heptapeptide DEL immobilized on beads so
that soluble protein targets can be directly selected;20 more
recently, Paegel and co-workers have built large-scale
peptide DELs on solid phase but with more sophisticated
multi-functional linkers.79-81 Moreover,
a variety of
strategies have also been developed to select solution-
phase DELs against non-immobilized proteins. Liu and co-
workers developed methods capable of targeting
endogenous proteins in cell lysates using the interaction-
dependent PCR.78, 82, 83 Vipergen reported a Binder Trap
DEL to become
a
major technology platform
complementing traditional HTS in near future.32, 35, 42, 64
Typically, selections of DELs are achieved with immo-
bilized protein targets based on binding affinity using the
Enrichment method based on water-oil emulsion.27,
84
Biophysical method such as kinetic
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Figure 1. The proposed “ligate-crosslink-purify” method: li-
brary DNA is ligated with a PC-DNA (photocrosslinker DNA)
at the end distal to the small molecule. The PC-DNA has a
short sequence complementary to the PBS (primer-binding
site), therefore a hairpin structure is formed. After target bind-
ing and light irradiation, the target-DNA conjugates formed
are gel-purified and separated from non-binders for hit iden-
tification by PCR amplification and DNA sequencing.
capillary electrophoresis (KCE) has also been used to
86
partition ligands from non-binders.85,
Recently,
Krusemark and co-workers reported a photo-crosslinking-
based selection method.87 In addition, libraries of PNA-
encoded small molecule libraries equipped with
chemically reactive groups have been used to profiling
endogenous enzymes in lysates.88-91
Figure 2. a) Selection scheme of hairpin 1 with avidin. Condi-
tions: 1, 2.5 µM; avidin: 2.5 µM; buffer: 0.1 M NaCl, 1x PBS; ir-
radiation: 365 nm for 30 sec. at 0 °C; b) Electrophoresis analy-
sis of the photo-crosslinking reaction (15% SDS-PAGE). M:
marker; lane 1 and lane 3-4: reactions are as marked; lane 2: a
mixture of avidin and avidin-1 standard sample, prepared by
direct conjugation.94 c) Denaturing PAGE (15% TBE-Urea)
analysis of the PCR products. M: marker; lane 1: PCR products
from the avidin-1 conjugate (lane 2 in b); lane 2: after HindIII
digestion; lane 3-4: same as lane 1-2 but using a 42-nt positive
control DNA with a HindIII digestion site. d) Mixtures of 1 and
2 were selected against avidin and digested with the same pro-
cedure as in a). PCR products were analyzed with 15% PAGE.
e) qPCR analysis of DNA recovery and enrichment in the
1:1000 sample in d). **: (post-selection 1/2 ratio)/(pre-selection
1/2 ratio). ***: (post-selection percentage of 1)/(pre-selection
percentage of 1). See Supporting Information and Table S1 for
details.
Previously we reported a method compatible with un-
modified and non-immobilized targets using a terminal
protection strategy.92 In this method, target-binding lig-
ands are crosslinked to the protein, and then exonuclease
I (ExoI) is used to digest the DNA tags of non-binders while
the tags of bound ligands are protected by the target pro-
tein. However, this method is limited to DELs with small
molecules conjugated at the 3’-end of the library DNA and
the use of exonuclease leads to over-digestion of the DNA
tags of binding ligands.92, 93 In order to address these issues,
here we report the development and application of a novel
selection method that solves these problems and can be di-
rectly applied to existing DELs without library redesign or
resynthesis.
due to the relatively lower molecular weight (Figure 1).
Therefore, the main purpose of the ligation step is to es-
tablish a stable covalent link between the target protein
and the DNA codes of the ligands selected by the target, so
that the target-DNA conjugates can be reliably separated
from non-binders by gel purification and subjected to fur-
ther sample processing under various experimental condi-
tions. Moreover, this method avoids the drawbacks of nu-
clease digestion and can be applied to DELs with either 3’-
or 5’-conjugated small molecules.
RESULTS AND DISCUSSION
Our proposed method is based on a “ligate-crosslink-
purify” strategy. As shown in Figure 1, for a given DEL, a
short DNA strand (PC-DNA: photocrosslinker-DNA) can
be enzymatically ligated to the library at the end distal to
the small molecule. The PC-DNA contains a sequence
complementary to the primer binding site (PBS) of the li-
brary DNA, thereby forming a hairpin structure and bring-
ing the photo-crosslinker close to the small molecule. The
hairpin-shaped library can then be selected against non-
immobilized target proteins. After light irradiation, im-
portantly, for binders, a covalent “protein-DNA” conjugate
is formed and can be purified and separated from non-
binders by electrophoresis, before typical PCR amplifica-
tion and DNA sequencing are applied for hit deconvolu-
tion. For non-binders, they are not crosslinked the target
protein and are removed during gel purification
As an initial test, we prepared a 42-nt hairpin DNA (1;
Figure 2a), modified with a 5’-desthiobiotin and 3’-phenyl-
azide crosslinker. 1 forms a stable hairpin structure with a
10-bp stem (Tm: 54.5 °C in 0.1 M NaCl; Figure S2); it also
contains a digestion site specific for restriction enzyme
HindIII (5’-AAGCTT-3’) in the loop. Desthiobiotin is a high
affinity ligand for avidin with a Kd of ~2.0 nM.95 1 was incu-
bated with avidin (2.5 µM), irradiated under 365 nm for 30
seconds, and then analyzed by electrophoresis. As shown
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in Figure 2b, a slower moving band matching the molecu- while the negative control 4 has a mixed sequence (A, C or
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lar weight of the avidin-1 conjugate was detected (lane 3).
No such band was observed without desthiobiotin or light
(lane 4 and 5), proving the formation of the conjugate is
specific. The band was excised, extracted, PCR-amplified,
and then digested by HindIII, and it is shown that the PCR
products can be completely digested, confirming the band
is the expected avidin-1 conjugate (lane 1 and 2; Figure 2c).
G) at the same site. 3 and 4 were mixed at 1:100 and then
selected against avidin following the same procedure as in
Figure 2. The gel-purified conjugates were analyzed by
Sanger sequencing and compared with pre-selection mix-
tures. As shown in Figure 3b, before selection, as expected,
the encoding site had a mixed sequence, while after selec-
tion, the “TTT” codon encoding desthiobiotin has been dis-
tinctly enriched, proving the capability of this method in
identifying specific binders from a large excess of non-
binding background. In addition, qPCR analysis showed a
similar recovery of 3 to the experiment in Figure 2d (Figure
3, side table; see details in Table S1).
Next, we mixed 1 with a control DNA without desthio-
biotin (2; Figure 2d) at various ratios. 2 does not have the
HindIII site, so that HindIII digestion of the PCR products
can reveal the ratio of 1 and 2 in the mixture.82, 96 Mixtures
of 1 and 2 were subjected to the same selection, purifica-
tion, PCR amplification, and digestion procedures as in
Figure 2a. It is worth noting that a relatively large range of
gel band was excised to ensure the recovery of the protein-
DNA conjugate (Figure S3), which is highly important for
large libraries where the shifted gel band may not clearly
visible. In addition, we have verified by qPCR and Sanger
sequencing that excision of a larger gel band did not signif-
icantly increase background signals (Figure S4 and Table
S2).
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After these preliminary studies, next we tested the
“ligate-crosslink-purify” strategy to introduce the hairpin
structure to a DEL encoded with linear DNA tags. We
prepared a 49-nt DNA modified with 5’-GLCBS, a
sulfonamide ligand specific for carbonic anhydrase-II (CA-
II) (5, Kd = 9 nM; Figure 4a);94 The GLCBS is encoded by a
“GCTT” codon. In addition, a hairpin DNA without the
ligand of GLCBS but with a “TCCC” codon was also
prepared as the negative control (6; Figure 4a). Mixtures of
5 and 6 (1:10 and 1:100) were ligated with a 20-nt PC-DNA
bearing a 3’-phenylazide and a 5’-phosphate group, respec-
tively, mediated by T4 DNA ligase and a 14- nt splint DNA
(Figure S5). The 5’-end of the PC-DNA is complementary
to the 5’-PBS of 5 so that a stable hairpin
As shown in Figure 2d, with avidin, the PCR products
of the gel-purified avidin-DNA conjugates were efficiently
digested by HindIII, even at the ratio of 1:1000, indicating
significant enrichment of 1. Without avidin, the digestion
products roughly reflected the original mixing ratio of 1
and 2. In corroboration, qPCR results showed that nearly
full enrichment was obtained for 1 in the 1:1000 sample (see
the Supporting Information and Table S1 for details). In ad-
dition, qPCR quantitation also showed that the DNA re-
covery efficiency from gel purification is comparable to
similar washing-based methods (Table S1).87
In order to further validate that the enriched DNA in-
deed encodes the small molecule ligand, two 42-nt hairpin
DNAs (3 and 4; Figure 3a) were prepared. In 3, the desthio-
biotin is encoded by a “TTT” codon in the loop,
Figure 4. a) Mixtures of 5 and 6 (1:10 and 1:100) were converted
to hairpins via enzymatic ligation with a PC-DNA. Conditions:
5 and 6, 1.0 µM each; T4 DNA ligase: 10 units in T4 ligase
buffer; ligation: 1 hour at 16 °C. Ligation products were gel-pu-
rified and selected against CA-II as shown in Figure 1. b)
Sanger sequencing results of the pre-selection mixtures and
gel-purified CA-II-DNA conjugates. Coding sites are high-
lighted. See full images in Figure S7 and S8. c) qPCR analysis
of DNA recovery and enrichment. **: (post-selection 5/6 ra-
tio)/(pre-selection 5/6 ratio). ***: (post-selection percentage
of 5)/(pre-selection percentage of 5). See Table S1 for details.
Figure 3. a) A 1:100 mixture of 3 and 4 was selected against
avidin with the same procedure as in Figure 2, except Sanger
sequencing was used to analyze the pre-selection mixtures
and gel-purified conjugates. b) Sequencing results before (left
panel) and after (right panel) the selection. Encoding sites are
highlighted. Side table: qPCR analysis results. n.d.*: not deter-
mined due to mixed sequences in 4. See the Supporting Infor-
mation for details (Figure S6 and Table S1).
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Figure 5. a) A 4,800-member DNA-encoded macrocycle li-
brary and a positive control 7 were mixed and ligated with a
PC-DNA. The ligated library was selected against CA-II with
the same procedure as in Figure 4, except high throughput se-
quencing was used for hit identification. b) Selection results
shown in a scatter plot: enrichment folds (y-axis) vs. post-se-
lection sequence counts (x-axis). Enrichment fold = (post-se-
lection fraction)/(pre-selection fraction).92, 97 See the Supple-
mentary Information for more details on experimental condi-
tions, macrocycle structures, library DNA sequences, se-
quencing, and data analysis. Side table: qPCR analysis results
for the positive control 7. Other DNAs were not determined.
See the Supporting Information for details.
structure can be formed after ligation (Figure 4a; Tm: 52.7
°C, see details in Figure S2). These “hairpin libraries” were
then selected against CA-II; crosslinked target-DNA conju-
gates were gel-purified to be separated from non-binders;
then selected compounds were deconvoluted by Sanger se-
quencing. Again, before selection, only the dominant
“TCCC” codon can be observed; after selection, clear en-
richment of the “GCTT” codon encoding the ligand of
GLCBS was observed in both experiments (Figure 4b). In
addition, qPCR analysis also showed nearly full enrichment
of 5 in both cases and similar efficiency of DNA recovery
(Figure 4c). This result has demonstrated that the “ligate-
crosslink-purify” strategy can be straightforwardly applied
to DELs by simply ligating a PC-DNA to enable formation
of the hairpin structure.
Figure 6. The 4,800-member DNA-encoded macrocycle
library was ligated with a 20-nt PC-DNA and then selected
against avidin with the same procedure as described in Figure
5. a) Selection results are shown in a 3D scatter plot. Each axis
represents a set of building blocks. Size of data points:
enrichment folds (larger dot size indicates higher enrichment
fold; cut-off: 3.5); color of data points: post-selection sequence
counts (cut-off: 250). b) 2D projection of the scatter plot in a)
along the y-axis to show the SAR pattern more clearly; c)
Structures of three selected “hit” macrocycles (M1-M3) and
two negative control macrocycles (not selected from the
selection; M4 and M5). SPR sensorgrams are shown in Figure
S9.
Finally, we tested our method with a DEL containing
actual chemical diversity. We prepared a 4,800-member
DNA-encoded macrocycle library in which each
macrocycle is encoded by a 49-nt DNA tag as previously
described.92, 98 This library was spiked with a DNA-tagged
GLCBS (7) as an internal positive control (Figure 5a). This
4,801-member library was ligated with a 20-nt PC-DNA to
enable hairpin formation, selected against target CA-II,
irradiated under 365 nm, and then subjected to gel
purification to isolate the CA-II-DNA conjugates. First,
qPCR analysis showed a 11% recovery of the positive
control 7, similar to model libraries (Figure 4c) without
chemical diversity (Figure 5b, side table). Next, hit
compounds were deconvoluted with high throughput
DNA sequencing and the results are shown in form of a
scatter plot (Figure 5b). Compounds with relatively high
enrichment folds and post-selection sequencing counts are
considered potential hits (data points leaning towards the
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upper right direction of the plot).92, 97, 99 As shown in Figure
limitations arising from exonuclease digestion and also en-
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5b, the positive control 7 was distinctly identified, proving
the capability of this method in enriching high affinity
binders from chemically diverse libraries. The enrichment
fold of 7 appeared to be significantly lower than the posi-
tive control 2 in the model system (Figure 2d and 2e), pre-
sumably due to the relatively lower affinity of GLCBS and
the presence of macrocycle compounds who may also bind
and be selected by the target CA-II.
abling the reliable separation of binders from non-binders
by the critical gel purification step.93 Importantly, this
method is suitable for DNA-encoded libraries with small
molecules conjugated at either end of encoding DNA
strands. Moreover, by ligating a short PC-DNA strand and
a subsequent gel purification step, the “ligate-crosslink-pu-
rify” strategy enables the selection of existing DELs, which
may be prepared and encoded with other methods, with-
out the need for library redesign and resynthesis. However,
it is worth noting that the formation of the stem-loop
structure with variable sequences in the single-stranded
region may cause aptamer formation that can be selected
by the target; therefore, careful codon design and a nega-
tive selection with a “blank library” having the same DNA
sequences but without small molecules is necessary to con-
trol for selection of aptamers. Since all DELs already have
built-in primer binding sites available, we expect wide ap-
plications of this method in the ligand discovery against
targets previously intractable to DELs.
9
In chemical library selections, moderate and low
affinity binders are also valuable since they can be used as
starting points for downstream optimization to obtain
high quality lead compounds. However, often they are
difficult to be identified due to repeated washing steps.
Photo-crosslinking, which establishes a stable covalent
bond between the target and ligand, may provide
opportunities for moderate and weak binders to emerge,
such as being demonstrated by the Krusemark group in a
recent report.87 In order to test this and also examine the
generality of our method, we selected the same 4,800-
member macrocycle library against avidin as the target.
We reason that, due to the lack of known avidin-binding
structural features in the library,100 binders with moderate
and weak affinities may be identified. After the macrocycle
library was selected against non-immobilized avidin and
hit deconvolution by high throughput sequencing, we
plotted the selection data into a 3D scatter plot (Figure 6a),
in which each of the axe represents one respective set of
amino acid building blocks in the macrocycle structure (R1,
R2, and R3 in Figure 5a). This type of 3D plot enables easy
identification of structure-activity relationship (SAR) by
observing the “planes” and “lines” formed by selection data
points.49, 52 As shown in Figure 6a, in the avidin selection,
several “planes” can be identified, suggesting that these
specific building blocks bind more favorably to the target
avidin. This pattern can be seen more clearly with the 2D
projection along the R2-axis (Figure 6b).
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In principle, this method may also be applicable to se-
lections against targets in cell lysates; however, method
improvements to better locate the desired protein-DNA
conjugates during gel purification (e.g. using a fluores-
cently labeled PC-DNA) and to differentiate ligand binding
by the target from background proteins (e.g. using nega-
tive control selections or cell lysates with overexpressed
target77, 92) would be instrumental. Finally, this method
cannot be directly applied to DELs encoded with double-
stranded DNAs (dsDNAs) due to the inability of DNA du-
plexes to form stem-loop structures; converting dsDNAs to
single-stranded DNAs using methods such as biotin-tag-
ging and affinity separation,101 strand displacement, and
Lambda exonuclease digestion,102, 103 may be explored.
We will report our progresses on these two directions in
due course.
EXPERIMENTAL PROCEDURES
Next, three macrocycles that are located at the
intersections of several “planes” with good enrichment
folds or sequence counts were selected (M1, M2, and M3;
Figure 6c). These “hits” were resynthesized without the
DNA tag using a reported method97 and then assayed for
their binding affinity with surface plasmon resonance
(SPR). Results show that they are, as expected, weak
binders for avidin with Kd ranging from 36 to 89 µM (Figure
6c and Figure S8). As a specificity control, we have also
synthesized and assayed two additional macrocycles (M4
and M5; Figure 6c) that are not selected by avidin; indeed,
their binding affinities with avidin are not detectable with
SPR (Figure S8). Collectively, the above results have
demonstrated the generality and the capability of this
selection method in identifying novel binders with
Syntheses of small-molecule-conjugated DNAs
5’-desthiobiotin labeling. Solid-phase DNA synthesis
was performed with the 3’-amino-modifier CPG and 5’-
amino-C6 modifier phosphoramidite. 5’-MMT was depro-
tected with 3% TCA in CH2Cl2; the beads were then washed
with CH2Cl2 and then dried under vacuum. Desthiobiotin
(8.7 mg, 40 mmol) was dissolved in 200 μL anhydrous DMF
along with 0.9 equiv. HBTU (13.7 mg, 36 mmol) and 1 equiv.
HOBt (5.4 mg, 40 mmol). After vortexing for 1 hour at room
temperature, the activation mixture was added to the CPG
along with 2.3 equiv. DIPEA (15.2 μL, 92 mmol). The sus-
pension was then incubated at 37 °C overnight with agita-
tion. The CPG was washed with DMF (3x 600 μL), CH3CN
(3x 600 μL), and then dried with gentle airstream. The 5’-
desthiobiotin-labeled DNA was cleaved with AMA (50:50;
40% aqueous ammonium hydroxide : methylamine, v:v; 55
°C, 55 min) over a dry bath and then purified by HPLC.
relatively weak affinity from chemically diverse DELs.
CONCLUSION
In conclusion, we have developed a novel selection
method for DNA-encoded libraries. This method estab-
lishes a stable covalent link between the target and the
DNA tags of the selected ligands, thereby obviating the
4-azidobenzoate-SE synthesis. 4-azidobenzoic acid
(76.7 mg, 0.47 mmol) and NHS (54 mg, 0.47 mmol) were
dissolved in 1.5 mL anhydrous DMF. N,N’-dicyclohexylcar-
bodiimide (115 mg, 0.56 mmol) was added to the solution
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at room temperature. After the mixture was stirred for 8 pmol of target protein was added. The mixture was supple-
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hours, precipitated urea was removed by filtration. The fil-
trate was dried by a rotary evaporator. After washing
briefly with diethyl ether, the obtained white powder was
used directly in DNA labeling.
mented with water to a final volume of 10 µL, incubated at
4 °C for 1 hour, and then subjected to light irradiation un-
der 365 nm at 0 °C (over ice) for 30 seconds.
PAGE analysis and gel extraction. After photo-cross-
linking, 2 µL 5x loading buffer was added to the mixture,
which was then heat-denatured at 95 °C for 10 minutes over
a dry bath before PAGE analysis (12%-15%). Product bands
were excised, extracted by 1x PBS buffer, and purified by
ethanol precipitation.
3’-phenylazide labeling. The 3’-amine, 5’-desthiobi-
otin-modified DNA was dissolved in 40 μL high purity wa-
ter and 40 μL phosphate buffer (pH = 7.2). 4-azidobenzo-
ate-SE (3.1 mg, 12 μmol) was dissolved in 40 μL DMSO (fi-
nal concentration: 0.3 M). The two solutions were mixed
and the reaction was incubated with sonication at 55 °C for
1 hour. After brief centrifugation, the reaction mixture was
desalted by a NAP-5 column (GE Pharmacia) before the
product was purified by HPLC.
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PCR analysis. After ethanol precipitation, the pellet
obtained was dissolved with 50 µL water. 10 µL of the solu-
tion was added with 5 µL 5 µM PCR primers, 10 µL 5x
Phusion HF Reaction Buffer, 1 µL 1 mM Deoxynucleotide
Solution Mix and 0.5 µL Phusion HF DNA Polymerase. The
solution was then again diluted to 50 µL before PCR ampli-
fication. The PCR product was purified by ethanol precipi-
tation.
5’-GLCBS labeling. GLCBS (4.5 mg, 12 μmol), DCC (2.5
mg, 12 μmol) and NHS (1.4 mg, 12 μmol) were dissolved in
40 μL anhydrous DMSO and then the solution was vor-
texed for 1 hour at room temperature. The formed urea pre-
cipitate was removed by filtration. The filtrate was added
to a solution containing 40 μL 5’-amine-modified DNA (20
nmol) and 40 μL phosphate buffer (pH 7.5). The mixture
was the maintained with sonication at 37 °C for 1 hour. Af-
ter centrifugation, the reaction mixture was desalted by a
NAP-5 column (GE Pharmacia) before the product was pu-
rified by HPLC.
HindIII digestion. After ethanol precipitation, the
pellet obtained was dissolved with 120 µL water. 60 µL of
the solution was transferred to a 600 µL centrifuge tube;
then 8 µL 10x HindIII buffer, 1 µL HindIII (20 units, 20
units/µL), and 11 µL water were added to reach a final vol-
ume of 80 µL. The reaction mixture was incubated at 37 °C
for 2 hours. After digestion, the proteins were heat-dena-
tured at 95 °C for 20 minutes over a dry bath and the DNA
products were purified by ethanol precipitation.
PC-DNA. The synthesis and purification procedure is
the same as described above in 3’-phenylazide labeling ex-
cept that the 5’-phosphate-, 3’-amine-modified DNA was
used to conjugate with 4-azidobenzoate-SE.
T4 DNA ligase-mediated ligation. 200 pmol DNA to
be ligated, 200 pmol PC-DNA, and 200 pmol splint DNA
were added to a 1.5 mL centrifuge tube, heated to 85 °C for
30 seconds, and cooled to room temperature prior to the
addition of 20 µL 10x T4 DNA ligase buffer. 1 μL (350 units)
of T4 DNA ligase was added to the reaction solution at 0
°C. Ligation reactions were maintained at 16 °C for 2 hours,
before subjected to ethanol precipitation and denaturing
PAGE analysis. Product bands were excised, extracted by 1x
TE buffer, and purified by ethanol precipitation.
See the supporting information for MS characterization
of DNA-small-molecule conjugates.
DNA sequences
1: 5’-desthiobiotin-CTG AAT TCT CCC GAA AGT AAG
CTT AGA TAG GCG AGA ATT CAG-azide-3’;
2: 5’-CTG AAT TCT CCC GAA AGT GAA TCC AGA TAG
GCG AGA ATT CAG-azide-3’;
3: 5’-desthiobiotin-CTG AAT TCT CCC GAA AGT AAG
TTT AGA TAG GCG AGA ATT CAG-azide-3’
Synthesis, DNA sequences and building block
structures of the macrocycle library. the macrocycle li-
brary used in this study is the same as the macrocycle li-
brary we described previously.92
4: 5’-CTG AAT TCT CCC GAA AGT AAG DDD AGA
TAG GCG AGA ATT CAG-azide-3’ (D = A, C or G)
High-throughput sequencing and data analysis.
After PCR amplification of the selected library, PCR prod-
ucts were recovered by gel extraction with a gel extraction
kit and then directly submitted for high-throughput se-
quencing. Sequencing experiments were performed on an
Illumina® NextSeq 500 sequencer using standard 2 x 75
pair-end sequencing reagent kits and hybridization pri-
mers. After sequencing, data were exported for processing.
Sequence counts for each library member before and after
the selection were tallied to calculate the enrichment fold
for each library compound, following the method of previ-
ous reports.49, 92, 97, 104 First, the number of encoding DNA
sequence reads for each library compound was counted.
Second, the number was divided by the total number of in-
terpretable sequence reads to give the abundance of that
library member. Finally, enrichment folds for each library
member were obtained by dividing the post-selection
5: 5’-GLCBS-CCT GAA TTC CAA AGC CCT CAC AAG
CTT CAC AAG ACC CTC CCA AAC TGC C-3’
6: 5’-CCT GAA TTC CAA AGC CCT CAC AAT CCC CAC
AAG ACC CTC CCA AAC TGC C-3’
7: 5’-GLCBS-CCT GAA TTC CAA AGC CCT CAC AAT
CCC CAC ATC CGT CTC CCA AAC TGC C-3’
Splint-DNA: 5’-TTC GGG AGG CAG TT-3’
PC-DNA: 5’- phosphate-TCC CGA AAG TGG AAT TCA
GG-azide -3’
See the supporting information for DNA sequences of
PCR primers and high throughput sequencing adapters.
Library selection and selection result deconvolution
Selection. In a typical experiment, 25 pmol of DNA or
DNA-encoded library, 1 µL 10x PBS buffer, and 1 µL 1 M
NaCl were added to a 200 µL centrifuge tube, before 25
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abundance by the pre-selection abundance. Enrichment
1
2
3
4
5
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8
folds were then plotted against the sequence number. In
Figure 6, processed data was plotted into a 3D cube using
MATLAB software.
Hit Compound Resynthesis. Selected macrocycles
(M1-M5) were synthesized without the DNA tag following
a reported method.98 See the supporting information for
characterization data of these compounds.
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Methods Mol. Biol. 805, 3-28.
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(19) Nielsen, J., Brenner, S., Janda, K.D. (1993) Synthetic
Methods for the Implementation of Encoded Combinatorial
Chemistry. J. Am. Chem. Soc. 115, 9812-9813.
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Kochersperger, L. M., Dower, W. J., Barrett, R. W., and Gallop, M.
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Supporting Information. More details of selection proce-
dures, structure of DNAs, characterization data, high-
throughput sequencing procedure, full Sanger sequencing
data, qPCR analysis and calculation methods, SPR sensor-
grams, and other experimental details. The Supporting Infor-
mation is available free of charge on the ACS Publications
website at XXX
AUTHOR INFORMATION
Corresponding Author
* Email: xiaoyuli@hku.hk
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by grants from the Research Grants
Council of the Hong Kong Special Administrative Region,
China (Project No.: AoE/P-705/16 and 106160203), seed fund-
ing for basic research (201601159018) and start-up fund from
The University of Hong Kong, and a grant from National Nat-
ural Science Foundation of China (NSFC, 21572014). The au-
thors thank Dr. Yan Cao at The 2nd Military Medical Univer-
sity, Shanghai, China for assistance on SPR analysis.
(21) Kleiner, R. E., Dumelin, C. E., and Liu, D. R. (2011) Small-
molecule discovery from DNA-encoded chemical libraries. Chem.
Soc. Rev. 40, 5707-5717.
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