Molecular logic gates based on DNA tweezers responsive to multiplex restriction endonucleases

Abstract

DNA logic gates have received significant attention as biocompatible building blocks of molecular circuits. Herein, we described the construction of DNA self-assembled molecular tweezers containing four different restriction endonuclease recognition sites and application of the tweezers in the construction of DNA logic gates. An open tweezer is formed by three DNA oligonucleotides, one of which is labelled with a fluorophore and a quencher at the two ends. Addition of the fourth oligonucleotide might close the tweezers, thus turning off the fluorescence signal. The quenched fluorescence of the closed tweezers can be turned on again by any one of the four restriction endonucleases, thus conferring the tweezers with the ability for multiplex detection of the four endonucleases. Based on the fluorescence responses to DNA oligonucleotide and restriction endonucleases, a set of DNA logic gates, including one-input NOT and YES logic gates, two-input IMPLICATION logic gates, two, three and four-input OR logic gates, were constructed.

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Introduction

Logic gates, which can perform Boolean logic by receiving one or more logical inputs and producing a single logical output, are the fundamental building blocks of any digital circuits, and play core roles in conventional silicon-based computers.¹ As an alternative computing approach to silicon-based logic operations, molecular logic gates, which use chemical or biological molecules as logical inputs, have attracted more and more attention due to the potential for calculations on the nanometer scale.² DNA, a biomacromolecule that encodes the genetic information of cellular organisms, has been considered to be a powerful biomaterial for information storage and data calculation.³ Since DNA computing technology was initially proposed by Adleman in 1994,⁴ nearly all kinds of logic operations have been achieved on the basis of DNA's Watson–Crick complementarity characteristic and its ability to combine with specific target molecules.⁵

Structural polymorphism is another important characteristic of DNA. Based on this, various kinds of DNA nano-machines have been reported.⁶ A well-known one is DNA tweezers, which can perform tweezers-like opening and closing operations under external stimulations.⁷ One promising application of DNA tweezers is the construction of DNA logic gates. For examples, Elbaz et al. constructed two “SET–RESET” logic gate systems using pH and aptamer substrate-activated DNA tweezers machines, respectively.⁸ Li et al. designed dual-functional tweezers for multiplex detection of thrombin and adenosine triphosphate. Based on this, an “AND” logic gate was constructed.⁹

Restriction endonucleases, which can hydrolyze phosphodiester bonds at specific DNA sites,¹⁰ have been demonstrated to be involved in many physiological processes, such as DNA replication, repair, recombination, molecular cloning, genotyping and gene mapping,¹¹ and are regarded as promising targets for antimicrobial and antiviral drugs.¹² In this work, a DNA tweezers containing four recognition sites of restriction endonucleases was designed. Such a tweezers could give fluorescent response to four different restriction endonucleases. Based on this, molecular logic gates with one, two, three or four inputs were constructed.

Experimental

Materials

All oligonucleotides were purchased from Sangon Biotech. Co. Ltd. (Shanghai, China) and their sequences were listed in Table S1 in ESI.† The concentrations of the oligonucleotides were represented as single-stranded concentrations. Single-stranded concentrations were determined by measuring the UV absorbance at 260 nm. Molar extinction coefficient was calculated using a nearest neighbor approximation (http://www.idtdan.com/analyzer/Applications/OligoAnalyzer). Restriction endonucleases (EcoR I, BamH I, Hind III and Not I) were obtained from New England Biolabs (Beijing, China). Tris(hydroxymethyl)aminomethane (Tris), MgCl₂, NaCl and HCl were obtained from Sigma. All chemical reagents were of analytical grade and used without further purification.

Methods

Construction of the DNA tweezers. The DNA tweezers in opened state was constructed by three DNA strands (Strands A, B and C, 100 nM each) in 50 mM Tris–HCl buffer (pH 7.4) containing 100 mM NaCl and 10 mM MgCl₂. The solution was heated at 95 °C for 5 min, cooled slowly to 25 °C and incubated at 25 °C for 60 min. The prepared opened tweezers gives high fluorescence and will be used for the construction of NOT and IMPLICATION logic gates. The opened tweezers can be switched to closed state by adding the fourth DNA strand (Strand D, 100 nM) and incubating at 25 °C for 30 min. The prepared closed tweezers will be used in the activity detection of restriction endonucleases and the construction of YES and OR logic gates.

Fluorescence measurement. All fluorescence measurements were carried out on a Shimadzu RF-5301PC fluorescence spectrometer (Shimadzu Ltd., Japan). The emission spectra were collected from 490 nm to 650 nm with an excitation wavelength of 480 nm at room temperature. The fluorescence intensity at 517 nm was used for quantitative analysis of restriction endonucleases or used as output signal of logic gates. The excitation and emission slit widths were both set at 10 nm. Error bars in corresponding figures were obtained from three parallel experiments.

Assay of restriction endonuclease activity. The above-prepared closed tweezers was used for activity analysis of EcoR I, BamH I, Hind III and Not I. Taking EcoR I activity assay as an example, the assay system was prepared by mixing 100 nM closed tweezers and different amounts of EcoR I. After incubating at 37 °C for 50 min, the fluorescence spectra were recorded in the range of 490–650 nm using 480 nm as the excitation wavelength, and the fluorescence intensity at 517 nm was used for EcoR I activity analysis.

Results and discussion

Assembly and operation of the DNA tweezers

The basic configuration and operation principle of the DNA tweezers are depicted in Scheme 1. It consists of three oligonucleotide strands (A, B and C, Table S1†). Strand A is a dual-labelled oligonucleotide, whose 5′ and 3′-ends are labelled with a fluorophore 6-carboxyfluorescein (FAM) and a black hole quencher (BHQ-2), respectively. The 5′ and 3′ parts of Strand A can hybridize with the 3′ part of Strand B and the 5′ part of Strand C, respectively, to form two double-stranded regions each has 18 base pairs.¹³ The formation of rigid double-stranded structures separates FAM far away from BHQ-2 though there is a four-base single-stranded hinge between the two parts. As a result, fluorescence signal of FAM can be observed. Strand D consists of two parts, whose sequences are complementary to the dangling ends of Strands B and C, respectively. The hybridization between Strand D and the dangling parts of Strands B and C might close the tweezer, accompanied by the closely contact between FAM and BHQ-2. As a result, the fluorescence of FAM is quenched. In this closed tweezers, four restriction endonuclease recognition sites were introduced at the regions nearby the fluorophore and the quencher. These four sites can be specifically recognized by restriction endonucleases EcoR I, Hind III, BamH I and Not I, respectively. Addition of anyone can destroy the closed tweezers structure, leading to the separation of FAM from BHQ-2 and the recovery of FAM fluorescence.

Scheme 1 Working mechanism of the DNA tweezers responsive to DNA oligonucleotide and restriction endonucleases.

Fluorescence response of the DNA tweezers

Above-mentioned working mechanism of the DNA tweezers was demonstrated by the fluorescence changes of FAM shown in Fig. 1. Strands A, B and C construct an opened tweezers, which is characterized by the high fluorescence of FAM. Addition of Strand D can close the tweezers, accompanied by the significantly decreased FAM fluorescence. Subsequent addition either EcoR I, BamH I, Hind III or Not I can result in the recovery of the fluorescence, though the recovery extents are not identical for the four restriction endonucleases. In certain concentration ranges, linear relationship between fluorescence intensity and restriction endonuclease concentration can be obtained, thus indicating that such a DNA tweezers can be used for the activity detection of restriction endonucleases (Fig. 1 and S1–S3†). Interestingly, the activity detection of four restriction endonucleases can be achieved using only one DNA tweezers, and such a detection strategy might be easily extended to the assay of other restriction endonucleases.

Fig. 1 (a) Fluorescence responses of the DNA tweezers to Strand D and 0.13 U μL⁻¹ EcoR I, 0.2 U μL⁻¹ BamH I, 0.3 U μL⁻¹ Hind III or 0.4 U μL⁻¹ Not I. (b) Fluorescence spectra of the closed tweezers in the presence of different concentrations of EcoR I. (c) The fluorescence change at 517 nm as function of EcoR I concentration. The inset shows the fluorescence change in the EcoR I concentration range of 0–12 U/100 μL. The solid line represents a linear fit to the data.

Construction of DNA logic gates

Construction of DNA logic gates using nucleic acids or/and enzymes as inputs has attracted recent interests due to the potential applications in DNA computing technology and clinical diagnosis.¹⁴ Above-described fluorescence changes of the DNA tweezers enabled the design of several molecular logic gates, in which Strand D and/or restriction endonucleases were used as inputs, and the fluorescence signal of FAM was used as output. The fluorescence intensity at 517 nm above the threshold of 100 was defined as “1” or “true”, otherwise defined as “0” or “false”. Based on this, several logic gates with one, two, three or four inputs were designed.

One-input NOT and YES logic gates

Firstly, we constructed the basic one-input NOT and YES logic gates. The NOT logic gate was constructed on the basis of Strand D-triggered the closure of the DNA tweezers formed by Strands A, B and C. Strand D was used as the input of the gate. In the absence of Strand D (the input is 0), the tweezers was in the opened state, giving an output signal of “1” (Fig. 2a and b). With the addition of Strand D (the input is 1), the tweezers was closed, and an output of “0” was given. Similarly, a YES logic gate was constructed based on the closed DNA tweezers. One restriction endonuclease was used as the input. The closed tweezers contained four intact recognition sites corresponding to endonucleases EcoR I, Hind III, BamH I and Not I, respectively. The double-stranded DNA cleavage reaction triggered by any endonuclease could cleave the closed DNA tweezers into two parts, thus resulting in the separation of FAM from BHQ-2. Correspondingly, the fluorescence of FAM is recovered, and the output signal is changed from “0” to “1”. Fig. 2c and d show the fluorescent results of the YES logic gate using EcoR I as the input. The results of the YES logic gates using either of other three endonucleases can be found in ESI (Fig. S4–S6†).

Fig. 2 (a) Fluorescence spectra of the opened tweezers before and after addition of Strand D. (b) The outputs of the NOT logic gate. (c) Fluorescence spectra of the closed tweezers before and after addition of EcoR I (0.13 U μL⁻¹). (d) The outputs of the YES logic gate.

Two-input DNA IMPLICATION logic gate

By combing above NOT and YES gates, an IMPLICATION logic gate can be constructed. In this gate, Strand D and one restriction endonuclease were used as inputs. With no input or with endonuclease input alone, the DNA tweezers was in the opened state, and an output of “1” was given. With Strand D input alone, the DNA tweezers was closed, accompanied by an output of “0”. When the system was subjected to the inputs together, the cleavage reaction at corresponding site gave an output of “1”. The fluorescent results and corresponding truth table of the gate using Strand D and EcoR I as inputs are shown in Fig. 3, and the results of the IMPLICATION logic gates using Strand D and either of other three endonucleases can be found in ESI (Fig. S7–S9†).

Fig. 3 Two-input DNA IMPLICATION logic gate system using Strand D and EcoR I as inputs. (a) Fluorescence spectra of the closed tweezers in the presence of different inputs. [Strand D] = 100 nM; [EcoR I] = 0.13 U μL⁻¹. (b) Fluorescence intensity of the closed tweezers at 517 nm in the presence of different inputs. (c) Scheme of the IMPLICATION logic gate. (d) Truth table of the IMPLICATION logic gate.

Two, three and four-input OR logic gates

Using the restriction endonucleases as inputs, two, three or four-input OR logic gates can be constructed based on the closed DNA tweezers formed by Strands A, B, C and D. As for two-input OR logic gates, random two endonucleases are selected from the four endonucleases as the inputs. Therefore, six two-input OR logic gates could be constructed using the proposed DNA tweezers. Fig. 4 shows the fluorescent results and the truth table of the representative logic gate using EcoR I and BamH I as the two inputs. Without any inputs, the DNA tweezers is in closed state and an output of “0” is given. Adding either EcoR I or BamH I or both of them could recover the fluorescence, thus resulting in true signals (output of “1”). The fluorescence signal of the system with both EcoR I and BamH I is obviously higher than those of the system containing only one endonuclease. The reason is that the addition of two endonucleases can cleave the tweezers at two sites, thus promoting the destruction of the DNA tweezers and the separation of FAM from BHQ-2. The fluorescent results and truth tables of the other five two-input OR logic gates can be found in ESI (Fig. S10–S14†).

Fig. 4 Two-input DNA OR logic gate system. (a) Fluorescence spectra of the closed tweezers in the presence of different inputs. [EcoR I] = [BamH I] = 0.06 U μL⁻¹. (b) Fluorescence intensity of the closed tweezers at 517 nm in the presence of different inputs. (c) Scheme of the two-input OR logic gate. (d) Truth table of the two-input OR logic gate.

In addition to two-input OR logic gates, we also reported a set of three-input OR logic gates, in which three endonucleases were selected as inputs. Four such three-input OR logic gates can be constructed using the closed DNA tweezers. As shown in Fig. 5 and S15–S17,† all of the seven possible input combinations (inputting one, two or three endonucleases) enable the recovery of the quenched fluorescence and generate a true output.

Fig. 5 Three-input DNA OR logic gate system. (a) Fluorescence spectra of the closed tweezers in the presence of different inputs. [EcoR I (E)] = [BamH I (B)] = [Hind III (H)] = 0.06 U μL⁻¹. (b) Fluorescence intensity of the closed tweezers at 517 nm in the presence of different inputs. (c) Scheme of the three-input OR logic gate. (d) Truth table of the three-input OR logic gate.

Even though molecular logic gates were first suggested more than a decade ago,¹⁵ and numerous studies have been conducted in the latest years, reports on multi-input molecular gates are rare despite their practical significance.¹⁶ Here, we demonstrated an approach to design a four-input OR logic gate, in which, all of the four endonucleases are used as inputs. The fifteen possible input combinations (inputting one, two, three or four endonucleases) enable the recovery of the quenched fluorescence and convert the output from “0” to “1”. The fluorescent results and corresponding truth table of the four-input OR logic gate are shown in Fig. 6.

Fig. 6 Four-input DNA OR logic gate system. (a) Fluorescence spectra of the closed tweezers in the presence of different inputs. [EcoR I (E)] = [BamH I (B)] = [Hind III (H)] = [Not I (N)] = 0.06 U μL⁻¹. (b) Fluorescence intensity of the closed tweezers at 517 nm in the presence of different inputs. (c) Scheme of the OR logic gate. (d) Truth table of the OR logic gate.

Conclusions

In summary, a DNA tweezers containing four restriction endonuclease recognition sites was designed. Such a DNA tweezers can produce fluorescence response to four restriction endonucleases simultaneously, thus making the multiplex detection of different restriction endonucleases possible. This detection strategy might be easily extended to other restriction endonucleases by simply changing the endonuclease recognition sequences in the DNA tweezers structure. Based on this, a set of one-, two-, three- and four-input DNA logic gates were constructed. The logic operation is easy to carry out in a single tube by simple mixing the enzymes and the DNA oligonucleotides.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 21322507, 21175072) and Fundamental Research Funds for the Central Universities.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra05132d

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