Activation of Aptamers with Gain of Function by Small-Molecule-Clipping of Intramolecular Motifs

Article abstract of DOI:10.1002/anie.202013570

Click reactions have the advantages of high reactivity, excellent orthogonality, and synthetic accessibility. Inspired by click reactions, we propose the concept of “clipped aptamers”, whose binding affinity is regulated by the “clip”-like specific intera

Full text of DOI:10.1002/anie.202013570

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Activation of Aptamers with Gain-of-Function by Small-Molecule-
Clipping of Intramolecular Motifs
Authors: Mengjiao Huang, Tingyu Li, Yuanfeng Xu, Xinyu Wei, Jia
Song, Bingqian Lin, Zhi Zhu, Yanling Song, and Chaoyong
James Yang
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202013570
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10.1002/anie.202013570
Angewandte Chemie International Edition
RESEARCH ARTICLE
Activation of Aptamers with Gain-of-Function by Small-Molecule-
Clipping of Intramolecular Motifs
Mengjiao Huang,[a][+] Tingyu Li,[a][+] Yuanfeng Xu,[a] Xinyu Wei,[a] Jia Song,[b] Bingqian Lin,[a] Zhi Zhu,[a]
Yanling Song,*[a] and Chaoyong Yang*[a][b]
[a]
Mengjiao Huang, Tingyu Li, Yuanfeng Xu, Xinyu Wei, Bingqian Lin, Prof. Zhi Zhu, Dr. Yanling Song, Prof. Chaoyong Yang
The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, the Key Laboratory of Chemical Biology of Fujian Province, State Key Laboratory
of Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering
Xiamen University
Xiamen, Fujian 361005, China
E-mail: ylsong@xmu.edu.cn; E-mail: cyyang@xmu.edu.cn.
Dr. Jia Song, Prof. Chaoyong Yang
Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine
Shanghai Jiao Tong University
Shanghai, 200127, China
These authors contributed equally.
[b]
[+]
Supporting information for this article is given via a link at the end of the document.
Abstract: Click reactions, which are commonly used in the
challenging task of biology-oriented modular conjugation, have the
advantages of high reactivity, excellent orthogonality, and synthetic
accessibility, and they accelerate the generation of molecules
possessing well-defined functions. Inspired by click reactions, we
propose the concept of “clipped aptamers,” whose binding affinity is
regulated by the "clip"-like specific interaction between a synthetic
DNA-mismatch-binding small molecule (molecular glue, Z-NCTS) and
the preset CGG/CGG sequences in nucleic acid sequences. In this
study, we investigated a Z-NCTS-mediated de novo selection of
“clipped aptamers” against epithelial cell adhesion molecule. The
generated “clipped aptamers” can achieve the efficient transition from
a binding inactive state to an active state by clipping of Z-NCTS with
two CGG sites, which otherwise would not hybridize. The
experimental and simulation results proved that the “clipped aptamer”
had ideal binding thermodynamics and the ability to regulate cellular
adhesion. Because of this superior activated mechanism and
structural diversity, “clipped aptamers” hold great potential in
biosensing, imaging, conditional gene- and cellular behavior-
regulation, and drug delivery.
strategy for conjugation of intramolecular motifs of biomolecules
is needed.
Biomolecules such as aptamers, exert their biological functions
by folding into unique structures via intramolecular interactions.[5]
Extensive research has been conducted on controlling the binding
function of aptamers, but an efficient and universal strategy is still
lacking.[6] Inspired by the click reaction, we propose a concept of
aptamer, whose binding properties are activated by “clipping”
together different intramolecular motifs that do not inherently
hybridize spontaneously, triggering the transition of aptamer
structures from a binding inactive state to an active state (Figure
1A).
In response to the limitations of the click reaction for
intramolecular conjugation, we made use of naturally occurring
sequences in nucleic acids as the “clickable” moieties, thereby
avoiding the use of chemically modified nucleosides.
A
naphthyridine carbamate tetramer with a Z-stilbene (Z-NCTS)
(Figure 1B), designated as DNA molecular glue, binds to the
tandem mismatch sites in DNA duplex with CGG/CGG sequence
with high affinity and selectively (Figure 1C).[7] Therefore, Z-NCTS
is expected to “clip” the preset individual CGG sites in the
aptamers together to generate novel three-dimensional structures,
thus achieving control of the aptamer binding properties. In
addition, Z-NCTS has the potential to help generate not only
hairpin shapes but also high-order structures, endowing the
“clipped aptamers” with increased structural diversity.[8]
Meanwhile, the “clip” process is actually the specific interaction
between Z-NCTS and the CGG/CGG sequences driven
thermodynamically, ensuring a favorable “clip” orthogonality.
Since no chemically modified nucleosides are involved in the
proposed “clipped aptamers”, they are fully compatible with PCR
amplification. Thus, the de novo selection of aptamers with gain-
of-binding function through aptamer selection is possible (Figure
1D). The “clipped aptamer” selection is a directed evolution under
strictly controlled conditions, that effectively eliminate sequences
that do not conform to the expected functionality, avoiding the
need for additional engineering steps to optimize the resulting
sequences.
Introduction
Click reactions have been accepted as a promising toolbox for
modular conjugation of various molecules by virtue of high
reactivity, excellent orthogonality, and synthetic accessibility.[1]
These essential characteristics have shown a profound impact
across the broader chemical sciences, functional polymer
synthesis, and biological coupling.[2] But, despite the great
success of the “click” based conjugation of biomolecules, the
challenging installation of “clickable” moieties into the
biomolecules has obstructed applications for intramolecular
conjugations. Tremendous effort is required to produce "clickable"
monomers that are compatible with the recognition of
biomolecules.[3] In addition, “click” efficiency has proven to be
disturbed by steric hindrance in the complicated biomolecular
environment.[4] Therefore, an effective and compatible “click”
1
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RESEARCH ARTICLE
Figure 1. (A) Schematic illustration of the functional regulation of “clipped aptamer”. (B) Chemical structure of the molecular glue. (C) Schematic of hydrogen
bonding between Z-NCTS and CGG/CGG sequence. (D) Schematic representation of screening strategy for “clipped aptamer”.
library, as four repeat CGG sites interspaced by eight nucleotides.
Based on sequence similarity, the sequences were divided into
Results and Discussion
six main groups. A representative aptamer from each group
(Table S1) was chosen for further characterization. As shown in
Figure 2B, the binding performance of five of the six
representatives against EpCAM increased significantly after
adding Z-NCTS. Among them, the XMU3 aptamer exhibited the
Enrichment of “Clipped Aptamers”. First, we evolved “clipped
aptamers” regulated by Z-NCTS against epithelial cell adhesion
molecule (EpCAM), which is involved in cell adhesion, migration,
and proliferation.[9] The his-tag EpCAM protein was modified with
nickel-beads via the interaction of the his-tag epitope and nickel,
leaving the EpCAM properly displayed on the microspheres. The
selection procedures, called “Clipped Aptamer SELEX” were
similar to classical SELEX for aptamer selection,[10] but several
changes were made to obtain “clipped aptamers” (Figure 1D). The
changes were as follows: 1) The initial DNA library included two
flanking primer regions and a 52-nt region consisting of four “CGG”
fragments and five “eight random bases” fragments. Compared
with the standard random library, this patterned library has a
stronger possibility to contain CGG/CGG sites which can be
clipped by the Z-NCTS. 2) Only sequences with target binding
ability in the presence of Z-NCTS were enriched, while those
sequences with non-target binding ability and those with binding
ability in the absence of Z-NCTS were eliminated.
The progress of the “clipped aptamer” enrichment was monitored
by flow cytometry. Using a fluorophore-labeled library, the
regulation ratio (fluorescence of the library with Z-NCTS divided
by the fluorescence of the library without Z-NCTS, FZ-NCTS (+)/FZ
-
(-)) gradually increased with the progress of the selection
NCTS
against EpCAM-beads (Figure 2A), and the 9th through 12th
libraries indicated that a plateau was reached. These results
suggested that the 12th library was successfully enriched in
“clipped aptamers” recognizing EpCAM. Thus, the 12th library
was cloned for sequencing.
Figure 2. The regulation ratios of enriched libraries (A) and six representative
aptamers (B) against EpCAM. (C) The secondary structures of full-length XMU3
and optimized XMU3-abcd aptamer were simulated using mfold software; bases
in blue are four conserved CGG fragments within the randomized region of the
aptamer. (D) Confocal imaging to investigate the binding of XMU3 and XMU3-
abcd aptamer with EpCAM beads in the presence and absence of Z-NCTS.
Identification and Optimization of “Clipped Aptamers”. Forty
monoclines were randomly selected for sequencing. All
sequences showed the same conserved pattern as the initial
2
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highest binding enhancement ratio (FZ-NCTS (+)/FZ-NCTS (-)) of 2.0 in
the presence and absence of Z-NCTS.
The enhancement of binding ability measurements by flow
cytometry, indicated that three sequences (XMU3-acd, XMU3-
Therefore, the XMU3 aptamer was chosen for further optimization, abd, XMU3-abc) lost their gain-of-function activity, because they
yielding a 72-nt truncated XMU3-abcd aptamer, which retained
the 2-hairpin structure of the full-length XMU3 sequence (Figure
2C). Each letter followed by a “-” dash in the name of XMU3-abcd
represents a CGG site (from 5’ to 3’ terminal). The XMU3-abcd
aptamer contained two stems (Figure 2C), in which the CGG in
sites a, b and d were on the first and second stem, respectively,
while CGG in site c was on the second loop region. The binding
performance regulated by Z-NCTS of XMU3-abcd aptamer was
measured by confocal imaging (Figure 2D), which revealed that
the gain-of-function of the truncated aptamer was maintained and
similar to that of the full-length XMU3 aptamer.
did not bind to EpCAM after the addition of Z-NCTS. Only the
XMU3-bcd sequence (Figure 3A) maintained an obvious binding
enhancement after addition of Z-NCTS, with an enhancement
ratio of 2.0 (Figure 3B). However, the further truncated XUM3-cd,
XUM3-bd, and XUM3-bc sequences exhibited no observable
EpCAM binding affinity or binding regulation effects. These results
suggested that the “clipped aptamer” must contain CGG sites of
b, c, and d to be activated by “clipping” together different motifs
triggering the functional transition of aptamer structures from an
inactive state to an active state.
Native polyacrylamide gel electrophoresis (native PAGE) and
melting temperatures (Tm) were used to further confirm the
interaction of XUM3-bcd aptamer and Z-NCTS. As shown in
Figure 3C, a new band with a slower mobility than the original
aptamer formed after the addition of Z-NCTS. Moreover, the Tm
values of the aptamers in the presence of Z-NCTS were about 10
degrees higher than those of the original corresponding aptamer
(Figure 3D & Figure S1). The increased Tm values were slightly
smaller than that of the standard interaction of Z-NCTS and DNA
duplexes containing CGG/CGG,[7] possibly due to conformational
changes in the selected “clipped aptamers” caused by
rearrangement after the binding to Z-NCTS.
Binding Sites of Molecular Glue on “Clipped Aptamer”. To
characterize how the Z-NCTS regulates the structural change of
XMU3-abcd aptamer, each CGG site was successively removed
to obtain a series of sequences containing three CGG sites. In
order to ensure the integrity of the overall structure of the
sequence as much as possible, some CGG sites of XMU3-abcd
aptamer were removed using only one base in the specific CGG
fragment (Table S1), yielding XMU3-bcd, XMU3-acd, XMU3-abd
and XMU3-abc.
Figure 3. (A) The secondary structure of XMU3-bcd aptamer simulated using the mfold software; bases in blue are four conserved CGG fragments within the
randomized region of the aptamer. (B) The allosteric regulation activity of the truncated aptamers against EpCAM in the presence and absence of Z-NCTS. (C)
Native PAGE results confirmed the binding of Z-NCTS to aptamers. “+” in the existence of Z-NCTS. “-” in the absence of Z-NCTS. (D) Thermal melting curves of
XMU3, XMU3-abcd and XMU3-bcd in the presence and absence of Z-NCTS, respectively. The melting curve in the absence of Z-NCTS is in black.
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Characterization of the Binding Stoichiometry of “Clipped
Aptamers” and Molecular Glue. To characterize the binding
stoichiometry of the aptamer-Z-NCTS complexes, electrospray
time-of-flight mass spectrometry (ESI-TOF-MS) experiments
were carried out. In the absence of Z-NCTS, a set of ion peaks
corresponding to the aptamers were the only detectable peaks
(Figure 4A, D). In the presence of Z-NCTS, new ion peaks
of the Ka of Z-NCTS and “clipped aptamers” were the same order
of magnitude as that of the Z-NCTS and standard DNA duplexes
containing CGG/CGG.[7]
Interaction Model of XMU3-bcd Aptamer and Molecular Glue.
To further illuminate the interaction model of Z-NCTS-aptamer
complexes, we attempted to determine the Z-NCTS binding
CGG/CGG sites in XMU3-bcd aptamer. For XMU3-bcd aptamer,
Z-NCTS may clip together the CGG on the loop region (c site) and
the CGG on the stem region (b or d site), or insert between two
CGGs on the stem (b and d sites) to stabilize the hairpin structure.
corresponding to aptamer-Z-NCTS complexes with
a 1:1
stoichiometry were observed. For instance, in the presence of Z-
NCTS an ion peak corresponding to {XMU3-bcd-Z-NCTS}23-
(calcd 976.44, found 976.87) appeared, together with lower
intensities of the free aptamer ion peaks corresponding to {XMU3-
bcd}22- (calcd 965.80, found 966.03) (Figure 4D). Meanwhile, no
We designed several aptamer sequences labeled with
a
fluorescence resonance energy transfer (FRET) pair containing
FAM as the donor and TAMRA as the acceptor (Table S2). The
FRET signal was defined as the fluorescence signal at 580 nm
(emission wavelength of TAMRA) when 488 nm (excitation
wavelength of FAM) was used as the excitation wavelength.
Aptamers XMU3-bcd-FRET-1 and XMU3-bcd-FRET-2 contained
the same sequences with different labeling sites of FRET pair
(Figure 5A and 5B). The FRET pair of XMU3-bcd-FRET-1 was
modified at the 5 terminal of the b site of CGG and the 3 terminal
of the c site of CGG, respectively, while the FRET pair of XMU3-
bcd-2-FRET was modified at the 5 terminal of the c site and 3
terminal of the d site. In these two sequences, XMU3-bcd-FRET-
1 showed a significant increase of 1.65-fold in the 580 nm FRET
signal after the addition of Z-NCTS (Figure 5A), while that of
XMU3-bcd-FRET-2 only increased by 1.34-fold (Figure 5B). The
results revealed that b and c sites which were originally far away
from each other become significantly closer in the presence of
ion peaks corresponding to complexes with
stoichiometry were found.
a different
The association constants (Ka) of the Z-NCTS for XMU3-abcd and
XMU3-bcd were evaluated by circular dichroism (CD) titration.
The achiral molecule Z-NCTS itself exhibited no CD signals, while
the insertion of Z-NCTS into the aptamer sequences produced
ICD (induced circular dichroic) signals in the region of 300 to 360
nm, in which a negative peak at 325 nm and a positive peak at
349 nm were observed (Figure 4B, E). The intensities of these
peaks increased with the concentration of Z-NCTS and gradually
reached a plateau, implying that there was a typical binding
equilibrium between the Z-NCTS-free and Z-NCTS-bound
aptamers. The data at 349 nm fit well to the 1:1 binding isotherm
using a least-squares curve, and the Ka values of XMU3-abcd and
XMU3-bcd found to be (5.15 ± 1.6) x 106 L/mol (Figure 4C) and
(1.45 ± 0.28) x 106 L/mol (Figure 4F), respectively. These values
Figure 4. ESI-TOF-MS of XMU3-abcd (A) and XMU3-bcd (D) in the absence (upper panels) and presence of Z-NCTS (lower panels), respectively. The black lines
indicate the peaks of the aptamers, while the blue lines indicate the peaks belonging to the aptamer-Z-NCTS complexes. The CD spectra of XMU3-abcd (B) and
XMU3-bcd (E) were measured while titrating with Z-NCTS at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8 μM, respectively. The signals at 349 nm were plotted against the
concentration of Z-NCTS and fitted to a 1:1 binding isotherm to obtain the Ka values of Z-NCTS binding with XMU3-abcd (C) and XMU3-bcd (F).
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Figure 5. (A) & (B) FRET spectral analysis of XMU3-bcd aptamer, (C) XMU3-bc aptamer, and (D) XMU3-cd aptamer. (A) & (C) FAM and TAMRA are labelled at
CGG (b site) and CGG (c site), respectively. (B) & (D) FAM and TAMRA are labelled at CGG (c site) and CGG (d site), respectively. Emission spectra obtained
when excited at 488 nm.
Z-NCTS, while the c and d sites remained relatively far apart,
even in the presence of Z-NCTS.
Molecular Docking and Molecular Dynamics Simulations. To
better understand the different interaction patterns at the
molecular level of the aptamer with EpCAM in the presence and
absence of Z-NCTS, molecular docking and molecular dynamics
simulations (MDS) were carried out. The secondary structure of
the XMU3-bcd aptamer was predicted by mfold software, and it
was converted into a 3D-structure using RNAcomposer (Figure
7A).[11] The 3D structure of Z-NCTS was modeled in Chem3D, and
the docking of the aptamer and Z-NCTS was conducted by
To further ascertain the location of the binding sites, we designed
another two sequences as XMU3-bc-FRET (retaining b and c,
Figure 5C) and XMU3-cd-FRET (retaining c and d, Figure 5D). As
Figure 5C shows, XMU3-bc-FRET had a significant increase of
1.93-fold in 580 nm FRET signal after the addition of Z-NCTS,
while that of XMU3-cd-FRET only increased by 1.11-fold (Figure
5D). These results again indicated that Z-NCTS triggered the
proximity of CGG at b and c sites. Based on the results of ESI-
TOF-MS, CD titration, and FRET experiments, the conformational
change of XMU3-bcd aptamer after binding to Z-NCTS was due
to the intermolecular clicking of CGG at the b and c sites by Z-
NCTS with a 1:1 binding stoichiometry. However, the previous
results showed that XMU3-bc had no allosteric response to
EpCAM (Figure 3B), which suggested that the d site of XMU3-bcd
aptamer was independent of Z-NCTS interaction, but contributed
to the binding of EpCAM.
Interaction of XMU3-bcd-Z-NCTS Complex and EpCAM. The
next step was to characterize the binding affinity of the aptamer-
Z-NCTS complex to the EpCAM protein (Figure 6A). The
dissociation constant (Kd) of the XMU3-bcd-Z-NCTS complex
against EpCAM was measured by flow cytometry analysis. As
shown in Figure 6B, free XMU3-bcd sequence did not show
obvious affinity to EpCAM in the absence of Z-NCTS, while the
XMU3-bcd-Z-NCTS complex showed a high binding affinity
against EpCAM protein with a Kd of (9.3 ± 1.3) nM, indicating that
aptamer-target binding can be activated with good
thermodynamics through the addition of Z-NCTS.
Figure 6. (A) Schematic illustration of the formation of aptamer-Z-NCTS-
EpCAM complex. (B) The binding affinity curves of XMU3-bcd aptamer with
EpCAM by flow cytometry in the presence and absence of Z-NCTS.
5
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expression”. Herein, the binding affinity against the functional
protein of the evolved “clipped aptamer” can be activated by Z-
NCTS. Thus, it is possible to directly regulate cell behavior
independent of changes of gene and protein expression (Figure
8A).
Since EpCAM is a key protein for cell adhesion, the cell adhesion
of EpCAM-positive SW480 cells was investigated under the
conditions of both the inactive and activated aptamer, respectively.
The binding abilities of free XMU3-bcd aptamer and XMU3-bcd-
Z-NCTS complex against SW480 cells were first investigated. As
shown in Figure S2, in the absence of Z-NCTS, XMU3-bcd
aptamer showed slight binding against SW480 cells, while in the
presence of Z-NCTS, the binding was significantly enhanced.
Then the effect of the XMU3-bcd-Z-NCTS complex on SW480 cell
adhesion was investigated. After the digested cells were cultured
for two hours, the number of cells attached to the cell culture dish
was used as an evaluation criterion. As shown in Figure 8 B&C,
compared to the untreated group, 98% of cells have alive and
adhered to the culture dish with Z-NCTS treatment, which
suggested that Z-NCTS has no obvious cytotoxicity under this
condition. It may be due to the high affinity between the Z-NCTS
and the aptamer, the free Z-NCTS is at a very low concentration.
While the XMU3-bcd treated group showed a slight reduction to
about 80%, probably due to the weak binding between the
aptamer and EpCAM. For treatment with the XMU3-bcd-Z-NCTS
complex, the cell adherence rate was significantly reduced to 57%,
much more than that of the aptamer obtained by conventional
selection (Figure S3). It is possible that the regulation
enhancement was due to the increase of library structural
diversity by the interaction of Z-NCTS and CGG/CGG.
Figure 7. The results of docking and molecular dynamics simulations. The
overall structure of XMU3-bcd (A), XMU3-bcd−Z-NCTS (B), XMU3-bcd−EpCAM
(C), and XMU3-bcd−Z-NCTS-EpCAM complexes (D). Histogram plot showing
the interaction energy between the EpCAM and XMU3-bcd nucleotide in the
absence (E) and (F) presence of Z-NCTS.
AutoDock 4.2 software. The simulation results showed that Z-
NCTS interacted with the XMU3-bcd aptamer through hydrogen
bonds with a binding free energy of (-29.33 ± 0.71) kJ/mol. Since
the b and c sites were clipped together by Z-NCTS, the overall
structure of the aptamer-Z-NCTS complex (Figure 7B) changed
significantly compared with the initial structure of XMU3-bcd
aptamer (Figure 7A). These results were consistent with the
experimental results that Z-NCTS could induce a conformational
change in the clipped aptamer.
The 3D structure of EpCAM was constructed according to ID
4MZV from the RCSB PDB data bank. Further docking and MDS
results of aptamer-protein and aptamer-Z-NCTS-EpCAM
suggested that the XMU3-bcd-Z-NCTS complex interacted with
EpCAM through a different binding interface compared with the
free XMU3-bcd aptamer. Moreover, the binding interface of
XMU3-bcd aptamer and Z-NCTS was totally different from that of
XMU3-bcd aptamer and target, thus confirming our previous
hypothesis that Z-NCTS can act as an effector molecule to
activate the binding capacity of clipped aptamer. In addition, the
XMU3-bcd-Z-NCTS complex displayed an approximately 2-fold
negative binding free energy against EpCAM (binding free energy
= (- 113.74 ± 1.42) kJ/mol, Figure 7D) compared to that of the
XMU3-bcd aptamer (binding free energy = (- 65.53 ± 2.07) kJ/mol,
Figure 7C). The interaction energies of the EpCAM protein to the
individual aptamer residues are presented as histogram plots
(Figure 7 E, F), showing the interaction strength of EpCAM to
each aptamer residue. It is apparent that the binding strength
between the aptamer and EpCAM increased when Z-NCTS was
present, which further confirmed that the recognition ability of the
XMU3-bcd aptamer was activated by Z-NCTS.
Figure 8. SW480 cell adhesion results. (A) Schematic diagram of aptamer-Z-
NCTS complex inhibiting cell adhesion. (B) The percentages of the attached
cells in plate expressed as the number of cells without treatment and with
treatment with Z-NCTS, XMU3-bcd, XMU3-bcd-Z-NCTS complex. The number
of attached cells in the wells of the untreated group was set to 100%. Each bar
represents the mean ± SEM. (C) Representative image of attached cell numbers
in plate without treatment and with treatment with Z-NCTS, XMU3-bcd, XMU3-
bcd-Z-NCTS complex. t-test: P=0.0126, differences in cell adhesion rates
between the untreated group and XMU3-bcd-Z-NCTS complex-treated group.
(Scale bar, 50 μm.)
Regulation of Cell Adhesion by “Clipped Aptamer”. In nature,
riboswitches assist in maintaining the normal physiological
function of cells in response to specific stimuli, by the regulation
process of “riboswitch confirmation-mRNA translation-protein
6
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RESEARCH ARTICLE
A. Idili, M. A. D. Nijenhuis, T. F. A. de Greef, F. Ricci, J. Am. Chem. Soc.
2018, 140, 14725-14734; e) Y. Biniuri, G.-F. Luo, M. Fadeev, V. Wulf, I.
Willner, J. Am. Chem. Soc. 2019, 141, 15567-15576; f) E. Del Grosso,
G. Ragazzon, L. J. Prins, F. Ricci, Angew. Chem., Int. Ed. 2019, 58,
5582-5586; g) L. Zhao, X. Qi, X. Yan, Y. Huang, X. Liang, L. Zhang, S.
Wang, W. Tan, J. Am. Chem. Soc. 2019, 141, 17493-17497; h) I. A. P.
Thompson, L. Zheng, M. Eisenstein, H. T. Soh, Nat. Commun. 2020, 11,
2946.
These results demonstrated that the selected “clipped aptamer”
not only has EpCAM binding ability, but also can regulate protein
function. Unlike the riboswitch regulation mechanism, which
follows the Central Dogma, the regulation mechanism of the
“clipped aptamer” is similar to that of natural allosteric enzymes
through the change of “structure-binding affinity-protein function”
pathway, avoiding excessive time consumption and potential
secondary effects of dynamic biological processes.
[7]
[8]
[9]
C. Dohno, I. Kohyama, C. Hong, K. Nakatani, Nucleic Acids Res. 2012,
40, 2771-2781.
C. Dohno, M. Kimura, K. Nakatani, Angew. Chem., Int. Ed. 2018, 57,
506-510.
Conclusion
P. A. Baeuerle, O. Gires, Br. J. Cancer 2007, 96, 1491-1491.
[10] Y. Song, Z. Zhu, Y. An, W. Zhang, H. Zhang, D. Liu, C. Yu, W. Duan, C.
J. Yang, Anal. Chem. 2013, 85, 4141-4149.
In conclusion, we have reported the concept of “clipped aptamer”
and its direct evolutionary process, which takes advantage of the
specific recognition between the DNA-mismatch-binding
molecular glue and the preset CGG/CGG sites of DNA sequences.
Z-NCTS can quickly “clip” together two CGG sites within the DNA
sequence, triggering the structural transition from the target
unbound state to the bound state with ideal thermodynamics. A
series of “clipped aptamers” against EpCAM were obtained, and
the “clip” mode of XMU3-bcd aptamer was investigated through
experiments and MDS, which indicated that Z-NCTS “clipped” the
b and c sites of the XMU3-bcd aptamer together with a 1:1
stoichiometry. Moreover, XMU3-bcd aptamer can directly
regulate the cell adhesion via the “structure-binding affinity-
protein function” pathway, rather than following the Central
Dogma. Therefore, this strategy not only adds an extra level of
molecular recognition for aptamers to enhance the structural
diversity and function of aptamers, but also holds great potential
in producing sensitive and selective outputs in response to
binding to a specific molecule, which is a significant capability for
biosensing, imaging, conditional gene- and cellular behavior-
regulatory, drug delivery, etc.
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Acknowledgements
We thank the National Natural Science Foundation of China
(22022409, 21735004, 21874089, 21705024, 21775128), and the
Program for Changjiang Scholars and Innovative Research Team
in University (IRT13036) for their financial support.
Keywords: aptamer • selection • regulation of binding affinity •
gain of function
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This article is protected by copyright. All rights reserved.

10.1002/anie.202013570
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
Small molecule mediated selection method was developed to obtain “clipped aptamer” whose binding ability toward target can be
activated by “clipping” two intramolecular motifs via a synthetic DNA-mismatch-binding small molecule.
8
This article is protected by copyright. All rights reserved.