Author’s Accepted Manuscript

Cellular environment-responsive intelligent DNA

logic circuits for controllable molecular sensing

Huihui Wang, Jiao Zheng, Yudie Sun, Tao Li

www.elsevier.com/locate/bios

PII: S0956-5663(18)30507-4
DOI: https://doi.org/10.1016/j.bios.2018.07.006
Reference: BIOS10595
To appear in: Biosensors and Bioelectronic
Received date: 7 April 2018
Revised date: 12 June 2018
Accepted date: 5 July 2018
Cite this article as: Huihui Wang, Jiao Zheng, Yudie Sun and Tao Li, Cellular
environment-responsive intelligent DNA logic circuits for controllable molecular
s e n s i n g , Biosensors and Bioelectronic,
https://doi.org/10.1016/j.bios.2018.07.006
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Cellular environment-responsive intelligent DNA logic circuits for controllable molecular sensing

Huihui Wang, Jiao Zheng, Yudie Sun and Tao Li*

Department of Chemistry, University of Science & Technology of China, Hefei, Anhui, 230026, China.

Fax: (+86)551-63601813; E-mail: tlitao@ustc.edu.cn

ABSTRACT

Here we report smart molecular logic circuits built on a well-designed H-shaped DNA nanostructure that can recognize cell-simulated
bioenvironments and modulate the operations of a DNA nanosensor. By assembling a wild-type ATP aptamer and a parallel G-
quadruplex into the H-shaped DNA scaffold, two intrinsic cellular components, ATP and K+, are utilized to activate the logic circuits,
enabling fluorescent detection of the target DNA via toehold-mediated strand displacement. In this way, two logic circuits consisting
of cascaded “AND–AND” and “OR–AND” gates are achieved, which are responsive to the ATP and/or K+ concentration change outside
and inside cells, and therefore control whether or not the downstream DNA sensor works. This work illustrates a novel concept for
developing new bioinspired DNA molecular devices for not only programmable molecular sensing but also targeted drug delivery.

Keywords

DNA logic circuits, environment recognizing, programmable control, ATP aptamer, G-quadruplex

1. Introduction

Programmable molecular computing has proven to be an important tool for medical and biological researches such as multi-
parameter sensing, intelligent diagnosis and information modulation.(Chen et al. 2015; Groves et al. 2016) Because of the
specificity and predictability of Watson-Crick base pairing (A-T and C-G), DNA molecules have been widely used to construct
diversity of computation devices such as logic gates,(Chen et al. 2018; Frezza et al. 2007a; Frezza et al. 2007b; Li et al. 2012; Li et
al. 2009; Prokup et al. 2012; Saghatelian et al. 2003; Stojanovic et al. 2002; Wang et al. 2017; Xia et al. 2010; Zhang et al. 2017a)
logic circuits,(Chirieleison et al. 2013; Genot et al. 2011; Kabza et al. 2017; Kishi et al. 2018; Li et al. 2014; Lilienthal et al. 2017;
Seelig et al. 2006; Zhang et al. 2013) calculators, (Elbaz et al. 2010; Gao et al. 2017; Genot et al. 2013; Green et al. 2017; Groves et al.
2016; He et al. 2014; Liu et al. 2015; Pei et al. 2012) and computing networks.(Green et al. 2017; Qian and Winfree 2011; Qian et al.
2011; Qu et al. 2017; Stojanovic and Stefanovic 2003) Among them, biocompatible DNA logic devices are of particular interest.
For example, some logic gates are designed to recognize endogenous inputs in cellular environment.(Hemphill and Deiters 2013;
Prokup et al. 2012) By recognizing cell-surface markers, intelligent logic devices can screen out various abnormal conditions on
the cell surface.(Qu et al. 2017; You et al. 2014; You et al. 2015) Since cells provide ideal environments for biocompatible studies
on DNA computing, smart DNA logic devices to distinguish extracellular and intracellular environments are of great
significance in the programmable control of executing downstream tasks inside cells. Here we report a simulated cellular
environment-responsive DNA logic circuit for controllable molecular sensing, with two intrinsic cellular components (i.e. ATP
and K+) as the inputs due to their remarkable concentration differences outside and inside cells.(Ameen et al. 1992; Imamura et
al. 2009; Mo et al. 2014a; Pardo et al. 1992; Qiang et al. 2015; Yang et al. 2016; Zheng et al. 2017)

2. Experimental section
2.1. Oigonucleotides and chemicals.

Tris were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China), Adenosine 5'-triphosphate (ATP), Potassium
acetate (KAc), magnesium acetate (MgAc2) were purchased from Sigma-Aldrich. All chemical reagents were of reagent grade.
All DNA sequences listed in Table SI were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) without further
purification. The oligonucleotides were dissolved in TE buffer (10 mM Tris–HCl, 0.1 mM EDTA, pH 7.4) and quantified by UV–
Vis absorption spectroscopy with corresponding extinction coefficients (ε 260 nm, M-1 cm-1). A=15,400, G=11,500, C=7,400,
T=8,700.
2.2. Native Polyacrylamide Gel Electrophoresis (PAGE).
For H-shaped structure, the oligonucleotides that composed of the H-shaped DNA nonostructure (0.5 M) were dissolved in
TA buffer (20 mM Tris–acetate, 1.5 mM MgAc2, pH 8.0). Then, heated at 90 °C for 10 min, gradually cooled to room temperature
to form a stable H-shaped DNA structure. Samples of both the control structure and target structure after triggering by stimuli
(3mM ATP, 20mM KAc, 0.5 M DNA21) were characterized by 12% native polyacrylamide gel electrophoresis. Electrophoresis
was carried out at 95 V for 10 h at 4℃. The gels were stained with GelRed for 30 minutes. The imaging of the gels were
performed using UV light with Tanon 1600 Gel Imaging System (Shanghai, China). For the optimization of WtABA, 5ɥM DNA
prepared in the TA buffer were heated at 90 °C for 10 min and gradually cooled to room temperature. After incubating with or
without 1mM ATP for 3 hours, the samples were analyzed by 18% native PAGE under 96V for 10 h. Then the gels were stained in
0.01% Stains-All solution for 4 h and destained in deionized water under light until bands became clear, finally photographed
with a camera.
2.3. The fluorescence spectra analysis.

The H-shaped structures were diluted to a concentration of 0.2 M in TA buffer. After triggering by stimuli (3mM ATP, 140mM
KAc, DNA21) at 37 °C for 3 h, the fluorescence spectra were recorded on an F-4600 fluorescence spectrometer (Hitachi, Japan).
The fluorescence groups Carboxyfluorescein (FAM), tetramethylrhodamine (TAMRA), 2-aminopurine (2AP), N-
methylmesoporphyrin IX (NMM) and Cy5 were excited at 485.0 nm, 550nm, 300nm , 399 nm and 640 nm respectively. The
corresponding emissions (505-620 nm, 568-700 nm, 320-450 nm 550-700 nm, 656-720 nm) were recorded in 1 nm increments.
Three scans were accumulated and averaged then normalized to the same initial fluorescence intensity value. To demonstrate
the WtABA has a good response to ATP, the DNA solutions of WtABA-2AP (0.2 M) in TA buffer in the presence of 3mM ATP
or in the absence of ATP were measured with an F-4600 fluorescence spectrometer (Hitachi, Japan) at room temperature. Three
scans from 320 to 450 nm at 1 nm intervals with excited at 300nm were accumulated and averaged. PWA2 (0.5 μM) in TA buffer
in the presence of 140mM KAc or in the absence of KAc were also analysed in the same conditions. Three scans from 550 to 700
nm at 1 nm intervals with excited at 499nm were accumulated and averaged.
2.4. The CD spectra analysis.

The DNA solutions of WtABA (15 M) in TA buffer (pH 8) in the presence of 1mM ATP or in the absence of ATP were measured
with a Jasco J-1500 spectropolarimeter (Tokyo, Japan) at room temperature. PWA2 (15 μM) in TA buffer (pH 8) in the presence
of 20mM KAc or in the absence of KAc were also analysed in the same conditions. The optical chamber (1 mm path length, 1 mL
volume) was deoxygenated with dry purified nitrogen (99.99 %) before being used and kept the nitrogen atmosphere during
experiments. Three scans (100 nm/min) from 210 to 320 nm at 0.1 nm intervals were accumulated and averaged. The
background of the buffer solution was subtracted from the CD data.
2.5. Cell Culture and Experiments Assay.

MCF-7 cells were cultured in basic DMEM (HyClone) supplemented with 10% fetal bovine serum(Zhejiang Tianhang
Biotechnology Co., Ltd. China) and 100U/ml of 2% antibiotics penicillin/streptomycin (Sangon Biotechnology Co., Ltd. China)
and maintained at 37 °C in a 100% humidified atmosphere containing 5% CO2/95% air incubator (SANYO, Japan). In our
experiments, MCF-7 cells were cultured in 6-well plates at 37 °C for 24 h. After the removal of initial medium and washing with
PBS, we added 200ɥL Cell lysis buffer. Considering lysis solution was added in for complete lysis of cells, we got the uperior
solutions after centrifugation. Medium or the obtained uperior solutions with different quantity of cells were mixed with the H-
shaped structure (0.2 M) in proportion. After triggering by stimuli (3mM ATP, 140mM KAc, 0.2 M DNA21) at 37 °C for 1 h.
The excitation wavelength is 485 nm and the emission wavelength was collected in the range of 505−620 nm.

3. Results and discussion

3.1 Design and principle of the cascaded “AND–AND” logic gate.

Scheme 1 depicts the concept and process of such a logic circuit that modulates a DNA sensor. To recognize cell-simulated
bioenvironments, a wild-type ATP aptamer (WtABA) and a parallel G-quadruplex (PWA2) were designed and characterized
(see Fig. S1-S4 in Supporting Information). WtABA and PWA2 are assembled into the four arms of an H-shaped DNA
nanostructure H1. Meanwhile, the BHQ1-labelled antisense strand of the target DNA21, the DNA version of a cancer-related
gene(He et al. 2016; Li et al. 2016a; Ying et al. 2017) is embedded in the central double-stranded region (Fig. S5), with two hidden
2-nt toeholds blocked by WtABA and PWA2. This is to prevent H1 from responding to DNA21 in the extracellular environment
where the concentrations of ATP and K+ both are relatively low.(Pardo et al. 1992) In the intracellular conditions, however, high
concentrations of ATP (>1 mM) (Idzko et al. 2007; Mo et al. 2014a) and K+ (>100 mM)(Nolin et al. 2013; Yang et al. 2016) can
induce the disassembly of four arms of H1 due to the formation of aptamer-ATP complex and G-quadruplex, accompanied by
the release of a product structure H1-AK with two exposed toeholds in the single-stranded regions (Scheme 1). Upon addition of
DNA21, it can hybridize with its antisense sequence in H1-AK to replace another FAM-tagged component strand via the
toehold-mediated strand displacement (TMSD) reaction.(Huang et al. 2013; Song and Liang 2012; Zhang and Seelig 2011; Zhang
and Winfree 2009) This makes the fluorophore separate from the quencher, and therefore causes a sharp increase in
fluorescence intensity. In this way, the fluorescent analysis of the target DNA21 is achieved.

Scheme 1 Construction and operations of a cascaded “AND-AND” logic circuit. The binding domains of ATP and K+ are colored red and seagreen, respectively.
The target DNA21 and its antisense strand are colored blue.

To demonstrate our design, the above cellular environment-responsive DNA assembly of H1 was simulated in the presence of
intracellular concentrations of ATP and K+, characterized by native polyacrylamide gel electrophoresis (PAGE). In contrast, a
reference structure (CH1) obtained by disordering the sequences of WtABA, PWA2 and the antisense strand was investigated
together. Fig. 1A shows different electrophoretic behaviors of H1 and CH1 under four different conditions (panels a-d). In the
absence of ATP and K+ (panel a), H1 keeps enough stable (lane 2) even induced by DNA21 (lane 4). Upon addition of K+ (panel
b), two double-stranded arms of H1 is

Fig. 1. Characterization of the response of H1 to external stimuli. (A) Gel electropherograms for the stimuli-responsive disassembly of H1 and CH1 under four
different conditions: (a) Tris-Ac (TA) buffer, (b) TA buffer + 20 mM KAc, (c) TA buffer + 3 mM ATP, (d) TA buffer + 20 mM KAc + 3 mM ATP. Lanes 1-4: CH1, H1,
CH1 + DNA21, H1 + DNA21. (B) Fluorescence spectra of 2-AP excited at 300 nm (a) and NMM excited at 499 nm (b) for a mixture of 0.2 M H1 and 3M NMM in
response to different stimuli: 1, blank; 2, ATP; 3, K+; 4, ATP + K+; 5, DNA21.
subject to disassociation induced by the folding of G-quadruplex,(Li et al. 2012) reflected by a new fast moving band
corresponding to the product structure H1-K (lane 2). In this case, DNA21 cannot hybridize with the antisense strand to trigger
the TMSD reaction (lane 4 vs. lane 2), as only one toehold is exposed. The similar phenomenon is also observed upon addition
of ATP alone (panel c). A new faster band corresponding to the product structure H1-A appears, indicating that other two arms
of H1 are dissociated due to the combination of ATP with the aptamer.(Monserud et al. 2016; Wang et al. 2016; Zhang et al. 2015)
Likewise, the TMSD reaction triggered by DNA21 does not occur in H1-A (lane 4 vs. lane 2). In the presence of ATP and K+
(panel d), however, the product structure H1-AK is able to respond to DNA21, indicated by an observable shift in the band
mobility (lane 4 vs. lane 2). In contrast, the band of CH1 always keeps unchanged in the whole progress (panels a-d, lanes 1, 3),
excluding the possibility of the band shifts caused by nonspecific interactions. These observations clearly demonstrate that the
exposure of two toeholds in the structure H1-AK induced by both ATP and K+ is a prerequisite for the response to DNA21,
namely, the downstream DNA analysis is in fact controlled by an AND logic gate, where ATP and K+ serve as the two inputs.

Since the arm disassociation of H1 results from the K+-promoted folding of PWA2 and ATP-induced release of WtABA, this
process was also monitored by fluorescence spectroscopy via using a fluorescent ligand N-methylmesoporphyrin IX (NMM)
specific for G-quadruplexes(Li et al. 2013; Li et al. 2016c; Ren et al. 2012) and meanwhile incorporating a bulged 2-aminopurine
(2-AP),(Li et al. 2016b; Zhou et al. 2017) a fluorescent analogue of adenine, into WtABA (see Fig. S1 in Supporting Information).

Fig. 2. Logic behaviors of H1 under different input conditions. (A) Fluorescence spectra of the BHQ1/FAM-reported logic system in response to different stimuli.
(B) Bar representation of the fluorescence intensity at 520 nm. The threshold for logic output is set as 0.7. (C) Truth table of the AND logic gate for DNA21 sensing.
(D) Logic symbol of the logic circuit consisting of cascaded AND–AND gates, where the first gate is for programmable control and the second one for target

sensing.

Fig. 1B shows that the addition of ATP and/or K+ induces a noticeable increase in fluorescence intensity of 2-AP (panel a, curves
2, 4) and NMM (panel b, curves 3, 4). However, there is no significant change in fluorescence signal of 2-AP and NMM upon
addition of the target DNA21 alone (curve 5). We found that ATP can slightly promote the fluorescence of NMM bound to the
folded PWA2 (panel b, curve 4 vs curve 3), possibly owing to a positive effect of ATP on the NMM-G-quadruplex interaction. A
similar phenomenon has been observed in the interactions of G-quadruplexes with another porphyrin ligand (hemin).(Kong et
al. 2010)

By labeling the antisense strand and its partly complementary sequence with BHQ1 and FAM, different behaviors of H1 in
response to ATP, K+ and DNA21 can be revealed by fluorescent readout, as shown in Fig. 2. It is observed that in the presence of
ATP and K+, the target DNA21 can cause a sharp increase in the fluorescence intensity of FAM (Fig. 2A), attributed to the
separation of the fluorophore from the quencher. This is indicative of DNA21 hybridizing with the antisense strand and
replacing the complementary sequence via the TMSD reaction. In the other cases, no significant change in fluorescence readout
is observed. Fig. 2B shows a bar representation of the fluorescence intensity at 520 nm under all input conditions, illustrating
that H1 in fact behaves as an AND logic gate in terms of DNA21 sensing with the FAM/BHQ1 system. The corresponding truth
table is shown in Fig. 2C. As a whole logic system, this sensing gate is cascaded with the controlling gate demonstrated by PAGE
(Fig. 1A), and therefore these two gates constitute a logic circuit,(Chatterjee et al. 2017) of which the logic symbol is shown in
Fig 2D.
3.2 Design and principle of the cascaded “OR–AND” logic gate.

Scheme 2 Schematic illustration of a cascaded “OR –AND” logical operation. The binding domain of ATP and K+ are colored red and seagreen, respectively.
The DNA 21 sequence and its complementary strand are colored blue.
Fig. 3. The H-shaped DNA molecular logic circuit integrates the first level “OR” gate with the second level “AND” gate to control the logic operation. (A) Native
PAGE gel analysis. The experiment was carried out in the TA buffer (a), in the TA buffer with 20mM KAc (b), in the TA buffer with 3mM ATP (c) and in the TA
buffer with 20mM KAc and 3mM ATP at the same time (d), respectively. Lanes 1-4 correspond to “CH2”, “H2”, “CH2” with DNA21 and “H2” with DNA21. (B)
Fluorescence spectra of the logic circuit system respond to different stimuli in the excitation of TAM. (C) Fluorescence changes of the logic circuit system in the
form of a bar presentation respond to different stimuli in the excitation condition of TAM. The threshold is set as 0.5. (D) Truth table of the logic circuit system.
(E) The logic scheme illustrate the operational design of the integrated logic circuit.

To test the generality of our developed strategy for logic circuits, we designed another H-shaped DNA nanostructure (H2) by

modifying the arms and toeholds (see Fig. S5 in Supporting Information), which can behave as a logic circuit consisting of
cascaded OR-AND gates (Zhu et al. 2014) upon input of ATP, K+ and DNA21 (see Scheme 2). In this design, one cellular
environmental component, ATP or K+, can activate H2 for DNA21 sensing, unlike H1.

Fig.3 depicts the programmable operation process. To validate this design, we performed native polyacrylamide gel
electrophoresis to monitor the DNA reaction(Fig. 3A). As shown in panel a, successive assembly of the H-shaped DNA structure
resulted in retarded mobility in the gel. We designed another contrast H-shaped DNA structure (termed as CH2) that has no
response for ATP, potassium ion and DNA by disturbing the order base sequence of H2.
The “Control structure” and “Target structure” have identical migration position in the absence of any stimuli and in the
presence of DNA21, suggesting that two toehold regions are well blocked in the H2. The disassembly of PW17-A occurred in the
presence of K+, accompanying with a faster electrophoretic mobility (lane 2, panel b. Interestingly, the exposed toehold provides
a strand point for DNA to bind to the structure. Subsequently DNA21 migrates along the complementary chains. The branch
migration induces the release of the other blocker and the complementary strand. Finally, the other blocker is released (lane 4,
panel b). Similarly, with the addition of ATP, ABA27 was disassembled from H2, bringing in the release of the other blocker and
the complementary stand in the presence of DNA21 (panel c). In the coexistence of ATP and K+, appeared a band that moved
much faster, indicating two blocker strands were released. Once DNA21 was added, the complementary strand was released
(panel d). From these results, we can conclude that a “OR –AND” DNA circuit could work pretty well to respond to ATP, K+ and
DNA21. In this logic circuit, we chose the originally selected ATP aptamer (ABA27) and PW17-A as blockers. Knowing the good
performance of the blockers, we simplified the readout and just gave the second layer output signal to show the fluorescence
phenomenon of the logic circuit. As shown in Fig.3B and Fig.3C, striking enhanced fluorescence signals would be observed with
the presence of DNA together with ATP or/ and K + . With the output threshold of 0.5, the logic behavior of the circuit is defined
as an “OR-AND” circuit. The truth table of the logic circuit is shown in Fig.3D. The logic scheme of the “OR-AND” circuit is
ploted in Fig.3E. Without changing the stimuli, we realized cascaded “AND–AND” logic gate and “OR–AND” logic gate let alone
changing the targets. We have designed and performed the required “OR-OR” and “AND-OR” logic gates via small variations on
DNA sequences of the H-shape scaffold ( see Fig. S6 and Fig. S7 in Supporting Information). So, diversity of logic gates (or
circuits) can be successfully constructed on our designed H-shape scaffold. Upon integrating different aptamer sequences into
the H-shaped DNA nanostructure, this molecular platform will be sensitive to various stimuli and applied in more extensive
areas.
3.3. Real sample analysis.

Since there is a remarkable concentration difference of ATP and K + outside and inside cells, our designed logic circuits are
promising for constructing intelligent DNA molecular devices to recognize simulated cellular environments and then
execute the programmed tasks. As a proof-of-concept experiment, we operated H1 as a logic circuit-controlled sensor for
DNA21 analysis in cell-mimicking bioenvironments, i.e. the lysates of MCF-7 cells. Fig. 4A shows that in the cell lysates, the
addition of the target DNA causes a sharp increase in the fluorescence intensity (curves 7 vs. 4) that is above the logic
threshold (0.7), which is almost not influenced by supplementary ATP and K + (curve 8). That is, ATP and K+ in cells lysates
are enough to activate the logic circuit and turn on the DNA sensor. As

Fig. 4. Operation of the DNA logic circuit in the cell-simulated environment. (A) Fluorescence spectra for operating H1 in the cell culture medium and lysates,
which mimics the situations before and after the DNA device enters cells. From bottom to top (curves 1-8): (1) H1 in culture medium; (2) 1 + ATP + K+; (3) 1 +
DNA21; (4) H1 in the lysates of ~20000 MCF-7 cells; (5) 4 + ATP + K+; (6) 1 + DNA21 + ATP + K+; (7) 4 + DNA21; (8) 4 + DNA21 + ATP + K+. (B) Fluorescence
intensity of H1 operated in the lysates of different number of MCF-7 cells.

reported previously that some DNA nanostructures (e.g. tetrahedrons) are easy to enter the target cells incubated in the culture
medium,(Li et al. 2011) here we employed the cell culture medium to simulate an extracellular environment. In this solution,
there is no significant change in fluorescence signal upon addition of DNA21 (curves 3 vs. 1), which is always below the logic
threshold (0.7). Nonetheless, supplementing ATP and K+ into it increases the fluorescence intensity over the threshold (curve 6),
just like in cell lysates (curves 7, 8). It suggests that the operations of H1 only depend on ATP and K+ in the cellular
environment, with little interference from other components. This is further confirmed by utilizing different number of MCF-7
cells for DNA analysis (Fig. 4B). The fluorescence intensity gradually increases with the number of used cells, owing to the
increasing concentrations of ATP and K+ in the lysates of the same volume. These observations

Fig. 5. Performance of the intelligent DNA logic circuits for controllable molecular sensing. (A) Fluorescence emission spectra tested with different
concentrations of DNA21. (B) Fluorescence intensity changes of H1 in the presence of no DNA (a), target DNA21 (b), single-base mismatched near the left end and
the right of DNA21, L-mis1 and R-mis1 (c and d), duplex-base mismatched DNA21, Mis2 (e), triple-base mismatched DNA21, Mis3 (f), completely mismatched
DNA21, Let-7d (g).

clearly demonstrate that our designed logic circuit consisting of cascaded AND-AND gates has the ability to distinguish the
internal and external environments of cells and therefore logically control the downstream tasks such as DNA sensing and drug
delivery.(Halstead et al. 2015; Wang et al. 2014; Zhang et al. 2017b)

To illustrate the applications of our logic circuits, we employed H1 for the quantitative and specific analysis of DNA21 in the cell
lysates (Fig. 5). As the concentration of DNA21 increases from 0 to 5000 nM, the fluorescence of H1 gradually increases (Fig. 5A).
It is observed that 2 nM target DNA can induce an obvious change in the fluorescence intensity, indicating a detection limit of 2
nM for DNA21 analysis. To test the selectivity, we employed mismatched DNAs in place of DNA21 to perform parallel
experiments, with no significant signal change (Fig. 5B). It reveals a good selectivity of our system for the target
DNA21.

4. Conclusions

In summary, we have reported H-shaped intelligent DNA logic circuits which are able to recognize the simulated cellular
environments and modulate the downstream DNA sensor. WtABA, PWA2 and the antisense strand of DNA21 are assembled
into the branched DNA scaffold H1. With two intracellular components ATP and K+ as the inputs, H1 behaves as an AND logic
gate, verified by native PAGE. This gate in fact serves as a modulator for the downstream DNA21 analysis with the FAM/BHQ1
system that also behaves as an AND logic gate. The cascaded two AND gates constitute a logic circuit with the ability to
distinguish the simulated environments outside and inside cells, illustrated by the utilization of H1 for controllable DNA21
analysis in the lysates of MCF-7 cells. Such a smart feature is of particular significance for the programmable control of
biochemical and biomedical operations (e.g. drug delivery) inside living cells (Awino et al. 2017; Mo et al. 2014b; Sun et al. 2014;
Yan et al. 2015) and molecular imaging,(He et al. 2016; Santangelo et al. 2009; Ying et al. 2017) For example, by mixing the H1
structure with doxorubicin (DOX), a widely-used duplex-interactive anticancer drug,(Perez-Arnaiz et al. 2014) our designed
logic-controlled platform will be employed to transport this drug into the targeted cancer cells, where the dissociation of H1 is
triggered by incellular K+ and ATP thereby in situ releasing the drug.

ACKNOWLEDGMENT

This work was supported by the National Key Research and Development Program of China [No. 2016YFA0201300], the National
Natural Science Foundation of China [No. 21575133], and the Recruitment Program of Global Experts.
Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version.

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Highlights
1. Without changing the stimuli, we realizedcascaded “AND–AND” logic gateand“OR–AND” logic
gate,andendowedDNA logic circuits with desired intelligence.
2.The well-designed H-shaped DNA nanostructure that can recognize cell-simulated bioenvironments
and modulate the operations of a DNA nanosensor.
3.No complex instruments and equipment are required.
4. It is of great significance for future researches onprogrammable control of biochemical and biomedical
operations inside living cells such as drug delivery.