A versatile DNA-supramolecule logic platform for multifunctional information processing

DNA-based logic units are rapidly being developed in molecular computation. However, because DNA cannot produce detectable signals, suitable signal reporters must be carefully selected,

which is challenging, especially in advanced, multifunctional devices. By introducing a supramolecular reporter MTC [3,3’-di(3-sulfopropyl)-4,5,4’,5’-dibenzo-9-methyl-thiacarbo-cyanine

triethylammonium salt], we developed a simplified DNA-supramolecule platform. Owing to the multiple assembly states of MTC, the platform contains only one reporter block but can provide

multiple parallel outputs and easily implement several types of information processing functions, including data filtration (binary and ternary INHIBIT gates), selection (multiplexer and

demultiplexer) and verification (parity checker and comparator). In addition to combinational circuits, a fundamental sequential logic circuit, counter, has also been fabricated at the

molecular level. With the advantages of high reconfigurability, flexibility and enormous parallelism, this DNA-supramolecule prototype may have a promising future in the field of molecular

computing and multiplex chemical analysis.

Molecular computation has attracted intense attention in widespread. scientific studies over the past few decades1,2,3. Unlike traditional digital circuitry whose logic units are physically

combined, molecular logic units are functional-combined by associating chemical or biochemical molecules4,5. In molecular logic devices, DNA is endowed with unique advantages due to its

strict base complementarity and controllable structural transitions. Hitherto, a series of advanced logic circuits, such as multiplexer/demultiplexer6, encoder/decoder7,8 and parity

checker9,10, have been constructed via DNA-based systems. These works not only promote the development of molecular computation but also show great application potential for biomedical

imaging11, data storage12, medical diagnostics13, and biochemical analysis14.

Although great advancements have been achieved in DNA computation, the inherent drawback that DNA cannot generate detectable signals still exists. Therefore, the development of DNA-based

circuits relies heavily on the design of ingenious signal reporters. Reported DNA-based logic systems mostly contain two independent components: (1) oligonucleotides in charge of logical

function and (2) extrinsic reporters responsible for monitoring the “state” of the logic devices. This strategy works very well in simple logic circuits, such as basic Boolean logic gates

and some single-output units9,15. However, for advanced, multi-output logic circuits, selecting suitable reporters with distinguishable signals is still a challenge. For example, the

DNA-based platform designed by Wang’s group8 employed four different fluorescent reporters with partially overlapping signals to construct a 2-to-4 decoder. Obviously, the higher the

complexity of the logic functions, the more difficult it is to find suitable reporters. To simplify the design of advanced logic circuits, we introduce a supramolecular reporter, which

involves only one molecular block but is capable of displaying multiple assembly states16,17 and, consequently, represents multiple output channels.

Cyanine dye, a common fluorochrome, has been applied in various devices, such as biosensors18, components for nonlinear optics19, and data storage devices20, owing to its unique

photophysical and photochemical properties. Cyanines can assemble into various aggregates21, and each aggregate presents distinguishable spectral features, implying that they have the

potential to simultaneously provide multiple independent output channels. In our previous work, we succeeded in constructing a 2-to-4 decoder based on a cyanine supramolecular system22.

Another advantage of cyanines is that their assembly behavior can be regulated by various stimuli, including pH, metal ions and biomacromolecules, especially specific DNA

structures23,24,25,26,27,28, and the assembly conversions could be reversed by eliminating the stimuli. Herein, we developed a multifunctional logic platform based on DNA D1

(5′-GGTGGTGGTGGT-3′), D2 (5′-CCACCACCACCACAACCACCACCACCAAA-3′),  and cyanine dye MTC [3,3’-di(3-sulfopropyl)-4,5,4’,5’-dibenzo-9-methyl-thiacarbocyanine triethylammonium salt] (Fig. 1). By

introducing stimuli that can regulate both the secondary structures of D1/D2 and the assembly states of MTC, the platform implemented several types of information processing functions,

including data filtration, selection, verification, and accumulation.

a The changes in the structures of D1/D2 and the assembly states of MTC triggered by various inputs. b The molecular structure and assembly states of MTC. c The DNA motifs of D1 and D2

DNA oligonucleotides D1 (5′-GGTGGTGGTGGT-3′) and D2 (5′-CCACCACCACCACAACCACCACCACCAAA-3′) were purchased from Sangon Biotechnology Co. Ltd. (Shanghai, China). The cyanine dye MTC was

synthesized according to the methods of Hamer29 and Ficken30, and the purity was determined by MS, elemental analysis, NMR and absorption spectroscopy (shown in Figs. S1–S4 and Tables 

S1–S3). Lead chloride (PbCl2), 18-crown-6-ether (CE) and potassium nitrate (KNO3) were purchased from Aldrich Ltd. (Shanghai, China). Silver nitrate (AgNO3) and l-cysteine (Cys) were

obtained from Sigma-Aldrich. All the chemicals are analytical reagent grade and used as received without further purification. Ultrapure water prepared by an ULUPURE (Chengdu, China)

ultrapure water system and was used throughout the experiments.

The stock solution of MTC was prepared by dissolving its powder in methanol and was stored in darkness. The DNA stock solutions were prepared by dissolving certain amounts of

oligonucleotides in 10 mM Tris-HCl buffer solution. The D1D2 duplex was prepared by mixing D1 and D2 in equimolar amounts and annealing the mixture in a thermocycler (first heated at 90°C

for 5 min and then cooled slowly to room temperature). The DNA concentrations were determined by measuring the absorbance at 260 nm.

The fluorescence spectra were collected with a F-4600 spectrophotometer (HITACHI, Japan) in a 10 mm quartz cell. Xenon arc lamp was used as the excitation light source. Both the excitation

and emission slits were 5 nm, the PMT voltage was 400 V, and the excitation wavelength was 530 nm. The absorption spectra were acquired with an EVOLUTION 201 spectrophotometer (Thermo

SCIENTIFIC, USA) at room temperature in a 10 mm quartz cell.

To better demonstrate the logic functions of the platform, the optimized input conditions were selected for each circuit. All samples were incubated in the dark at room temperature for 20 

min before the measurement.

For the binary INHIBIT gate, 4 μM D1 and 4 μM MTC in 10 mM Tris-HCl buffer solution (pH 7.0) were used as the initial state, and 10 μM PbCl2 and 4 μM D2 were employed as the inputs. With

respect to the ternary INHIBIT gate, 4 μM MTC in 10 mM Tris-Ac buffer solution (pH 7.4) was used as the initial state, and 1 μM D1, 1 μM D1 with 0.15 μM PbCl2, 1 μM D2, and 1 μM D2 with 3 μM

AgNO3 were employed as the inputs.

For the 2:1 MUX, 4 μM D1 and 4 μM MTC in 10 mM Tris-HCl buffer solution (pH 7.0) were used as the initial state; 10 μM PbCl2 and 10 mM Tris-HCl buffer solution (pH 4.0) were employed as the

data inputs; and 4 μM D2 with 30 mM KCl was employed as the address input.

For the DEMUX and parity checker, 6 μM MTC in 10 mM Tris-HCl buffer solution (pH 8.5) was used as the initial state; 60 mM KCl was employed as the data input; and 50 mM CE and 6 μM D1 were

used as the address inputs in the 1:3 DEMUX. 10 mM Tris-HCl buffer solution (pH 4.0), 60 mM KCl, 10 μM PbCl2 and D1 were employed as the four binary bits of the examined numbers in the

parity checker.

For the comparator, 6 μM MTC and 6 μM D1D2 in 10 mM Tris-HCl (pH 8.5) were used as the initial state, and 30 mM KCl and 10 mM Tris-HCl buffer solution (pH 4.0) were employed as the inputs.

For the counter, 5 μM MTC in 10 mM Tris-HCl buffer solution (pH 8.5) was used as the initial state; 40 mM KCl and 70 mM CE were selected as the cumulating and descending pulses,

respectively.

The human serum samples were supplied by Solarbio (China). The serum was centrifuged at 10,000 rpm for 3 min to remove precipitates, and the supernatants were diluted ten times using

Tris-HCl (10 mM, pH 7.0) buffer before the logic operations. For the logic operations, all the samples contained 1000-fold diluted serum, and the input stimuli were consistent with the above

description.

As a typical cyanine, MTC presents several assembly behaviors under different conditions. In alkaline buffer solutions (such as pH 8.5, 10 mM Tris-HCl), it has a dominating absorption peak

at 528 nm assigned to dimer (termed the D-band), while in acidic conditions (such as pH 4, 10 mM Tris-HCl) it self-assembles into J-aggregates (typical absorption peak at approximately 655 

nm, termed the J-band)31, as shown in Supplementary Figs. S5 and S6. Some cations can also facilitate the self-assembly of MTC. For example, MTC can assemble into either J- or H-aggregates

(absorption peak in the range of 440–480 nm, termed the H-band) in the presence of potassium (Supplementary Fig. S7). In addition, we previously reported that certain DNA G-quadruplex

structures can specifically disassemble MTC aggregates into monomers, accompanied by a dramatic fluorescence enhancement (maximum emission wavelength at 600 nm, termed the FI)32,33. The

unique assembly behavior of MTC can provide at least four distinguishable output signals (D-band, J-band, H-band, and FI), which makes it convenient for the construction of multi-output

logic circuits.

In this paper, we defined the threshold of the output signals (normalized MTC J-band, H-band or FI) at 0.3 in all the binary logic devices. Any signal greater than the threshold was defined

as “1”, and those less than the threshold as “0”. Similarly, the thresholds in the ternary gate were defined at 0.3 and 1.5. It is worth mentioning that all the inputs in this work can be

eliminated by adjusting the complexing agents, so all the logic operations are reversible. For example, Pb2+ and Ag+ can be complexed by cysteine (Cys), K+ can be complexed by 18-crown-6

(CE), and the G-quadruplexes can be unwound into a stable complex (Supplementary Figs. S8 and S9).

The INHIBIT gate (Fig. 2a) is one of the most widely used Boolean logic gates. A binary INHIBIT gate can only output the high level by inputting special one of the two inputs individually.

This filtering function makes it to be applied widely in biochemical detection13. As mentioned above, MTC can recognize specific G-quadruplex structures against single-/double-stranded

oligonucleotides. The G-rich oligonucleotide D1 in a random single strand (under metal ion-free conditions) can induce only weak fluorescence signal of MTC, while its parallel G-quadruplex

formation (induced by metal ions, such as K+ or Pb2+) can cause a strong fluorescence enhancement. In addition, the D1D2 complex can hardly induce fluorescence (Supplementary Fig. S8). By

utilizing distinctive fluorescence patterns, both binary and ternary logic gates were constructed.

a Diagram of the electronic INHIBIT logic circuit. b The schematic truth table of the binary INHIBIT gate. The fluorescence spectra of MTC with different input conditions (c) and the

normalized fluorescence intensity (FI) of MTC monomers as the output (d) in the binary INHIBIT gate. e The schematic truth table of the ternary INHIBIT gate. The fluorescence spectra of MTC

with different input conditions (f) and the normalized MTC FI as the output (g) in the ternary INHIBIT gate

To construct a binary INHIBIT gate, we chose MTC and D1 as the initial state and Pb2+ and D2 as the input stimuli (Fig. 2b). Pb2+ can facilitate the formation of the D1 G-quadruplex, while

D2 has the opposite effect. As shown in Fig. 2d, in the initial state, MTC shows a weak fluorescence signal with single-stranded D1, while it emits a strong signal with PbCl2 input. The

addition of D2 complements D1 to D1D2 complex, which is stable and cannot be unwound by Pb2+, and reduces the signal to near zero. The normalized MTC FI values are plotted as a bar chart in

Fig. 2c, and the result is accordance with the proper execution of the INHIBIT gate.

In binary logic systems, each bit is coded by “0” or “1”, corresponding to a low and high signal, respectively. However, due to the complexity of information processing, sometimes it is

difficult to identify certain and accurate states based on this low/high system. In contrast, multi-valued logic systems, which involve a switch between multiple states, show an overwhelming

advantage in dealing with uncertain information. Ternary logic gates, as a typical representation of multi-valued logic systems, have three different output levels: low, medium and high,

which correspond to logic values of “0”, “1”, and “2”, respectively34. With respect to MTC, its fluorescence signals can be induced to three different levels by D1 with certain structures,

i.e., (1) a strong signal induced by the G-quadruplex (termed D1-G4), (2) a weak signal induced by the single-stranded structure (termed D1-ss) and (3) almost no signal induced by the D1D2

complex. Therefore, a ternary logic unit can be constructed based on this platform, and the outputs can be defined as “2”, “1”, and “0”, according to the fluorescence strength.

As shown in Fig. 2e, MTC is defined as the initial state. With respect to the input X, D1 is defined as “1” and further addition of Pb2+ as “2”. With respect to the input Y, the mixture of

D2 and Ag+ is defined as “1” and D2 alone as “2”. In this system, there are five DNA motifs, including D1-ss, D1-G4 induced by Pb2+, single-stranded D2 (termed D2-ss), i-motif D2 induced by

Ag+ (termed D2-i) and the D1D2 complex, and their stabilities are in the following order: D2-i > D1D2 > D1-G4 > D1-ss/D2-ss. The confirmation of each DNA structure and the thermal stability

experiment results are shown in Supplementary Figs. S10–S13.

As shown in Fig. 2f and Supplementary Fig. S12, in the case of input X = “0”, the system contains no DNA, D2-i or D2-ss with different input Y situations. Since neither D2-i nor D2-ss can

induce MTC to generate a fluorescence signal, all the outputs are “0”. In the case of input X = “1”, owing to the absence of Pb2+, D1-ss causes a weak signal (output = “1”) in the (1,0)

input situation. For the input of (1,2), D1 is totally complemented by D2, and the D1D2 complex decreases the output signal to “0”. For the input of (1,1), since D2-i is more stable than

D1D2, part of D1D2 is unwound by Ag+, and the released D1-ss causes a weak signal, which is similar to the (1,0) input situation (output = “1”). In the case of input channel X = “2”, the

D1-G4 induced by Pb2+ causes a strong signal (output = “2”) in the (2,0) input situation. For the input (2,2), since D1D2 is more stable than D1-G4, D1-G4 is totally complemented by D2,

which leads to no signal (output = “0”). For the input (2,1), which is similar to the case of (1,1), partial D1 is released and forms the G-quadruplex in the presence of Pb2+. Since only

some D1-G4 is formed, the signal of MTC is still weak compared with that observed in the case of (2,0), and the output is “1”. The normalized MTC FI values are plotted as a bar chart in Fig.

 2g, and the results are accordance with the proper execution of a ternary INHIBIT gate.

The multiplexer (MUX) and demultiplexer (DEMUX) are controlled rotary switches and play key roles in electronic, telecommunication, and signal processing systems35. They involve a couple of

data input/output channels and an additional address (A) control. Depending on the state of A, data can be transmitted between certain input and output channels. To date, several MUX/DEMUX

systems have been designed based on organic molecules, enzyme reactions, or DNA platforms8,35,36,37. Among them, an oligonucleotide-triggered DNA conformational change is a common strategy.

However, the interference among DNA strands increases the difficulty of the implementation process and limits the operational performance. In this work, owing to the multiple assembly states

of MTC, a series of MUX and DEMUX was constructed in a simple manner.

The simplest MUX contains two data inputs, one output channel, and an A control (termed 2-to-1 MUX). The function of the 2-to-1 MUX is selecting one certain of the two data inputs according

to the “state” of A (Fig. 3d). Figure 3a presents the schematic illustration of the constructed 2-to-1 MUX. The mixture of MTC and D1 is considered the initial state, and D2 is the A

control. To balance the output signal, the D2 sample contains 30 mM KCl, which can facilitate the formation of D1-G4. Two ions, i.e., Pb2+, which can induce D1 to fold into the G-quadruplex,

and H+, which can induce D2 to fold into the i-motif, are chosen as the two data input stimuli. In the case of A = “0”, inputting Pb2+ promotes the formation of D1-G4 and turns on the

fluorescence of MTC, while inputting H+ cannot change the structure of D1 nor the MTC fluorescence (Fig. 3b). In this way, the input of Pb2+ is transmitted into the MTC fluorescence signal,

and the H+ channel has no influence on the output. In the case of A = “1”, the presence of D2 induces the formation of the D1D2 complex, which can be unwound by H+ but not Pb2+. Therefore,

only the acidic condition can release D1. The D1-G4 induced by KCl can turn on the MTC fluorescence, while the Pb2+ channel cannot influence the output. The conformational changes in MUX

were confirmed by circular dichroism (CD) and native polyacrylamide gel electrophoresis (PAGE) experiments (Supplementary Figs. S14 and S15). The normalized MTC FI values are plotted as a

bar chart in Fig. 3c, which satisfy the logic requirement of the 2-to-1 MUX. (The truth table is shown in Fig. 3e).

a Schematic illustration of the implementation of the 2-to-1 MUX. b The fluorescence spectra of MTC with different input conditions. c The normalized MTC FI as the output of the 2-to-1 MUX.

d The equivalent switching device of the 2-to-1 MUX. e The truth table of the 2-to-1 MUX logic operation

Any ion pairs capable of inducing the corresponding structural changes of D1/D2 can be considered as inputs the 2-to-1 MUX. To demonstrate the adaptability of the platform, we also

constructed another 2-to-1 MUX by changing H+ to Ag+. The result is shown in Supplementary Fig. S16, and it demonstrates that the platform can be applied for parallel detection of

heavy-metal Pb2+ and Ag+.

In contrast to MUX, the function of DEMUX is detangling data streams from a compressed signal. In this work, a 1-to-3 demultiplexer was also fabricated in a similar strategy (Fig. 4).

a Schematic illustration of the implementation of the 1-to-3 DEMUX. b The equivalent switching device of the 1-to-3 DEMUX. The absorption (c) and fluorescence (d) spectra of MTC with

different input conditions. e The normalized MTC J-band, H-band, and FI as the outputs of the 1-to-3 DEMUX. f The truth table of the 1-to-3 DEMUX logic operation

Potassium can induce MTC to form several aggregations by increasing its effective dielectric constant38, for example, 60 mM KCl can induce MTC to form H-aggregates (Supplementary Fig. S7).

Herein, KCl is chosen as the data input and the H-band is considered the first output channel. CE, which can chelate K+ and reduce its active concentration, is chosen as one address control

(A1). When A1 = “1”, inputting KCl leads to the formation of MTC J-aggregates. The featured J-band is considered the second output channel. In the same way, D1, which can be transformed into

the G-quadruplex by KCl and disassemble MTC aggregates into monomers, is considered another kind of address control (A2), and MTC FI is defined as the third output channel. The absorption

and fluorescence spectra are shown in Fig. 4c, d, respectively. The normalized outputs are plotted as bar charts (Fig. 4e), and the obtained truth table (Fig. 4f) presents the input data

leads to three different output channels according to the A conditions.

In data transmission, an inevitable problem is incorrect bits39, which have serious effects on the stable performance of logic computing, especially in sophisticated circuits. A parity

checker is a logic unit that can determine erroneous bits and maintain data validity. To develop a parity checker on this platform, we exploited the logic devices mentioned above with slight

modifications to distinguish odd and even numbers from natural numbers between 0 and 9 (Fig. 5). The decimal numbers are encoded into four-bit binary numbers (N1, N2, N3, and N4), and H+,

K+, Pb2+, and D1 are assigned to the corresponding bits for computation (Fig. 5e). Only the input situations containing D1 can produce the MTC fluorescence signal (Fig. 5b, c). Therefore,

the corresponding fluorescence signals of all the odd numbers are extremely low, i.e., false outputs, while the even numbers produce dramatic fluorescence signals, i.e., true outputs. It is

worth mentioning that the parity of a natural number, regardless of odd or even, is determined by the digit in the unit position. Thus, the logic system fabricated here can distinguish all

the even numbers from the odd ones.

a Schematic illustration of the implementation of the parity checker. b The fluorescence spectra of MTC with different input conditions. c The normalized MTC FI as the output of the parity

checker. d Diagram of electronic parity checker logic circuit. e The truth table of the parity checker

The comparator is another important device in signal processing systems, and it is widely used in telecommunication interfaces, analog–digital converters and sensory circuits. In this work,

K+ and H+ are chosen as the two numbers for comparison (A and B), and a digital comparator is constructed based on the structural change of D1D2 and corresponding assembly state of MTC (Fig.

 6).

a Schematic illustration of the implementation of the comparator. b The absorption spectra of MTC with different input conditions. c The normalized MTC J-band and H-band as the outputs of

the comparator. d Diagram of the electronic comparator logic circuit. e The truth table of the comparator logic operation

Under our experimental conditions, KCl at certain concentrations can induce MTC to form a mixture of H- and J-aggregates, while H+ can induce MTC to form J-aggregates. The two assembly

states of MTC (J- and H-aggregates) are employed as the two output channels (O2 and O1), respectively. The former indicates whether A ≠ B, and the latter indicates whether A > B. There are

two input situations, i.e., (0, 0) and (1, 1), that lead to the result A = B. For the (0, 0) situation, the D1D2 complex shows little interaction with MTC and MTC remains in the dimer form.

For the (1, 1) situation, H+ unwinds D1D2, and the released D1 is induced into the G-quadruplex by K+. Consequently, MTC is disassembled into monomers by D1-G4. In both situations, J- or

H-aggregates do not form, indicating that both outputs are “0”. In the case where A and B are not equal, there is no formation of D1-G4, and the assembly states of MTC are determined by the

cations in the system. If A > B, there is only K+ in the system, and MTC exists as a mixture of J- and H-aggregates, thus, both outputs are “1”. If A  B and (O1 = 1, O2 = 0) presents A