A DNA molecular logic gate based on nucleic acid hybridization

Abstract

The present invention relates to a DNA molecular logic gate based on nucleic acid hybridization. Specifically, the present invention provides a DNA molecular logic gate based on nucleic acid hybridization, comprising: an input signal, a signal conversion unit, and an output signal, wherein the input signal includes at least two DNA input strands; the signal conversion unit includes a substrate strand; and the output signal includes a nucleic acid amplification result. Based on DNA self-assembly, the present invention provides a simple and feasible solution for the construction of the basic principle of DNA computers.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular computing, and particularly relates to a DNA molecular logic gate based on nucleic acid hybridization. Background Art

[0002] The creation of computers based on molecular biology has attracted wide attention, especially in the field of synthetic biology.

[0003] A computing device mainly includes an algorithm logic unit, a control unit, a memory, input and output devices. Boolean logic and logic gates are the core of its operation. If a biological computer is to become a reality, the construction of biological molecular logic gates is necessary.

[0004] Currently, with the development of complex biological tools, logic systems based on nucleic acids and proteins have emerged, and DNA- and RNA-based catalysts and logic gates have been proposed as general components for synthetic chemical circuits and applied in aspects such as medical treatment, nanotechnology, and embedded control of chemical reactions. The progress in this direction will depend on the progress in three fields: 1. Developing input / output interfaces between DNA circuits and biologically relevant molecules, DNA nanomachines, and conventional chemistry; 2. Developing DNA circuit construction techniques to scale up in order to systematically create large circuits; 3. Extending DNA programming methods beyond spatial structures at the molecular and macroscopic scales.

[0005] An algorithm logic unit is a complex device that performs logical operations. It includes three parts with detailed functions: sensing input, processing input information for decision-making, and executing output.

[0006] For this reason, a biology-based algorithm logic unit has built-in sensors that can receive input signals generated by various environmental factors. Specifically, the plasma membrane and its integrated receptors can sense pressure, osmotic pressure, intracellular contact, temperature, and chemicals. At the same time, reactive oxygen species, pH, nutrients, signal transduction factors, and other internal state indicators are recorded by internal receptors. The degree of change in a single environmental input or many combinations of them is presented to the unit at any given time, resulting in a large set of input information. Cells continuously process a large number of input signals to decide their appropriate responses, leading to changes in gene expression, enzyme activity, and signal network reconnection. This decision-making process is manifested in the form of output information such as migration, growth, or division and programmed cell death. In addition to these elements, DNA can also constitute a logic unit.

[0007] DNA is a stable biomolecule that can be used to construct molecular computing systems. In particular, DNA logic circuits have shown good performance in terms of scalability and computational correctness. However, previous DNA logic circuit architectures had two limitations. First, the computational speed was very slow, usually taking several hours to compute a simple function. Second, the circuit was highly complex with respect to the number of DNA strands. For example, Guarnieri was the first to propose DNA addition, and he completed 2-bit binary addition using primer extension reactions. Subsequently, other DNA addition algorithm operation schemes emerged one after another, such as the addition and unified identification method scheme by Wasiewicz, the recursive scheme by Barua, etc. These algorithms all had a flaw: their experimental steps were linearly superimposed. This means that the experimental steps would increase with the increase in the number of addition bits, and ultimately it would lead to the inability to proceed in the laboratory. Of course, there were also some schemes with unchanged experimental steps, but they were more complex experimentally and could only be realized theoretically. Later, Hug et al. proposed a DNA computing method that operated on a DNA chip and could be implemented in parallel, but this experiment would take a considerable amount of time and it was not certain that carry addition could be completed on the chip surface. Recently, some schemes have been designed to speed up DNA logic circuits, including using local DNA circuits and leak-free strand displacement. However, both of these methods require a lot of extra work on DNA origami gates, which increases the complexity of design, operation, and strands. Therefore, it is important to design a DNA addition that can perform multi-bit binary operations without increasing the experimental steps. Summary of the Invention

[0008] To this end, the object of the present invention is to provide a DNA molecular logic gate based on nucleic acid hybridization.

[0009] The first aspect of the present invention provides a DNA molecular logic gate based on nucleic acid hybridization, including: an input signal, a signal conversion unit, and an output signal.

[0010] The input signal includes: at least two DNA input strands.

[0011] The signal conversion unit includes: a substrate strand, which contains an addend strand A0 strand, an A i strand, an A e strand, and an augend strand B i strand, where i = 1, 2, 3…n, and n is a positive integer greater than 1. The input signal is logically operated through the hybridization and amplification of nucleic acids (substrate strand and input strand), and the logical operation result is output.

[0012] The output signal includes: the nucleic acid amplification result.

[0013] Among them,

[0014] The 3' end a of A i ​2i One sequence is complementary to the 3'-end of B i a sequence at the 3'-end of 2i One sequence is complementary to the 5'-end of B i a sequence at the 5'-end of 2i+1 One sequence is complementary to the 5'-end of A i+1 a sequence at the 5'-end of 2i+1 One sequence is complementary to the 5'-end of B i-1 a sequence at the 5'-end of 2i-1 One sequence is complementary to the 5'-end of A i a sequence at the 5'-end of 2i-1 One sequence is complementary to the 5'-end of B n c sequences at the 5'-end are complementary to the 5'-end of A e c sequences at the 5'-end are complementary, such that the addend chain A i chain, A e chain and the addend chain B i chain can cross-complement to form a nicked double-strand in the order of i = 1, 2, 3…n

[0015] The 5'-end of A0 and the 3'-end of A e are respectively complementary to the primers

[0016] The value c and each a are each independently a positive integer greater than or equal to 5, and a 2i-1 + a 2i ≤ the total number of bases in A i and a 2i + a 2i+1 ≤ the total number of bases in B i the total number of bases

[0017] In one or more embodiments, a 2i + a 2i+1 is 0, 1, 2, 3, 4 or 5 less than the total number of bases in B i the total number of bases

[0018] In one or more embodiments, a 2i-1 + a 2i is 0, 1, 2, 3, 4 or 5 less than the total number of bases in A i the total number of bases

[0019] In one or more embodiments, the A0 chain is conjugated to a detectable label, preferably conjugated to biotin

[0020] In one or more embodiments, c and each a are each independently a positive integer from 5 to 40, preferably a positive integer from 10 to 20, more preferably 20

[0021] In one or more embodiments, in the above DNA molecular logic gate, the DNA molecular logic gate is an AND gate, and in the AND gate:

[0022] a0 sequences at the 3'-end of A0 are complementary to a0 sequences at the 3'-end of the first input strand. b sequences at the 5'-end of the first input strand are complementary to b sequences at the 3'-end of the second input strand. a1 sequences at the 5'-end of the second input strand are complementary to a1 sequences at the 5'-end of A1. The input signals are subjected to logical operations through hybridization and amplification of the substrate strand and the input strands, and the logical operation results are output.

[0023] In one or more embodiments, a0, a1, and b are each independently positive integers from 5 to 40, preferably positive integers from 10 to 20.

[0024] In one or more embodiments, in the above DNA molecular logic gate, the DNA molecular logic gate is an OR gate, and in the OR gate:

[0025] The substrate strand further includes linker strands L0 and L1, and A0 is divided into A0I and A0II.

[0026] a0 sequences at the 3'-end of A0I are complementary to a0 sequences at the 3'-end of the first input strand. b sequences at the 5'-end of the first input strand are complementary to b sequences at the 3'-end of L0. a1 sequences at the 5'-end of L0 are complementary to a1 sequences at the 5'-end of A1.

[0027] a0' sequences at the 3'-end of A0II are complementary to a0' sequences at the 3'-end of the second input strand. b' sequences at the 5'-end of the second input strand are complementary to b' sequences at the 3'-end of L1. a1' sequences at the 5'-end of L1 are complementary to a1' sequences at the 5'-end of A1.

[0028] The first input strand is not complementary to L1, and the second input strand is not complementary to L0.

[0029] The input signals are subjected to logical operations through hybridization and amplification of the substrate strand and the input strands, and the logical operation results are output.

[0030] In one or more embodiments, a0, a0', a1, a1', and b are each independently positive integers from 5 to 40, preferably positive integers from 10 to 20.

[0031] In one or more embodiments, in the above DNA molecular logic gate, the DNA molecular logic gate is a NAND gate, and in the NAND gate:

[0032] The substrate strand further includes addend strands B0I and B0II, and A1 is divided into A1I and A1II.

[0033] a0 sequences at the 3'-end of B0 are complementary to a0 sequences at the 3'-end of A0, a0' sequences at the 3'-end of B0II are complementary to a0' sequences at the 3'-end of A0, and the a0 sequences at the 3'-end of B0I and the a0' sequences at the 3'-end of B0II have at least 90% identity (preferably 99% or identical),

[0034] a1 sequences at the 5'-end of B0I are complementary to a1 sequences at the 5'-end of A1I, a1' sequences at the 5'-end of B0II are complementary to a1' sequences at the 5'-end of A1II, and the a1 sequences at the 5'-end of A1I and the a1' sequences at the 5'-end of A1II have at least 90% identity (preferably 99% or identical),

[0035] a 2i sequences at the 3'-end of A1I are complementary to a 2i sequences at the 3'-end of B1,

[0036] a 2i ' sequences at the 3'-end of A1II are complementary to a 2i ' sequences at the 3'-end of B1,

[0037] B0I preferentially hybridizes with the first input strand rather than with A0 and A1I, and B0I does not hybridize with the second input strand; preferably, B0I is at least 70% (preferably 80%, 90%, more preferably 98%) complementary to the first input strand,

[0038] B0II preferentially hybridizes with the second input strand rather than with A0 and A1II, and B0II does not hybridize with the first input strand; preferably, B0II is at least 70% (preferably 80%, 90%, more preferably 98%) complementary to the second input strand,

[0039] Logical operations are performed on the input signals through hybridization and amplification of the substrate strand and the input strand, and the logical operation results are output.

[0040] In one or more embodiments, a0, a0', a1, a1', a 2i、 a 2i ' are each independently a positive integer from 5 to 40, preferably a positive integer from 10 to 20.

[0041] In one or more embodiments, a0 is equal to or different from a0'.

[0042] In one or more embodiments, a1 is equal to or different from a1'.

[0043] In one or more embodiments, a 2i is equal to or different from a 2i '.

[0044] In one or more embodiments, the signal conversion unit further comprises one or more reagents selected from the following: phosphorylase, ligase, polymerase, dNTP, and buffer.

[0045] The ligase can link the addend strands together and / or link the augend strands together.

[0046] The second aspect of the present invention further provides a DNA circuit comprising the DNA molecular logic gate described in any one of the embodiments herein.

[0047] The third aspect of the present invention further provides the application of the DNA molecular logic gate or DNA circuit described in any one of the embodiments herein in biological detection, molecular computing, or the preparation of circuit nano-devices.

[0048] The fourth aspect of the present invention further provides a method for constructing the DNA molecular logic gate described in any one of the embodiments herein, comprising the following steps:

[0049] Optionally, 1) phosphorylate the substrate strand, and optionally phosphorylate the input strand and / or the ligation strand,

[0050] 2) mix the input strand and the substrate strand in equal proportions and anneal them to assemble into a nicked DNA double strand,

[0051] 3) fill in the nick of the DNA double strand to obtain a complete DNA double strand,

[0052] 4) amplify using primers, and the output signal is the amplified DNA double strand.

[0053] In one or more embodiments, the input strand and the substrate strand are as described in any one of the embodiments of the first aspect of the present invention.

[0054] The beneficial effects of the present invention are as follows:

[0055] Based on DNA self-assembly, the experimental steps of the present invention are simple and easy to operate, providing a simple and feasible solution for the construction of the basic principle of DNA computers. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Schematic diagram for AND gate construction. Where Endgroup indicates strand A e strand.

[0057] Figure 2 Schematic diagram for OR gate construction. Where Endgroup indicates strand A e strand.

[0058] Figure 3 Schematic diagram for NAND gate construction. Where Endgroup indicates strand A e strand. DETAILED DESCRIPTION OF THE INVENTION

[0059] To mitigate the limitations described in detail above, the architecture of a biocomputer requires certain characteristics. First, the logic gates should have a very low error tolerance (unexpected reactions) so that the steps of signal amplification are minimized; second, logic gates with multiple DNA complexes should be avoided because such DNA complexes increase the complexity of the strands and are more prone to errors due to imperfect sequence design and purification compared to simple DNA structures (such as single strands).

[0060] The present invention first provides a DNA molecular logic gate based on nucleic acid hybridization, comprising: an input signal, a signal conversion unit, and an output signal,

[0061] The input signal includes: at least two DNA input strands ss1 and ss2;

[0062] The signal conversion unit includes: a substrate strand, comprising an addend strand A0 strand, A i strand, A e strand, and an addend strand B i strand, where i = 1, 2, 3…n, and n is a positive integer greater than 1. Logical operations are performed on the input signal through nucleic acid hybridization and amplification (such as hybridization and amplification of the substrate strand and the input strand), and the result of the logical operation is output;

[0063] The output signal includes: the nucleic acid amplification result,

[0064] wherein,

[0065] The 3'-end a i sequences of A 2i are complementary to the 3'-end a i sequences of B 2i ; the 5'-end a i sequences of B 2i+1 are complementary to the 5'-end a i+1 sequences of A 2i+1 ; the 5'-end c sequences of B n are complementary to the 5'-end c sequences of A e such that the addend strands A i strand, A e strand, and the addend strand B i strand can be alternately complementary to form a nicked double strand in the order of i = 1, 2, 3…n,

[0066] The 5'-end of A0 and the 3'-end of A e are respectively complementary to the primers.

[0067] The respective values of a and c are each independently a positive integer greater than or equal to 5, such as a positive integer from 5 to 40, preferably a positive integer from 10 to 20, and more preferably 20.

[0068] Generally, a 2i-1 +a 2i ≤A i is the total number of bases in full length, and a 2i +a 2i+1 ≤B i is the total number of bases in full length.

[0069] In logical operation, due to the complementary relationship between sequences, the addend chain and the augend chain will hybridize to form a double-stranded form with staggered pairing and gaps after mixing. One strand of the double-stranded is the addend chains A1, A2…A n and A e arranged in sequence in order, with gaps between adjacent addend chains; the other strand is the augend chains B0 (if any), B1, B2…B n arranged in sequence in order, with gaps between adjacent addend chains. A i hybridizes complementarily with B i-1 (if any) and B i respectively to form a structure of staggered hybridization. As shown in the result chain in Figures 1-3 . The "gap" is the unconnected part in the single strand, which can be the disconnection between adjacent but unconnected nucleic acid molecules, or the deletion of 1-5 (preferably 1-3, more preferably 1) nucleotides, that is, a 2i +a 2i+1 is 0, 1, 2, 3, 4 or 5 less than the total number of bases in full length of B i , and a 2i-1 +a 2i is 0, 1, 2, 3, 4 or 5 less than the total number of bases in full length of A i .

[0070] At this time, through the ligase in the system, the gaps in each single strand in the double-stranded are filled by forming a linkage bond (such as 3',5'-phosphodiester bond) between nucleotides. Before ligation, each strand (at least including the addend chain) is optionally phosphorylated, for example, by phosphorylase. In some embodiments, at least the addend chain is phosphorylated. Of course, other strands can also be phosphorylated.

[0071] In the embodiment where the gap is the deletion of nucleotides, the system also contains DNA polymerase and dNTP, so as to fill the gap according to the DNA base pairing principle in the gap.

[0072] Accordingly, the signal conversion unit further comprises one or more reagents selected from the following: phosphorylase, ligase, polymerase, dNTP, and buffer. The various enzymes and buffers applicable herein are those commonly used in the field of biochemistry for DNA phosphorylation, hybridization, ligation, or PCR, and they are all commercially available. Their concentrations or ratios can be adjusted by those skilled in the art according to the situation of the experimental materials (such as the content of DNA strands). Preferably, the phosphorylase is a nucleic acid kinase (such as T4 nucleic acid kinase), the ligase is a DNA ligase (such as T4 DNA ligase), and the polymerase is a DNA polymerase.

[0073] Through an AND gate, OR gate, or NAND gate described below, A0 with a primer pairing sequence is connected or not connected to the above double-stranded structure, so that DNA amplification (such as PCR) is carried out through the primer pairing sequences of A0 and A e to achieve result output.

[0074] The result is based on whether there is an amplified result strand. Methods for detecting the amplified result strand are well-known in the art, such as agarose gel electrophoresis and sequencing.

[0075] AND gate

[0076] As Figure 1 shown, the input unit (input strand) is ss1 and ss2, and the conversion unit contains a substrate strand. The a0 sequences at the 3' end of A0 are complementary to the a0 sequences at the 3' end of the first input strand ss1, the b sequences at the 5' end of the first input strand ss1 are complementary to the b sequences at the 3' end of the second input strand ss2, and the a1 sequences at the 5' end of the second input strand ss2 are complementary to the a1 sequences at the 5' end of A1. Logical operations are performed on the input signals through hybridization and amplification of the substrate strand and the input strands, and the logical operation results are output. Similar to the values of each a and c above, a0, a1, and b are each independently a positive integer greater than or equal to 5, such as a positive integer from 5 to 40, preferably a positive integer from 10 to 20, and more preferably 20.

[0077] When only ss1 (0,1) is input, the addition operation cannot occur and no signal is output; when only ss2 (1,0) is input, the addition operation cannot occur and no signal is output; when both ss1 and ss2 are input (1,1), the condition for the addition operation is formed, a result strand is generated, a signal is output, and the signal can be amplified by PCR. When only ss1 (0,1) is input, the addition operation cannot occur and no signal is output; when only ss2 (1,0) is input, the addition operation cannot occur. The truth table is shown as follows:

[0078]

[0079] In some embodiments, each of the two input strands ss1 and ss2 comprises a substrate strand (adder strands A0 and A1) binding portion (H or CS) and two portions connecting complementarily (P); ss1(0,1) = H 0 - P1, ss2(1,0) = P 1 - C1S0.

[0080] In an embodiment where n = 3, the substrate strands include: A0 strand (A0 = PrimerL - H0), adder strand A1 = C 1 S 0 - H1, A2 = C 2 S 1 - H2, A3 = C 3 S 2 - H3, ENDGROUP (i.e., A e ) = C 4 S 3 - PrimerR, addend strand B1 = H 1 - C2S1, B2 = H 2 - C3S2, B3 = H 3 - C4S3.

[0081] In a preferred embodiment, the substrate strands include: A0 strand (A0 = PrimerL - H0[0]), adder strand A1 = C 1 S 0

[00] - H1[0], A2 = C 2 S 1

[00] - H2[0], A3 = C 3 S 2

[00] - H3[1], ENDGROUP (i.e., A e ) = C 4 S 3

[10] - PrimerR, addend strand B1 = H 1[0] - C2S1

[00] , B2 = H 2[0] - C3S2

[00] , B3 = H 3[1] - C4S3

[10] . Exemplary specific sequences are shown in Table 1.

[0082] In this article, the assignments in the square brackets "[]" after H and CS are exemplary and are only used to distinguish different sequences, and do not constitute a limitation on each strand and its sequence.

[0083] After PCR amplification using primers, the presence or absence of the result strand is detected (e.g., by detecting the band position through agarose gel electrophoresis) to achieve the output of the AND gate.

[0084] OR gate

[0085] As Figure 2 shown, the input units (input chains) are ss3 and ss4. The conversion unit contains substrate chains. Different from the AND gate, two connecting chains L0 and L1 are added to ensure the operation can run, and A0 is divided into two chains A0I and A0II.

[0086] The a0 sequences at the 3'-end of A0I are complementary to the a0 sequences at the 3'-end of the first input chain ss3. The b sequences at the 5'-end of the first input chain ss3 are complementary to the b sequences at the 3'-end of L0. The a1 sequences at the 5'-end of L0 are complementary to the a1 sequences at the 5'-end of A1. The a0' sequences at the 3'-end of A0II are complementary to the a0' sequences at the 3'-end of the second input chain ss4. The b' sequences at the 5'-end of the second input chain ss4 are complementary to the b' sequences at the 3'-end of L1. The a1' sequences at the 5'-end of L1 are complementary to the a1 sequences at the 5'-end of A1. The first input chain ss3 is not complementary to L1, and the second input chain is not complementary to L0. Logical operations are performed on the input signals through hybridization and amplification of the substrate chains (including connecting chains) and the input chains, and the logical operation results are output. Similar to the values of each a and c above, a0, a0', a1, a1', b are each independently a positive integer greater than or equal to 5, such as a positive integer of 5 - 40, preferably a positive integer of 10 - 20, and more preferably 20.

[0087] When only ss3 (0,1) is input, an addition operation can occur and a signal output is generated; when only ss2 (1,0) is input, an addition operation can occur and a signal output is generated; when both ss1 and ss2 are input (1,1), an addition operation can also occur and a signal output is generated. The truth table is as follows:

[0088]

[0089]

[0090] In some embodiments, each of the two input chains (ss3 and ss4) contains two parts: a substrate chain (A0I and A0II) binding part (H or CS) and a connection complementary region (P) with the connecting chains (L0 and L1), ss3(0,1) = H 0[0]-P1, ss4(1,0) = H 0[1]-P0.

[0091] In the embodiment where n = 3, the substrate chains include: 2 types of A0 chains (A0I = PrimerL - H0[0] and A0II = PrimerL - H0[1]), the addend chain A1 = C 1 S 0 - H1, A2 = C 2 S 1 - H2, A3 =C 3 S 2-H3, ENDGROUP (i.e., A e = C 4 S 3-PrimerR), the addend chain B1 = H 1-C2S1, B2 = H 2[0]-C3S2, B3 = H 3-C4S3, the connecting chain: L0 = P 1-C1S0, L1 = P 0-C1S0.

[0092] In a preferred embodiment, the substrate chain includes: 2 types of A0 chains (A0I = PrimerL-H0[0] and A0II = PrimerL-H0[1]), the addend chain A1 = C 1 S 0

[00] -H1[0], A2 = C 2 S 1

[00] -H2[0], A3 = C 3 S 2

[00] -H3[1], ENDGROUP (i.e., A e = C 4 S 3

[10] -PrimerR), the addend chain B1 = H 1[0]-C2S1

[00] , B2 = H 2[0]-C3S2

[00] , B3 = H 3[1]-C4S3

[10] , the connecting chain L0 = P 1-C1S0

[00] , L1 = P 0-C1S0

[00] . Exemplary specific sequences are shown in Table 2.

[0093] Because the substrate contains two types of A0 chains (A0I and A0II), when the input chains ss3 and ss4 are added separately or both are added, DNA double strands (structural chains) will be generated. After PCR amplification using primers, the presence or absence of the resulting chains is detected (e.g., by detecting the band position through agarose gel electrophoresis), achieving the output of an OR gate.

[0094] NAND gate

[0095] As Figure 3 shown, the input unit (input chains) are ss5 and ss6, the conversion unit contains the substrate chain for performing operations, two types of B0 chains (B0I and B0II) are added to ensure that the addition operation can run, and A1 is divided into two chains A1I and A1II.

[0096] a0 sequences at the 3'-end of B0I are complementary to a0 sequences at the 3'-end of A0, a0' sequences at the 3'-end of B0II are complementary to a0' sequences at the 3'-end of A0, and the a0 sequences at the 3'-end of B0I and the a0' sequences at the 3'-end of B0II have at least 90% identity (preferably 99% or identical). a1 sequences at the 5'-end of B0I are complementary to a1 sequences at the 5'-end of A1I, a1' sequences at the 5'-end of B0II are complementary to a1' sequences at the 5'-end of A1II, and the a1 sequences at the 5'-end of A1I and the a1' sequences at the 5'-end of A1II have at least 90% identity (preferably 99% or identical). The a 2i sequences at the 3'-end of A1I are complementary to a 2i sequences at the 3'-end of B1. The a 2i ' sequences at the 3'-end of A1II are complementary to a 2i ' sequences at the 3'-end of B1. Logical operations are performed on the input signal through hybridization and amplification of the substrate strand and the input strand, and the result of the logical operation is output.

[0097] B0I preferentially hybridizes with the first input strand rather than with A0 and A1I, and B0I does not hybridize with the second input strand; preferably, B0I is at least 70% (preferably 80%, 90%, more preferably 98%) complementary to the first input strand. Similarly, B0II preferentially hybridizes with the second input strand rather than with A0 and A1II, and B0II does not hybridize with the first input strand; preferably, B0II is at least 70% (preferably 80%, 90%, more preferably 98%) complementary to the second input strand.

[0098] Similar to the above values of each a and c, a0, a0', a1, a1', a 2i 、a 2i ' are each independently a positive integer greater than or equal to 5, such as a positive integer from 5 to 40, preferably a positive integer from 10 to 20, and more preferably 20. a0 and a0', a1 and a1', a 2i and a 2i ' may be equal or unequal respectively.

[0099] When only ss5(0,1) is input, ss5 binds to B0I (B0 = 0), but the B0II (B0 = 1) strand can perform an addition operation to generate a signal output; when only ss6(1,0) is input, ss6 binds to B0II (B0 = 1), but the B0I (B0 = 0) strand can perform an operation to generate a signal output; when ss5 and ss6 are input simultaneously (1,1), ss5 and ss6 bind to the two B0 strands respectively and no operation can occur, and no signal output is generated; when the input signal is (0,0), that is, neither ss5 nor ss6 is added, the operation in the substrate can run and a signal output can be generated. The truth table is as follows:

[0100]

[0101] In some embodiments, ss5 is fully complementary to B0I, i.e., ss5(0,1) = H0- C 1 S 0

[00] ; ss6 is fully complementary to B0II, i.e., ss6(1,0) = H0- C 1 S 0

[01] .

[0102] In an embodiment where n = 3, the substrate chain includes: A0 = PrimerL-H0, addition chain A1 (A1I = C 1 S 0

[00] -H1 and A1II = C 1 S 0

[01] -H1), A2 = C 2 S 1-H2, A3 = C 3 S 2-H3, ENDGROUP (i.e., A e = C 4 S 3-PrimerR), added chain B0 (B0I = H 0-C1S0

[00] and B0II = H 0-C1S0

[01] )、B1 = H 1-C2S1、B2 = H 2-C3S2、B3 = H 3-C4S3.

[0103] In a preferred embodiment, the substrate chain includes: A0 = PrimerL-H0[0], addition chain A1 (A1I = C 1 S 0

[00] -H1[0] and A1II = C 1 S 0

[01] -H1[0])、A2 = C 2 S 1

[00] -H2[0]、A3 = C 3 S 2

[00] -H3[1], ENDGROUP (i.e., A e = C 4 S 3

[10] -PrimerR), added chain B0 (B0I = H 0[0]-C1S0

[00] and B0II = H 0[0]-C1S0

[01] )、B1 = H 1[0]-C2S1

[00] 、B2 =H 2[0]-C3S2

[00] , B3 = H 3[1]-C4S3

[10] . An exemplary specific sequence is shown in Table 3.

[0104] When ss5 and ss6 are added simultaneously, since ss5 and ss6 are fully complementary to two B0 strands respectively, they will preferentially bind and cannot bind to other substrate strands, making it impossible to perform addition operations and thus impossible to form the result strand. Therefore, a result will only occur when neither ss5 nor ss6 is added or only one of them is added. After PCR amplification using primers, the presence or absence of the result strand is detected (for example, by detecting the band position through agarose gel electrophoresis) to achieve the output of the NAND gate.

[0105] In the process of performing addition operations using the above logic gates, each A0 (including A0I and A0II) or B0 (including B0I and B0II) may include at least one of two strands A0 and A0', where A0 and A0' correspond to binary 0 and 1 respectively; each addend strand includes at least one of two strands A i and A i ', where A i and A i ' correspond to binary 0 and 1 respectively; each augend strand includes at least one of two strands B i and B i ', where B i and B i ' correspond to binary 0 and 1 respectively.

[0106] For ease of detection, any one or more addend strands (such as A0 strands and / or A e strands) can be conjugated with a detectable label, and the detectable label can be a visualizable label such as a fluorescent molecule or a conjugation label such as biotin. Nucleic acids can be linked to a solid-phase carrier (such as magnetic beads) through biotin. The magnetic beads are preferably commercially available magnetic beads.

[0107] The concentration of each nucleic acid strand is usually less than or equal to 1 mM, for example, less than or equal to 10 uM. The present invention does not have special limitations on the specific sequences of the input strands, substrate strands, ligation strands, biotin strands, etc., as long as they can meet the above requirements of the present invention.

[0108] The present invention also provides a method for constructing a DNA molecular logic gate, including the following steps: Optionally, 1) phosphorylate the substrate strand, 2) mix the input strand and the substrate strand in equal proportion and anneal them to assemble into a nicked DNA double strand, 3) fill in the nick of the DNA double strand to obtain a complete DNA double strand, and 4) perform amplification using primers, and the output signal is the amplified DNA double strand. The input strand and the substrate strand are as described in any one of the specific embodiments herein. Step 1) may further include phosphorylating the input strand and / or the ligation strand.

[0109] A number of input strands, substrate strands, nucleic acid kinase, DNA ligase, and buffer are mixed, annealed, and purified to obtain the logic gate. The buffer is preferably a DNA ligation buffer. During the preparation of the circuit, it also includes volume-fixing to a fixed volume with water. In the present invention, the volume of the input strand is preferably 10 μL; the volume of the substrate strand is preferably 10 μL; the volume of the ligation solution is preferably 20 μL; the volume of the nucleic acid kinase is preferably 1 - 10 μL, and the volume of the DNA ligase is preferably 1 - 10 μL. In the present invention, the annealing temperature is preferably 10 - 60 °C; the annealing time is preferably 10 min - 2 h, more preferably 30 min. In the present invention, the purification preferably uses a DNA purification kit. In the specific implementation process of the present invention, preferably, the number of input strands, substrate strands, nucleic acid kinase, and buffer are first mixed for phosphorylation to obtain a mixed strand; then the mixed strand is mixed with DNA ligase for annealing and ligation to form a DNA logic gate substrate. In the present invention, preferably, the DNA logic gate substrate is purified, and the present invention has no special limitation on the purification method, and conventional DNA purification methods can be used.

[0110] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

[0111] Example

[0112] Three logic gates - AND gate, OR gate, and NAND gate - were constructed based on linear DNA self-assembly without fluorescence detection. The input unit consists of two 40-bp DNA single strands as signal input strands 1 and 2; the conversion unit is an addition operation based on DNA self-assembly, which contains DNA single strands providing the addition operation as substrate strands; the 200-bp long strand obtained from the addition operation is used as the output signal of the output unit, and the biotin-labeled 200-bp DNA double strand is separated by T1 magnetic beads, and PCR amplification is used to amplify the signal.

[0113] Experimental Materials

[0114] The DNA strands were prepared by Shanghai Diwin Biotech Co., Ltd.

[0115] The T4 Polynucleotide Kinase kit was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0116] The T4 DNA Ligase kit was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0117] The Taq Mix kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0118] Experimental methods

[0119] 1. Phosphorylation

[0120] 1) Dilution: Each single-stranded DNA was diluted to a certain volume according to the instructions (Shanghai Diwin Biotechnology Co., Ltd.), and the final concentration was 10 uM. The A0 strand was linked with biotin so that it could be coupled with magnetic beads.

[0121] 2) Divide all the single-stranded DNAs corresponding to the 10 uM input strand and the substrate strand into two groups (Group1 and Group2), and perform phosphorylation reactions separately at 37 °C for 30 min.

[0122] 2. Hybridization

[0123] Hybridize the two groups of phosphorylated products according to the hybridization reaction system shown in each example. After 30 s at 94 °C, place them at room temperature for 30 min to obtain Group3.

[0124] 3. Ligation

[0125] Perform ligation reaction on the hybridized solution according to the ligation reaction system shown in each example at 4 °C overnight (about 16 h). Obtain Group4.

[0126] 4. Magnetic bead adsorption

[0127] 1) Magnetic bead washing:

[0128] a) Take T1 magnetic beads, place the magnetic bead bottle on a vortex oscillator for 20 s, oscillate to resuspend the magnetic beads, and place them at room temperature for 30 min;

[0129] b) Take 50 ul of T1 magnetic beads into a new centrifuge tube, add 200 ul of washing solution 1, shake and mix well, place it on a magnetic rack, and remove the supernatant after 1 min;

[0130] c) Repeat step b, wash the magnetic beads 3 times in total;

[0131] d) After removing the supernatant for the last time, add 200 ul of washing solution 1 to resuspend the magnetic beads;

[0132] 2) Magnetic bead adsorption

[0133] a) Add 200 ul of Group4 to the T1 magnetic beads containing 200 ul of washing solution 1, fully oscillate to resuspend the magnetic beads, place the centrifuge tube on a rotary mixer, and rotate and mix at room temperature for 30 min;

[0134] b) Remove the centrifuge tube, place it on a magnetic rack for 2 - 3 min, discard the supernatant, and wash the magnetic beads 3 times with 200 ul of washing solution 1;

[0135] 3) Magnetic bead elution: Add 20 μl of ddH2O to the washed magnetic beads, resuspend the magnetic beads, place them on a magnetic stand, and take the supernatant.

[0136] 5. PCR

[0137] Take 5 μl of the eluted ligation product and perform PCR (25 μl system) according to the reaction system shown below.

[0138]

[0139] PCR parameter settings:

[0140]

[0141] 6. Detection by agarose gel electrophoresis

[0142] Detect the PCR product using 1.5% agarose gel. The loading volume is 10 μl, and D2000 is used as the marker; the size of the target band is 200 bp.

[0143] Example 1

[0144] 1. Introduction to AND gate:

[0145] As Figure 1 shown, the input units are ss1 and ss2, and the conversion unit contains substrate strands for performing addition operations. When only ss1 (0,1) is input, the addition operation cannot occur and no signal is output; when only ss2 (1,0) is input, the addition operation cannot occur and no signal is output; when both ss1 and ss2 are input (1,1), the conditions for the addition operation are formed, a result strand is generated, a signal output is formed, and the signal can be amplified by PCR.

[0146] 1) Truth table

[0147]

[0148] 2) Design of input strand ssDNA: According to the design method of the substrate strand, each of the two input strands contains a substrate strand binding part (H or CS) and a ligation complementarity (P) part, and each part is 20 bp; ss1(0,1) = H 0[0]-P1, ss2(1,0) = P 1-C1S0

[00] .

[0149] 3) Substrate strand: Biotinylated A0 strand (A0 = PrimerL-H0[0]), addend strand A1 = C 1 S 0

[00] -H1[0], A2 = C 2 S1

[00] -H2[0], A3 = C 3 S 2

[00] -H3[1], ENDGROUP = C 4 S 3

[10] -PrimerR, the addend chain B1 = H 1[0]-C2S1

[00] , B2 = H 2[0]-C3S2

[00] , B3 = H 3[1]-C4S3

[10] .

[0150] 4) Sequence

[0151] Table 1

[0152]

[0153]

[0154] 2. Experimental procedures

[0155] 1) Phosphorylation

[0156] a) Add two input chains

[0157]

[0158] b) When only input chain SS1 is added, Group1 remains unchanged

[0159]

[0160]

[0161] c) When only input chain SS2 is added, Group1 remains unchanged

[0162]

[0163] d) When no input chain is added, Group1 remains unchanged

[0164]

[0165] 2) Hybridization

[0166]

[0167] 3) Ligation

[0168]

[0169]

[0170] After the resulting chain is amplified by PCR, the position of the band is detected by agarose gel electrophoresis. Sequencing can also be performed for further detection.

[0171] Example 2

[0172] Introduction to OR Gate

[0173] As Figure 2 shown, the input units are ss3 and ss4, and the conversion unit contains substrate chains for addition operations. Different from the AND gate, two additional linker chains are added to ensure the addition operation can run. When only ss3(0,1) is input, the addition operation can occur and a signal output is generated; when only ss2(1,0) is input, the addition operation can occur and a signal output is generated; when both ss1 and ss2 are input (1,1), the addition operation can also occur and a signal output is generated.

[0174] 1) Truth Table

[0175]

[0176] 2) Design of input ssDNA strands: According to the design method of the substrate strands, each of the two input strands contains a substrate strand binding part (H or CS) and a linker complementarity (P) part. ss3(0,1) = H 0[0]-P1, ss4(1,0) = H 0[1]-P0.

[0177] 3) Substrate strands: Two types of A0 strands with different biotin assignments (A0 = 0, A0 = 1), adder strands A1, A2, A3, ENDGROUP, and addend strands B1, B2, B3.

[0178] 4) Linker strands: L0 = P 1-C1S0

[00] , L1 = P 0-C1S0

[00]

[0179] 5) Sequences:

[0180] Table 2

[0181]

[0182]

[0183] Experimental procedures:

[0184] 1) Phosphorylation

[0185] a) Add two input strands

[0186]

[0187]

[0188] b) When only the input strand SS3 is added, Group1 remains unchanged.

[0189]

[0190] c) When only the input strand SS4 is added, Group1 remains unchanged.

[0191]

[0192] d) When no input strand is added, Group1 remains unchanged.

[0193]

[0194]

[0195] 2) Hybridization

[0196] a) Add two input strands

[0197]

[0198] b) When only SS3 is added, it is the same as a)

[0199] c) When only SS4 is added, it is the same as a)

[0200] d) No input strand is added

[0201]

[0202] 3) Ligation

[0203] a) Add two input strands

[0204]

[0205] b) When only the SS3 strand is added, it is the same as a)

[0206] c) When only the SS4 strand is added, it is the same as a)

[0207] d) No input strand is added

[0208]

[0209] Because the substrate contains two types of A0 strands, when the input strands ss3 and ss4 are added separately or both are added, a 200-bp DNA double strand will be generated, which can be detected by agarose gel electrophoresis, proving that the OR gate is successfully constructed.

[0210] Example 3

[0211] Introduction to NAND gate

[0212] As shown Figure 3 in the figure, the input units are ss5 and ss6. The conversion unit contains substrate chains for performing addition operations. Two types of B0 chains (B0 = 0, B0 = 1) are added to ensure that the addition operation can run. When only ss5(0,1) is input, ss5 binds to B0 = 0, but the B0 = 1 chain can perform the addition operation to generate a signal output; when only ss6(1,0) is input, ss6 binds to B0 = 1, but the B0 = 0 chain can perform the addition operation to generate a signal output; when both ss5 and ss6 are input simultaneously (1,1), ss5 and ss6 bind to the two types of B0 chains respectively, and the addition operation cannot occur, resulting in no signal output; when the input signal is (0,0), that is, neither ss5 nor ss6 is added, the addition operation in the substrate can run and a signal output can be generated.

[0213] 1) Truth table:

[0214]

[0215] 2) Design of input ssDNA chains: According to the design method of the substrate chains, ss5 is completely complementary to B0 = 0, that is, ss5(0,1) = H0[0]- C 1 S 0

[00] ; ss6 is completely complementary to B0 = 1, that is, ss6(1,0) = H0[0]- C 1 S 0

[01] .

[0216] 3) Substrate chains: A0 chain with biotin (A0 = PrimerL - H0[0]), addend chains A1, A2, A3, ENDGROUP, augend chains B0, B1, B2, B3.

[0217] 4) Sequences

[0218] Table 3

[0219]

[0220]

[0221] Experimental procedures

[0222] 1) Phosphorylation

[0223] a) Add two input chains

[0224]

[0225]

[0226] b) When only input chain SS5 is added, Group1 remains unchanged

[0227]

[0228] c) When only the input strand SS6 is added, Group1 remains unchanged.

[0229]

[0230] d) When no input strand is added, Group1 remains unchanged.

[0231]

[0232] 2) Hybridization

[0233] a) Add two input strands

[0234]

[0235] b) Add only the input strand SS5, same as a)

[0236] c) Add only the input strand SS6, same as a)

[0237] d) Do not add any input strand

[0238]

[0239] 3) Ligation

[0240] a) Add two input strands

[0241]

[0242]

[0243] b) Add only the input strand SS5, same as a)

[0244] c) Add only the input strand SS6, same as a)

[0245] d) Do not add any input strand

[0246]

[0247] When both ss5 and ss6 are added, since ss5 and ss6 are respectively completely complementary to two types of B0 strands, they will preferentially bind and cannot bind to other substrate strands, making it impossible to perform addition operations and thus impossible to form the result strand. Therefore, the result strand will only appear when neither ss5 nor ss6 is added or only one of them is added. A correct band is detected at the proper position by agarose gel electrophoresis, proving the successful construction of the NAND gate. SEQUENCE LISTING <110> Shanghai Institute of Technology Shanghai Jiao Tong University <120> A DNA Molecular Logic Gate Based on Nucleic Acid Hybridization <130> 219930 1CNCN <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate strand <400> 1 aagtgttagc cgataggagg ccgaaggtca gcgattcgcg 40 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate strand <400> 2 atacagcctc gatgaggtct agaaaggcaa tcttcgcagc 40 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate strand <400> 3 gttaatatcg ccaaccgcct tctgctggat tcctggccgc 40 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate strand <400> 4 tcacggttat gcaaagcgag ctgattctag gtcgtccgcg 40 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 5 aggcggttgg cgatattaac gctgcgaaga ttgcctttct 40 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 6 ctcgctttgc ataaccgtga gcggccagga atccagcaga 40 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 7 tatagcaata ctcggatctg cgcggacgac ctagaatcag 40 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 8 cagatccgag tattgctata actttcacca gcgtttctct 40 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Input strand <400> 9 tgcattccaa gctagtacat cgcgaatcgc tgaccttcgg 40 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Input strand <400> 10 agacctcatc gaggctgtat atgtactagc ttggaatgca 40 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate strand <400> 11 aagtgttagc cgataggagg ccgaaggtca gcgattcgcg 40 <210> 12 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate strand <400> 12 aagtgttagc cgataggagg gcttacgaca agaaaaatac 40 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate strand <400> 13 atacagcctc gatgaggtct agaaaggcaa tcttcgcagc 40 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 14 gttaatatcg ccaaccgcct tctgctggat tcctggccgc 40 <210> 15 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 15 tcacggttat gcaaagcgag ctgattctag gtcgtccgcg 40 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 16 aggcggttgg cgatattaac gctgcgaaga ttgcctttct 40 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 17 ctcgctttgc ataaccgtga gcggccagga atccagcaga 40 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 18 tatagcaata ctcggatctg cgcggacgac ctagaatcag 40 <210> 19 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 19 cagatccgag tattgctata actttcacca gcgtttctct 40 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Input chain <400> 20 tgcattccaa gctagtacat cgcgaatcgc tgaccttcgg 40 <210> 21 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Input chain <400> 21 ctggaccgtt cgtagccctt gtatttttct tgtcgtaagc 40 <210> 22 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Linking chain <400> 22 agacctcatc gaggctgtat atgtactagc ttggaatgca 40 <210> 23 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Linking chain <400> 23 agacctcatc gaggctgtat aagggctacg aacggtccag 40 <210> 24 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 24 aagtgttagc cgataggagg ccgaaggtca gcgattcgcg 40 <210> 25 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 25 atacagcctc gatgaggtct agaaaggcaa tcttcgcagc 40 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 26 gcttctcgcc ttgtacgcga agaaaggcaa tcttcgcagc 40 <210> 27 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 27 gttaatatcg ccaaccgcct tctgctggat tcctggccgc 40 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 28 tcacggttat gcaaagcgag ctgattctag gtcgtccgcg 40 <210> 29 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 29 agacctcatc gaggctgtat cgcgaatcgc tgaccttcgg 40 <210> 30 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 30 tcgcgtacaa ggcgagaagc cgcgaatcgc tgaccttcgg 40 <210> 31 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 31 aggcggttgg cgatattaac gctgcgaaga ttgcctttct 40 <210> 32 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 32 ctcgctttgc ataaccgtga gcggccagga atccagcaga 40 <210> 33 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 33 tatagcaata ctcggatctg cgcggacgac ctagaatcag 40 <210> 34 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Substrate chain <400> 34 cagatccgag tattgctata actttcacca gcgtttctct 40 <210> 35 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Input chain <400> 35 ccgaaggtca gcgattcgcg atacagcctc gatgaggtct 40 <210> 36 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Input chain <400> 36 ccgaaggtca gcgattcgcg gcttctcgcc ttgtacgcga 40

Claims

1. A DNA molecular logic gate based on nucleic acid hybridization, comprising: Input signal, signal conversion unit, and output signal The input signal includes: at least two kinds of DNA input strands The signal conversion unit includes: a substrate strand, including an addend strand A0 strand, an A i strand, an A e strand, and an addend strand B i strand, where i = 1, 2, 3... n, and n is a positive integer greater than 1. Logical operations are performed on the input signal through nucleic acid hybridization and amplification, and the logical operation result is output; The output signal includes: nucleic acid amplification result wherein A i The 3'-end a 2i sequences of which are complementary to the 3'-end a of B i The 3'-end a 2i sequences of which are complementary to the 3'-end a of B i The 5'-end a 2i+1 sequences of which are complementary to the 5'-end a of A i+1 The 5'-end a 2i+1 sequences of which are complementary to the 5'-end a of A i-1 The 5'-end a 2i-1 sequences of which are complementary to the 5'-end a of A i The 5'-end a 2i-1 sequences of which are complementary to the 5'-end a of A n The 5'-end c sequences of which are complementary to the 5'-end c sequences of A, such that the addend chain A e chain, the A i chain, and the addend chain B e chain can cross-complement to form a nicked double strand in the order of i = 1, 2, 3... n. The 5'-end of A0 and the 3'-end of A i are respectively complementary to the primers. The values of c and each a are each independently a positive integer greater than or equal to 5, and a e + a 2i-1 ≤ the total number of bases of A 2i and a i + a 2i ≤ the total number of bases of B 2i+1 i ​​ 2. The DNA molecular logic gate according to claim 1, characterized in that a 2i +a 2i+1 B i The number of bases in the full length of the sequence is 0, 1, 2, 3, 4 or 5 less, and / or a 2i-1 +a 2i Ratio A i The number of bases in the full length of the sequence is 0, 1, 2, 3, 4 or 5 less, and / or The numerical value c and each a are each independently a positive integer from 5 to 40 3. The DNA molecular logic gate according to claim 1, characterized in that The A0 chain or the A chain can be conjugated with a detection label, where the detection label is biotin. e The chain can be conjugated with a detection label, where the detection label is biotin.

4. The DNA molecule logic gate according to any one of claims 1-3, characterized in that, The signal conversion unit further includes one or more selected from the following: phosphorylase, ligase, polymerase, dNTP, and buffer 5. The DNA molecule logic gate according to any one of claims 1-3, characterized in that The DNA molecular logic gate is an AND gate. In the AND gate The a0 sequences at the 3' end of A0 are complementary to the a0 sequences at the 3' end of the first input strand. The b sequences at the 5' end of the first input strand are complementary to the b sequences at the 3' end of the second input strand. The a1 sequences at the 5' end of the second input strand are complementary to the a1 sequences at the 5' end of A1. The input signal is subjected to a logical operation through hybridization and amplification of the substrate strand and the input strand, and the logical operation result is output 6. The DNA molecule logic gate according to claim 5, wherein The DNA molecular logic gate is an AND gate. In the AND gate: a0, a1, and b are each independently a positive integer greater than or equal to 5 7. The DNA molecule logic gate according to any one of claims 1-3, characterized in that, The DNA molecular logic gate is an OR gate. In the OR gate The substrate strand further includes linker strands L0 and L1, and A0 is divided into A0I and A0II The a0 sequences at the 3' end of A0I are complementary to the a0 sequences at the 3' end of the first input strand. The b sequences at the 5' end of the first input strand are complementary to the b sequences at the 3' end of L0. The a1 sequences at the 5' end of L0 are complementary to the a1 sequences at the 5' end of A1 The a0' sequences at the 3' end of A0II are complementary to the a0' sequences at the 3' end of the second input strand. The b' sequences at the 5' end of the second input strand are complementary to the b' sequences at the 3' end of L1. The a1' sequences at the 5' end of L1 are complementary to the a1' sequences at the 5' end of A1 The first input strand is not complementary to L1, and the second input strand is not complementary to L0 The input signal is subjected to a logical operation through hybridization and amplification of the substrate strand and the input strand, and the logical operation result is output 8. The DNA molecule logic gate according to claim 7, wherein The DNA molecular logic gate is an OR gate. In the OR gate: a0, a0', a1, a1', and b are each independently a positive integer greater than or equal to 5 9. The DNA molecular logic gate according to any one of claims 1 to 3, characterized in that, The DNA molecular logic gate is a NAND gate. In the NAND gate The substrate strand further includes addend strands B0I and B0II, and A1 is divided into A1I and A1II The a0 sequences at the 3' end of B0I are complementary to the a0 sequences at the 3' end of A0. The a0' sequences at the 3' end of B0II are complementary to the a0' sequences at the 3' end of A0. The a0 sequences at the 3' end of B0I and the a0' sequences at the 3' end of B0II have at least 90% identity The a1 sequences at the 5' end of B0I are complementary to the a1 sequences at the 5' end of A1I. The a1' sequences at the 5' end of B0II are complementary to the a1' sequences at the 5' end of A1II. And the a1 sequences at the 5' end of A1I and the a1' sequences at the 5' end of A1II have at least 90% identity a at the 3'-end of A1I 2i sequences are complementary to a 2i sequences at the 3'-end of B1 a at the 3'-end of A1II 2i ' sequence is complementary to a 2i ' sequence at the 3'-end of B1 B0I preferentially hybridizes with the first input strand rather than with A0 and A1I, and B0I does not hybridize with the second input strand; B0I is at least 70% complementary to the first input strand. B0II preferentially hybridizes with the second input strand rather than with A0 and A1II, and B0II does not hybridize with the first input strand; B0II is at least 70% complementary to the second input strand. Logical operations are performed on the input signals through hybridization and amplification of the substrate strand and the input strands, and the results of the logical operations are output.

10. The DNA molecule logic gate according to claim 9, characterized in that, The DNA molecule logic gate is a NAND gate. In the NAND gate, a0, a0’, a1, a1’, and a 2i、 a 2i ’ are each independently a positive integer greater than or equal to 5.

11. The DNA molecular logic gate according to claim 10, wherein a0 is equal to a0', and / or a1 is equal to a1', and / or a 2i is equal to a 2i ’ is equal to 12. A DNA circuit comprising the DNA molecular logic gate according to any one of claims 1-10.

13. Use of the DNA molecular logic gate according to any one of claims 1-10 in the preparation of a device for biological detection, a device for molecular computing or a circuit nano-device.

14. A method for constructing the DNA molecular logic gate according to any one of claims 1-10, comprising the following steps: 1) Phosphorylate the substrate strand and phosphorylate the input strands. 2) Mix the input strands and the substrate strand in equal proportions and anneal them to assemble into a nicked DNA double strand. 3) Fill in the nick of the DNA double strand to obtain a complete DNA double strand, and 4) Use primers for amplification, and the output signal is the amplified DNA double strand.

Images