Pip-HoGu: An Artificial Assembly with Cooperative DNA Recognition Capable of Mimicking Transcription Factor Pairs

Article abstract of DOI:10.1021/jacs.7b13275

Cooperation between pairs of transcription factors (TFs) has been widely demonstrated to play a pivotal role in the spatiotemporal regulation of gene expression, but blocking cooperative TF pair-DNA interactions synergistically has been challenging. To achieve this, we designed programmable DNA binder pyrrole-imidazole polyamides conjugated to host-guest assemblies (Pip-HoGu) to mimic the cooperation between natural TF pairs. By incorporating cyclodextrin (Cyd)-adamantane (Ada), we synthesized Ada1 (PIP1-Ada) and Cyd1 (PIP2-Cyd), which were evaluated using T_m, EMSA, competitive, and SPR assays and molecular dynamics studies. The results consistently demonstrated that Pip-HoGu system formed stable noncovalent cooperative complexes, thereby meeting key criteria for mimicking a TF pair. The system also had a longer recognition sequence (two-PIP binding length plus gap distance), favorable sequence selectivity, higher binding affinity, and in particular, a flexible gap distance (0-5 bp). For example, Ada1-Cyd1 showed thermal stability of 7.2 °C and a minimum free energy of interaction of -2.32 kcal·mol^-1 with a targeting length of 14 bp. Furthermore, cell-based evaluation validated the capability of Pip-HoGu to exhibit potent cooperative inhibitory effects on gene expression under physiological conditions by disrupting TF pair-DNA function. In conclusion, the modular design of Pip-HoGu defines a general framework for mimicking naturally occurring cooperative TF pair-DNA interactions that offers a promising strategy for applications in the precise manipulation of cell fate.

Full text of DOI:10.1021/jacs.7b13275

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Communication
Pip-HoGu, an artificial assembly with cooperative DNA
recognition capable of mimicking transcription factor pairs
Zutao Yu, Chuanxin Guo, Yulei Wei, Kaori Hashiya, Toshikazu Bando, and Hiroshi Sugiyama
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b13275 • Publication Date (Web): 02 Feb 2018
Downloaded from http://pubs.acs.org on February 4, 2018
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Pip-HoGu, an artificial assembly with cooperative DNA recognition
capable of mimicking transcription factor pairs
Zutao Yu†, Chuanxin Guo†, Yulei Wei†, Kaori Hashiya†, Toshikazu Bando†, Hiroshi Sugiyama*†‡
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†Department of Chemistry, Graduate School of Science and ‡Institute for Integrated CellꢀMaterial Sciences (iCeMS), Kyoꢀ
to University, KitashirakawaꢀOiwakecho, Sakyoꢀku, Kyoto 606ꢀ8502, Japan
Supporting Information Placeholder
disrupting those of both TFs will result in complex biological
ABSTRACT: Cooperation between pairs of transcription factors
outcomes. Therefore, novel strategies are needed to address these
challenges to the implementation of deliberate and precise manipꢀ
ulation of gene expression patterns.
(TFs) has been widely demonstrated to play a pivotal role in the
spatiotemporal regulation of gene expression, but blocking coopꢀ
erative TF pair–DNA interactions synergistically has been chalꢀ
lenging. To achieve this, we designed programmable DNA binder
pyrroleꢀimidazole polyamides conjugated to host–guest assemꢀ
blies (Pip-HoGu) to mimic the cooperation between natural TF
pairs. By incorporating cyclodextrin (Cyd)–adamantane (Ada), we
synthesized Ada1 (PIP1ꢀAda) and Cyd1 (PIP2ꢀCyd), which were
evaluated using Tm, EMSA, competitive, and SPR assays and
molecular dynamics studies. The results consistently demonstratꢀ
ed that Pip-HoGu system formed stable noncovalent cooperative
complexes, thereby meeting key criteria for mimicking a TF pair.
The system also had a longer recognition sequence (twoꢀPIP bindꢀ
ing length plus gap distance), favorable sequence selectivity,
higher binding affinity, and in particular, a flexible gap distance
(0–5 bp). For example, Ada1–Cyd1 showed thermal stability of
7.2 °C and a minimum free energy of interaction of –2.32
kcal·mol–1 with a targeting length of 14 bp. Furthermore, cellꢀ
based evaluation validated the capability of Pip-HoGu to exhibit
potent cooperative inhibitory effects on gene expression under
physiological conditions by disrupting TF pair–DNA function. In
conclusion, the modular design of Pip-HoGu defines a general
framework for mimicking naturally occurring cooperative TF
pair–DNA interactions that offers a promising strategy for appliꢀ
cations in the precise manipulation of cell fate.
PIPs are the best characterized programmable DNA minorꢀ
groove binders that can compete with TF binding, with the bindꢀ
ing rules that Py/Im recognizes C/G, Im/Py recognizes G/C, and
Py/Py recognizes A/T and T/A11. Despite substantial progress,
there is still a long way to go before these molecules can be apꢀ
plied clinically7,12. The short recognition sequences of PIPs lead
to high offꢀtarget rates, but the extension of PIP length significantꢀ
ly impairs its cell permeability13,14. Critically, the fixed PIPsꢀ
binding motif (4–8 bp) without elasticity, is unsuitable for blockꢀ
ing flexible TF binding, especially that of TF pairs15.
O
N
O
NH
O
N
n
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N
H
H
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H
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H
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H
H
O
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Host-guest
interaction
N
H
N
Ada1: n=1
O
N
Ada2
: n=2
O
OH
O
O
N
HN
O
O
O
O
OH
OH
HO
O
HO
OH
O
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N
HO
N
H
O
H
N
Cyd1
O
N
HO
O
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O
HO
H
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OH
H
O
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N
N
OH
N
H
O
O
H
N
H
H
O
OH
Manipulating spatiotemporally variable gene expression has
been the goal of generations of scientists1,2. In mammals, there are
approximately 1000 transcription factors (TFs) that extensively
regulate gene expression patterns, and 55–70% of these TFs may
be functioning as cooperative TF pairs via homoꢀ/heterodimers to
N
O
N
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OH
N
N
O
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H
H
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O
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HO
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H
HO
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OH
HO
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OH
HO
O
Figure 1. Overview of cooperative interactions of a TF pair tarꢀ
geting a sequence associated with two components of Pip-HoGu
assembly, Ada1 and Cyd1. n = gap distance. (Bottom) Chemical
structures of Ada1, Ada2, and Cyd1.
ensure high binding affinity and extended recognition sequence3,4
.
Programmable molecules, e.g., nucleic acid analogues and pyrꢀ
roleꢀimidazole polyamides (PIPs), can disrupt individual TF–
DNA interactions5ꢀ7, but cannot block interactions between colꢀ
laborative TF pairs and DNA. More specifically, the gap sequencꢀ
es between the two binding motifs of the TF pair are not conꢀ
served and the gap distances are relatively flexible, ranging from
–1–5 bp8. Most significantly, by switching cooperative partners,
TF pairs can exert divergent biological functions. For example,
Sox2/Oct4 instigates pluripotent gene activation, but Oct4/Sox17
functions as a HEX activator and Sox2/Pax6 plays a pivotal role
in ocular lens development9,10. Accordingly, disrupting the indiꢀ
vidual binding sites of Sox2 or Oct4, rather than synergistically
In this context, we envisaged the integration of PIPs with a coꢀ
operative system to mimic the homoꢀ or heterodimer binding sysꢀ
tems of TF pairs. There are several classic noncovalent cooperaꢀ
tive systems, including nucleic acid analogues, metal ion–ligand,
and host–guest systems (e.g., cyclodextrin (Cyd), cucurbit[n]uril,
and carcerands with guests)16ꢀ21. Among these cooperative sysꢀ
tems, Cyd–adamantane (Ada) has been extensively studied as an
exemplary host–guest system both in vitro and in cells22,23. By
replacing a leucineꢀzipper dimerization domain with Cyd/Ada,
Morii and colleagues designed an artificial system in which the
cooperative Cyd–Ada interaction highly stabilized the interaction
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of DNA with the DNAꢀbinding domains of GCN4 homodimer24,25
.
positiveꢀbinding ODNs with 0–5 bp gap distances displayed coꢀ
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The work of Mascarenas and colleagues of Cyd–Ada assisted
DNAꢀbinding peptide–distamycin derivatives represented a step
forward in the development of smaller, selective, and ligandꢀ
responsive systems26. Accordingly, the design of programmable
DNA binder/host–guest scaffold for mimicking cooperative TFꢀ
pair systems and especially their cellꢀbased applications is highly
attractive.
To achieve this, we designed PIPs conjugated to a host–guest
Cyd–Ada scaffold, i.e., Pip-HoGu. We first evaluated them in
vitro using the DNAꢀbinding sequences of the Tax/CREB heteroꢀ
dimer, which functions by cooperative recruitment of p300 that is
essential for HLTVꢀ1 virus amplification27,28 Ada1 (PIP1ꢀAda)
consists of a PIP moiety to target the Tax binding site (5′ꢀ
WWGGCWꢀ3′) conjugated to Ada via a miniꢀPEG linker (Figure
1)24. Host conjugate Cyd1 (PIP2ꢀCyd) contains a Cyd moiety and
a CREBꢀcompetitiveꢀbinding PIP (5′ꢀWGWCGWꢀ3′). We generꢀ
ated series of positiveꢀ and negativeꢀbinding sequences, and the
difference in binding originates from the relative positions of the
Ada1 and Cyd1 binding sites (Figure 2A). In positiveꢀbinding
sequences, Cyd–Ada covers only a short distance (equal to the
gap distance) (Figure S1). In contrast, in the negativeꢀbinding
mode, Ada must bridge two PIPꢀbinding sites plus the gap disꢀ
tance, making it impossible for Ada to interact with Cyd.
operative binding function, and no cooperative effect was obꢀ
served with gap distances ≥6 bp (Figure 2C). These results highꢀ
light the gapꢀdistance dependency of cooperative binding energies.
ODN2P with a 2ꢀbp gap distance demonstrated the highest level
of cooperation (ꢀꢀTm = 7.2 °C). Of note, a 1ꢀbp mismatch Tm
assay showed that Pip-HoGu exhibited high sequence specificity
with a ꢀꢀTm of 8.3 °C (Table S2)32.
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Figure 3. EMSA illustrating the cooperativity of Pip-HoGu. (A)
The gelꢀshift behavior of all the positiveꢀbinding sequences with
Ada1–Cyd1. Concentrations are shown in figure. (B) Quantitative
EMSA of ODN2P with Cyd1 at various concentrations (top) and
Cyd1 supplemented with saturated Ada1 (bottom). ODNs conꢀ
centration: 0.1 ꢁM. (C) Equilibrium association constants and free
energies for ODN2P with Ada1–Cyd1.
Parallel to the Tm assay, electrophoretic mobility shift assays
(EMSAs) were conducted to visualize bandꢀshift behavior upon
formation of stable complexes33. The band upshifts for Ada1,
Cyd1, and Ada1–Cyd1 with ODN2P can be clearly distinguished
(Figure S3). Next, fixed concentrations of Ada1–Cyd1 were alꢀ
lowed to equilibrate with all ODNs. In agreement with the results
of the Tm assay, the appearance of an upshifted band showed that
ODNs with positiveꢀbinding mode and 0–5 bp gap distances disꢀ
play cooperative binding (Figure 3A). Ada1–Cyd1 shows subꢀ
stantially weaker band shift with ODN0P, for which steric hinꢀ
drance might be partially responsible. In comparison, no comꢀ
plexes were observed for DNA sequences of –1 bp and >6 bp.
Moreover, no negativeꢀbinding mode DNA sequences could form
upshifted band indicative of cooperative complexes, and there was
also no upshifted band of negative ODNs with Ada1 or Cyd1
individually, suggesting cooperative complex mediated sequence
selectivity (Figure S4)3. In addition, competitive EMSA assays
showed that cooperation was weakened in the presence of a guest
competitor (Figure S5)34.
Quantitative EMSAs were performed to analyze the magnitude
of cooperativity and the equilibrium association constant was
determined by fitting to the Langmuir binding isotherm35. The
increase in the upshifted band for ODN2P at various concentraꢀ
tions of Cyd1 alone and in the presence of Ada1 at excess conꢀ
centration demonstrates the cooperative effect (Figure 3B)36. Speꢀ
cifically, the data generated an equilibrium association constant of
2.50 × 105 M–1 (K1) for Cyd1 alone, and promisingly increased to
1.25 × 107 M–1 (K1,2) in the presence of Ada1 (Figure 3C). Using
the equation for the free energy of binding, the free energies of
binding for Cyd1 alone and in the presence of Ada1 were –7.36
and –9.68 kcal·mol–1, respectively, giving a minimum free energy
of interaction (G2ꢀ1 – G2) of –2.32 kcal·mol–137. Therefore, Pip-
HoGu has superior cooperationꢀstabilization effects to the previꢀ
ously reported 8ꢀbp DNA duplex (–2.2 kcal·mol–1) and the natural
phage λ repressor system (–2.0 kcal·mol–1).37,38. Concomitantly, a
surface plasmon resonance (SPR) assay was further validated
quantitatively the cooperative effects of Pip-HoGu assembly
(Table S3, Figure S7)39.
Figure 2. Tm assay illustrating the cooperativity of Pip-HoGu. (A)
The DNA oligomers (ODNs) used in the Tm assay, including posiꢀ
tive (ODN1′P–ODN6P) and negative (ODN1′N–ODN6N) bindꢀ
ing sequences. The gap distance (green) is the number of bp beꢀ
tween the binding sites of Ada1 (blue) and Cyd1 (red). The chart
only shows the forward DNA strand. (B) Tm profiles of positive
ODNs (TmP, light blue), negative ODNs (TmN, gray), positive
ODNs/Ada1–Cyd1 (TmPA, blue), and negative ODNs/Ada1–
Cyd1 (TmNA, black). (C) ꢀꢀTm profiles of cooperativity of Ada1–
Cyd1 assemblies. ꢀTm = Tm (ODNs/PIPs) – Tm (ODNs); ꢀꢀTm =
ꢀTmP – ꢀTmN. Error bars indicate the standard deviation of three
replicates.
A thermal stabilization assay (Tm assay) was performed to
evaluate the cooperative binding potency and how it was influꢀ
enced by the gap distance29. In the positiveꢀbinding mode, the
overall thermal stability of Ada1–Cyd1 had a ꢀTmP value of 9–15
°C (ꢀTmP = TmP – Tm) in a gapꢀdistanceꢀdependent manner (Figure
2B, Table S1). In negativeꢀbinding mode, however, there were no
gapꢀdistanceꢀdependent effects on the thermal stability of Ada1–
Cyd1 with ꢀTmN values around 9–10 °C (Table S1). In the control
experiment with mixture of PIP1 and PIP2, there was no signifiꢀ
cant difference of thermal stability between positiveꢀ and negaꢀ
tiveꢀbinding sequences (Figure S2). Therefore, the discrepancy of
thermal stabilization effect between positiveꢀ and negativeꢀ
binding modes should mainly attributable to the cooperative interꢀ
action of the Cyd–Ada complexes30,31. The results showed that
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tion of Ada3 and Cyd2, 4–5ꢀfold enhancement of the inhibitory
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effect was observed, which strongly correlated with the cooperaꢀ
tive interactions of the host–guest system. The use of endoporter
ensured that Ada3 and Cyd2 reached homodimerꢀbinding sites at
the same time, and it is plausible that Pip-HoGu could also work
well for heterodimer binding sites in the absence of endoporter44.
This cellꢀbased assay system demonstrated for the first time that
the individual host–guest interactions of Pip-HoGu could effecꢀ
tively stabilize the PIP pair–DNA cooperative interaction and
potently inhibit natural TFꢀpair binding in cells.
Figure 4. Mechanistic studies of cooperative binding. (A) Gelꢀ
shift behavior Ada2–Cyd1. (B) Tm profiles of all positiveꢀbinding
sequences in the presence of Ada1–Cyd1 (TmPA, blue, same as
Figure 2B) and Ada2–Cyd1 (TmPB, red).
Cooperation between TF pairs is ubiquitous in cells8. Our proꢀ
totype Pip-HoGu defines a general framework for mimicking
cooperative TF pair–DNA interactions through the integration of
programmable DNA binders and a host–guest system. In vitro
assays showed that Ada1–Cyd1 assemblies formed stable cooperꢀ
ative binding complexes with target DNA sequences with 0–5 bp
gap distances. In essence, the cellꢀbased assay demonstrated that
our artificial Ada3–Cyd2 assemblies formed highly stable coopꢀ
erative complexes that competed with naturally occurring cooperꢀ
ative TF pair–DNA systems. Therefore, Pip-HoGu could be apꢀ
plied to the regulation of spatiotemporally variable gene expresꢀ
sion patterns.
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Continually, we studied the influence of linker length on coopꢀ
erative binding since the underlying mechanisms of gapꢀdistanceꢀ
dependent cooperativity (≤5 bp) are not totally clear19. For this,
we used Ada2, which has a long, double miniꢀPEG linker (Figure
1). In the EMSA and Tm assay, Ada2–Cyd1 showed similar coopꢀ
erative patterns to Ada1–Cyd1, i.e., only those DNA sequences
with gap distances of 0–5 bp could form cooperative complexes
(Figure 4A, B). Specifically, Ada2–Cyd1 showed lower stability
for gap distances of 0–4 bp in the Tm assay. This demonstrated
that an extraꢀlong linker might destabilize the binding affinity of
the complex over short gap distances. Interestingly, when the gap
distance was extended to 5–6 bp, Ada2–Cyd1 displayed slightly
higher stability than Ada1–Cyd1 because the longer and more
flexible linker can reduce the tension of complex formation. In
conclusion, the cooperative energy of Pip-HoGu was highly disꢀ
tance dependent and that gap distances of >5 bp diminish the
cooperation, even when the linker region was long enough to
allow the encounter of host–guest moieties. This can be explained
by the hydrophobic and van der Waals interactions of Cyd–
ASSOCIATED CONTENT
Supporting Information
Experimental details including synthesis, evaluation method and
supporting data. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Ada16,40
.
*hs@kuchem.kyotoꢀu.ac.jp
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work was supported by JSPS KAKENHI Grant NO.
JP16H06356, “Basic Science and Platform Technology Program
for Innovative Biological Medicine by Japan Agency for Medical
Research and Development (AMED)” , and “the Platform Project
for Supporting Drug Discovery and Life Science Research funded
by AMED”. We thank China Scholarship Council support Z. Y.
Figure 5. Cellꢀbased assay of Pip-HoGu. (A) Crystal structure of
ERα homodimer and DNA sequence. (B) Schematic diagram of
EREꢀdriven luciferase in T47DKꢀBLuc cells. (C) The structural
design of Ada3 and Cyd2 targeting ERE sites (Figure S8). (D)
Luciferase activity assay after normalization to the total protein
concentration.
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