A reagent with three-input DNA logic gate function and its preparation method and use

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a reagent with a three-input DNA logic gate function and a preparation method and application thereof. The reagent with the three-input DNA logic gate function is constructed based on a tetrahedral framework nucleic acid structure, fluorescence intensity change excited thereby conforms to a logic operation result of a three-input logic gate, multi-target recognition and logic detection are realized, and meanwhile, in the application, a clever structural design enables each logic gate to have a simple tetrahedral structure when complex three-input logic operation is realized, excellent cell entry ability and extremely high scalability are maintained, and good application prospect is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a reagent with a three-input DNA logic gate function, its preparation method, and its uses. Background Technology

[0002] Due to the complexity of signal transduction within the human body, multi-target detection is crucial for determining pathological and physiological states. However, in clinical diagnosis and treatment, comprehensive analysis of multiple biological factors is often broken down into independent detection of individual factors and additional correlation analysis. This not only increases unnecessary reagent consumption and time costs but also leads to higher technical sensitivity in comprehensive analysis. When designing automated responsive therapeutic drugs with multiple functions, single-signal detection and analysis cannot meet the needs of precise diagnosis and treatment. Therefore, multi-signal analysis has become a hot research direction in the field of biological detection in recent years. For example, dual-signal recognition of telomerase and miR-21 in living cells is used for early tumor diagnosis; the release of active drugs is further stimulated after acidic pH, miR-21, and miR-155 signals are all satisfied. These studies have all achieved their respective automated responses by incorporating multi-signal recognition. However, functional enrichment leads to excessively large biomaterial structures, reduced biocompatibility, and weakened structural stability, resulting in problems such as low cell penetration and detection efficiency, and cytotoxicity. Therefore, there is an urgent need to develop new multi-signal recognition strategies.

[0003] DNA nanomaterials are a class of two-dimensional or three-dimensional structures built based on the complementary pairing rules of DNA bases. Among a large number of DNA nanomaterials, tetrahedral DNA nanostructures (TDNs) stand out for their efficient cell entry and tissue penetration capabilities, making them widely studied in drug delivery, tissue repair, and target detection. The unique tetrahedral structure endows TDNs with excellent cell entry performance, with 21 bp side length TDNs exhibiting the highest cell entry efficiency. In addition to possessing the general advantages of DNA nanomaterials, TDNs also feature high yield, efficient synthesis, and simple framework. Studies have shown that functionalization modification can achieve a structural transformation of TDNs from signal recognition to automatic response, while maintaining excellent cell entry performance and biocompatibility.

[0004] Logic gates were originally fundamental components of integrated circuits, performing logical operations such as OR, AND, NOR, and NAND. Any complex logic circuit can be further designed from these basic logic gates. Based on the high information density characteristics of nucleic acids, applying the concept of logic gates to biocomputing using DNA materials has attracted considerable attention from scholars. In the research of Mi Xianqiang, Wang Yaojun, and others, an electrochemical detection system based on DNA logic gates and DNA nanostructures was provided. This system, based on an electrochemical sensor of nucleic acid within a DNA framework and combined with DNA logic gate technology, achieves intelligent diagnosis and differentiation of bodily fluid samples from three common cancers: pancreatic cancer, breast cancer, and lung cancer. It boasts advantages such as integrated multiple detection and logic analysis functions.

[0005] However, combining the concept of logic gates with the DNA tetrahedral framework of nucleic acids for target identification through the detection of electrochemical signals suffers from problems such as low sensitivity and susceptibility to interference from other electroactive substances in the sample and environmental factors. Therefore, there is an urgent need to find a multi-target detection reagent with high sensitivity, high specificity, and minimal susceptibility to environmental factors. Summary of the Invention

[0006] To address the above problems, this invention provides a reagent with a three-input DNA logic gate function, its preparation method, and its uses.

[0007] A reagent with three-input DNA logic gate function, wherein the reagent is a tetrahedral framework nucleic acid with DNA logic gate operation function; the tetrahedral framework nucleic acid includes three single-stranded probe DNAs for recognizing three input strands respectively;

[0008] The tetrahedral framework nucleic acid with DNA logic gate operation function is selected from at least one of the following: OR logic gate tetrahedral framework nucleic acid, AND logic gate tetrahedral framework nucleic acid, MAJ logic gate tetrahedral framework nucleic acid, OA logic gate tetrahedral framework nucleic acid, NOR logic gate tetrahedral framework nucleic acid, NAND logic gate tetrahedral framework nucleic acid, and XOR logic gate tetrahedral framework nucleic acid.

[0009] The OR logic gate tetrahedral framework nucleic acid is modified with fluorescent and quenching groups. When the single-stranded probe DNA recognizes at least one input strand, the fluorescent and quenching groups are separated through a strand displacement reaction, releasing a fluorescent signal, and the OR logic gate outputs 1.

[0010] The AND logic gate tetrahedral framework nucleic acid is modified with fluorescent groups and quenching groups. When the single-stranded probe DNA recognizes three input strands at the same time, the fluorescent group and quenching group are separated through a strand displacement reaction, releasing a fluorescent signal, and the AND logic gate outputs 1.

[0011] The MAJ logic gate tetrahedral framework nucleic acid is modified with fluorescent groups and quenching groups. When the single-stranded probe DNA recognizes at least two input strands, a strand displacement reaction is initiated to cause one or two fluorescent groups to detach from the quenching group, releasing a fluorescent signal, and the MAJ logic gate output is 1.

[0012] The OA logic gate tetrahedral framework nucleic acid is modified with fluorescent groups and quenching groups. When the single-stranded probe DNA recognizes at least one other input strand in addition to one specified input strand, the fluorescent group and quenching group are separated through a strand substitution reaction, releasing a fluorescent signal, and the OA logic gate outputs 1.

[0013] The NOR logic gate tetrahedral framework nucleic acid is modified with fluorescent resonance energy transfer group pairs. When the single-stranded probe DNA recognizes at least one input strand, the fluorescent resonance energy transfer group pairs are separated through a strand displacement reaction, the fluorescence intensity decreases, and the NOR logic gate output is 0.

[0014] The NAND logic gate tetrahedral framework nucleic acid is modified with fluorescent resonance energy transfer group pairs. When the single-stranded probe DNA recognizes three input strands at the same time, the fluorescent resonance energy transfer group pairs are separated through a strand displacement reaction, the fluorescence intensity decreases, and the NAND logic gate output is 0.

[0015] The XOR logic gate's tetrahedral framework nucleic acid is modified with a fluorescent resonance energy transfer group pair and a quenching group. When the single-stranded probe DNA does not recognize any input strand or recognizes three input strands simultaneously, the fluorescent resonance energy transfer group pair is quenched by the quenching group, and the XOR logic gate outputs 0. When the single-stranded probe DNA recognizes one or two input strands, a strand displacement reaction causes the fluorescent resonance energy transfer group pair to leave the quenching group, releasing a fluorescent signal, and the XOR logic gate outputs 1.

[0016] Preferably, the tetrahedral framework nucleic acid with DNA logic gate operation function includes a first vertex, a second vertex, a third vertex, and a fourth vertex; wherein, the first vertex is composed of a first single-stranded DNA, a second single-stranded DNA, and a third single-stranded DNA; the second vertex is composed of a first single-stranded DNA, a third single-stranded DNA, and a fourth single-stranded DNA; the third vertex is composed of a first single-stranded DNA, a second single-stranded DNA, and a fourth single-stranded DNA; and the fourth vertex is composed of a second single-stranded DNA, a third single-stranded DNA, and a fourth single-stranded DNA.

[0017] One end of any three of the first single-stranded DNA, the second single-stranded DNA, the third single-stranded DNA, and the fourth single-stranded DNA includes a first single-stranded probe DNA, a second single-stranded probe DNA, and a third single-stranded probe DNA for recognizing the input strand.

[0018] In the OR logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA, the second single-stranded DNA, and the third single-stranded DNA respectively includes a first single-stranded probe DNA, a second single-stranded probe DNA, and a third single-stranded probe DNA; a portion of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA extends to a second vertex, a third vertex, and a fourth vertex, respectively; the remaining fragments of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA completely cover the region between the first vertex and another vertex; one of the single-stranded DNA strands at the first vertex has a DNA breakpoint; the two ends of the DNA breakpoint are modified with a fluorescent group and a quenching group, respectively.

[0019] And / or, in the AND logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA includes a first single-stranded probe DNA, one end of the second single-stranded DNA includes a second single-stranded probe DNA, one end of the fourth single-stranded DNA includes a third single-stranded probe DNA, a portion of the first single-stranded probe DNA extends into a tetrahedral framework nucleic acid, the second single-stranded probe DNA is complementary to a portion of the first single-stranded probe DNA, a portion of the third single-stranded DNA, and a portion of the fourth single-stranded DNA in sequence, respectively, a portion of the third single-stranded probe DNA extends into a tetrahedral framework nucleic acid, a portion of the third single-stranded probe DNA is complementary to a portion of the first single-stranded DNA and a portion of the second single-stranded DNA in sequence, the starting position of the second single-stranded probe DNA in the second single-stranded DNA is denoted as point A, the starting position of the third single-stranded probe DNA in the fourth single-stranded DNA is denoted as point B, and fluorescent groups and quenching groups are modified at points A and B, respectively;

[0020] And / or, in the MAJ logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA, the second single-stranded DNA, and the third single-stranded DNA respectively includes a first single-stranded probe DNA, a second single-stranded probe DNA, and a third single-stranded probe DNA; a portion of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA extends to a second vertex, a third vertex, and a fourth vertex, respectively; the remaining fragments of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA completely cover the region between the first vertex and another vertex; each of the three single-stranded DNAs at the first vertex is provided with a thymine-containing deoxyribonucleotide, the thymine-containing deoxyribonucleotide is denoted as a spacer, wherein two spacers are modified with a fluorescent group and a quenching group, respectively, and the thymine at a distance of 2 bases from the remaining spacer is modified with a fluorescent group;

[0021] And / or, in the OA logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA, the second single-stranded DNA, and the third single-stranded DNA respectively includes a first single-stranded probe DNA, a second single-stranded probe DNA, and a third single-stranded probe DNA; a portion of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA extends to a second vertex, a third vertex, and a fourth vertex, respectively; the remaining fragments of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA completely cover the region between the first vertex and another vertex; each of the three single-stranded DNA strands at the first vertex is provided with a spacer, wherein two of the spacers are modified with a fluorescent group and a quenching group, respectively;

[0022] And / or, in the NOR logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA, the second single-stranded DNA, and the third single-stranded DNA respectively includes a first single-stranded probe DNA, a second single-stranded probe DNA, and a third single-stranded probe DNA; a portion of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA respectively extends to a second vertex, a third vertex, and a fourth vertex; the remaining fragments of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA respectively completely cover the region between the first vertex and another vertex; one of the single-stranded DNAs at the first vertex has a DNA breakpoint; the two ends of the DNA breakpoint are modified with fluorescent resonance energy transfer group pairs.

[0023] And / or, in the NAND logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA includes a first single-stranded probe DNA, one end of the second single-stranded DNA includes a second single-stranded probe DNA, one end of the fourth single-stranded DNA includes a third single-stranded probe DNA, a portion of the first single-stranded probe DNA extends into the tetrahedral framework nucleic acid, the second single-stranded probe DNA is complementary to a portion of the first single-stranded probe DNA, a portion of the third single-stranded DNA, and a portion of the fourth single-stranded DNA in sequence, a portion of the third single-stranded probe DNA extends into the tetrahedral framework nucleic acid, a portion of the third single-stranded probe DNA is complementary to a portion of the first single-stranded DNA and a portion of the second single-stranded DNA in sequence, the starting position of the second single-stranded probe DNA in the second single-stranded DNA is denoted as point A, the starting position of the third single-stranded probe DNA in the fourth single-stranded DNA is denoted as point B, and fluorescent resonance energy transfer group pairs are modified at points A and B;

[0024] And / or, in the XOR logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA includes a first single-stranded probe DNA, and the other end of the first single-stranded DNA is denoted as endpoint A; one end of the second single-stranded DNA includes a second single-stranded probe DNA, and the other end of the second single-stranded DNA is denoted as endpoint B; one end of the fourth single-stranded DNA includes a third single-stranded probe DNA, and the other end of the fourth single-stranded DNA is denoted as endpoint C; a portion of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA extends to a first vertex, a third vertex, and a second vertex, respectively; the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA... The remaining fragments of the single-stranded probe DNA completely cover the region between its extended vertex and another vertex. Endpoints A, B, and C are located at the first vertex, the third vertex, and the second vertex, respectively. All three endpoints are modified with quenching groups. The first and third single-stranded probe DNA at the first vertex are modified with fluorescent resonance energy transfer group pairs. The third and first single-stranded probe DNA at the second vertex are modified with fluorescent resonance energy transfer group pairs. The second and first single-stranded probe DNA at the third vertex are modified with fluorescent resonance energy transfer group pairs.

[0025] Preferably, the fluorescent group is selected from at least one of Cy5, Cy3, and FAM groups; the quenching group is selected from at least one of BHQ1 and BHQ2 groups.

[0026] And / or, in MAJ logic gate tetrahedral framework nucleic acids, the thymine-containing deoxyribonucleotide does not form complementary hydrogen bonds with other bases; the fluorescent group modified on the thymine at a distance of 2 bases from the remaining spacer needs to be the same as the fluorescent group modified on the spacer;

[0027] And / or, the fluorescence resonance energy transfer group pair is selected from Cy5 and Cy3, or the fluorescence resonance energy transfer group pair is selected from Cy5 and FAM;

[0028] And / or, in the XOR logic gate tetrahedral framework nucleic acid, the fluorescence resonance energy transfer group pair is selected from at least two of Cy5, Cy3, and FAM groups; the quenching group is selected from at least one of BHQ1 and BHQ2 groups.

[0029] Preferably, the OR logic gate tetrahedral framework nucleic acid is assembled from 5 DNA single strands through base complementary pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively;

[0030] And / or, the AND logic gate tetrahedral framework nucleic acid is assembled from four DNA single strands through complementary base pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively;

[0031] And / or, the Majority logic gate tetrahedral framework nucleic acid is assembled from four DNA single strands through complementary base pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively;

[0032] And / or, the OA logic gate tetrahedral framework nucleic acid is assembled from four DNA single strands through complementary base pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.12, and SEQ ID NO.13, respectively;

[0033] And / or, the NOR logic gate tetrahedral framework nucleic acid is assembled from 5 DNA single strands through complementary base pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.14, and SEQ ID NO.15, respectively;

[0034] And / or, the NAND logic gate tetrahedral framework nucleic acid is assembled from four DNA single strands through complementary base pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.9, and SEQ ID NO.16;

[0035] And / or, the XOR logic gate tetrahedral framework nucleic acid is assembled from four DNA single strands through complementary base pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, and SEQ ID NO.20.

[0036] The present invention also provides a method for preparing the above-mentioned reagent, comprising the following steps: adding the DNA single strand corresponding to each logic gate tetrahedral framework nucleic acid to a buffer solution, mixing to obtain a logic gate solution, and incubating to obtain the reagent; the ratio of the amount of DNA single strands corresponding to each logic gate tetrahedral framework nucleic acid is 1:1:1:1 or 1:1:1:1:1.

[0037] The present invention also provides the use of the above-mentioned reagent with three-input DNA logic gate function in multi-target recognition.

[0038] Preferably, the reagent is used for logical detection of multiple targets.

[0039] The present invention also provides a kit for target detection, comprising an input strand and the above-mentioned reagent having a three-input DNA logic gate function.

[0040] Preferably, the input strand consists of three DNA single strands; when the tetrahedral framework nucleic acid with DNA logic gate operation function is any one of OR logic gate tetrahedral framework nucleic acid, MAJ logic gate tetrahedral framework nucleic acid, OA logic gate tetrahedral framework nucleic acid, NOR logic gate tetrahedral framework nucleic acid, or XOR logic gate tetrahedral framework nucleic acid, the nucleotide sequence of the target DNA single strand is as shown in SEQ ID NO.21, SEQ ID NO.22, and SEQ ID NO.23;

[0041] And / or, when the tetrahedral framework nucleic acid with DNA logic gate operation function is any one of AND logic gate tetrahedral framework nucleic acid or NAND logic gate tetrahedral framework nucleic acid, the nucleotide sequence of the target DNA single strand is as shown in SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.26.

[0042] The present invention also provides a method for target detection using the above-mentioned reagent kit, comprising the following steps: mixing the logic gate solution with the input chain and incubating to obtain the target.

[0043] This invention combines the concept of logic gates with tetrahedral framework nucleic acids to design a tetrahedral structure that conforms to the operations of DNA logic gates (OR, AND, MAJ, OA, NOR, NAND, and XOR). By designing (1) the breakpoint position of the initial structure of the logic gate and (2) the type, position, and number of fluorescent groups, the change in the excitation fluorescence intensity conforms to the logical operation result of the three-input logic gate, realizing multi-target recognition and logical detection. It has high sensitivity, strong specificity, and is less affected by environmental factors, and has good application prospects.

[0044] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0045] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0046] Figure 1 A schematic diagram of a tetrahedral framework nucleic acid with seven logic gate operations.

[0047] Figure 2 This is a characterization diagram of the synthesis of nucleic acids using the OR logic gate tetrahedral framework.

[0048] Figure 3 This is a characterization diagram of the synthesis of nucleic acids using the AND logic gate tetrahedral framework.

[0049] Figure 4 This is a characterization diagram of the synthesis of MAJ logic gate tetrahedral framework nucleic acids.

[0050] Figure 5 This is a characterization diagram of the synthesis of nucleic acids with an OA logic gate tetrahedral framework.

[0051] Figure 6 This is a characterization diagram of the synthesis of NOR logic gate tetrahedral framework nucleic acids.

[0052] Figure 7 This is a characterization diagram of the synthesis of nucleic acids in the tetrahedral framework of NAND logic gates.

[0053] Figure 8 This is a characterization diagram of the synthesis of nucleic acids using the XOR logic gate tetrahedral framework.

[0054] Figure 9 This represents the logical reaction result of a tetrahedral framework nucleic acid with an OR logic gate.

[0055] Figure 10 The result is the logical reaction of a nucleic acid with an AND logic gate tetrahedral framework.

[0056] Figure 11 This represents the logical reaction result of MAJ logic gate tetrahedral framework nucleic acids.

[0057] Figure 12 The logical reaction result of the OA logic gate tetrahedral framework nucleic acid.

[0058] Figure 13 The result is the logical reaction of a nucleic acid with a NOR logic gate tetrahedral framework.

[0059] Figure 14 This represents the logical reaction result of a NAND logic gate tetrahedral framework nucleic acid.

[0060] Figure 15 This represents the logical reaction result of a tetrahedral framework nucleic acid with an XOR logic gate.

[0061] Figure 16 This is a diagram of the logical operations of OR logic gate tetrahedral framework nucleic acids within MCF cells.

[0062] Figure 17 Image of logical operations of AND logic gate tetrahedral framework nucleic acids in MCF cells.

[0063] Figure 18 This image shows the logic operations of MAJ logic gate tetrahedral framework nucleic acids within MCF cells.

[0064] Figure 19 This is a diagram of the logical operations of OA logic gate tetrahedral framework nucleic acids within MCF cells. Detailed Implementation

[0065] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0066] Example 1: Tetrahedral framework nucleic acid based on DNA logic gate operations and its preparation method

[0067] The tetrahedral framework structure includes four vertices, which are referred to in this invention as "first vertex", "second vertex", "third vertex" and "fourth vertex". Each vertex is a trifle structure composed of three single-stranded DNA molecules through complementary base pairing. Specifically, the first vertex is composed of a first single-stranded DNA, a second single-stranded DNA, and a third single-stranded DNA; the second vertex is composed of a first single-stranded DNA, a third single-stranded DNA, and a fourth single-stranded DNA; the third vertex is composed of a first single-stranded DNA, a second single-stranded DNA, and a fourth single-stranded DNA; and the fourth vertex is composed of a second single-stranded DNA, a third single-stranded DNA, and a fourth single-stranded DNA.

[0068] One end of any three of the first single-stranded DNA, the second single-stranded DNA, the third single-stranded DNA, and the fourth single-stranded DNA includes a first single-stranded probe DNA, a second single-stranded probe DNA, and a third single-stranded probe DNA for recognizing the input strand.

[0069] In this invention, a "DNA breakpoint" is a gap formed when the endpoints of two DNA strands are adjacent to each other but not connected. Two DNA strands with DNA breakpoints are considered as the same single-stranded DNA used to form a tetrahedral framework nucleic acid in this invention.

[0070] In this invention, when describing the structure of tetrahedral framework nucleic acids, the extension of single-stranded probe DNA beyond the vertices of the tetrahedral framework nucleic acid, or the extension of double-stranded DNA with complementary pairing between the vertices of the tetrahedral framework nucleic acid, refers to the single-stranded probe DNA existing entirely or partially in single-stranded form without complementary pairing. Unless specifically stated that the single-stranded probe DNA extends to a certain position, it exists in a complementary pairing manner within the tetrahedral framework nucleic acid, rather than extending beyond a vertex or extending into double-stranded DNA with complementary pairing between two vertices.

[0071] In describing the structure of tetrahedral framework nucleic acids, the term "coverage" means that in a specific region of the described tetrahedral framework nucleic acid, all bases of one of the single-stranded DNA strands of the double-stranded DNA with complementary base pairing belong to the single-stranded probe DNA.

[0072] In the description of this invention, the use of serial numbers such as "first," "second," "third," and "fourth" is for descriptive purposes only, indicating the relative position or order of different technical features, and is not intended to limit the importance, function, or role of these features. The use of these serial numbers is to facilitate understanding and explanation of the technical solutions of this invention and does not represent any form of priority or functional difference. Those skilled in the art should understand that the use of these serial numbers is for simplification and does not constitute any limitation on the scope of protection of this invention.

[0073] 1. Tetrahedral framework nucleic acids based on OR logic gate operations

[0074] In the described OR logic gate tetrahedral framework nucleic acid: a first single-stranded DNA, a second single-stranded DNA, and a third single-stranded DNA are respectively attached to one end of each of the three single-stranded DNAs; a portion of each of the three single-stranded DNAs extends to a second vertex, a third vertex, and a fourth vertex; the remaining portions of each single-stranded DNA are located between the first vertex and another vertex. One of the single-stranded DNAs at the first vertex has a DNA breakpoint, with a fluorescent group (Cy5) and a quencher group (BHQ2) modified at both ends of the breakpoint. The three single-stranded probe DNAs can serve as three input terminals, specifically recognizing three single-stranded DNA segments (i.e., input strands as shown in SEQ ID NO.21, SEQ ID NO.22, and SEQ ID NO.23). When a single-stranded probe DNA recognizes at least one input strand, a strand substitution reaction triggers the separation of the fluorescent group and the quencher group, releasing a fluorescent signal, and the OR logic gate outputs 1.

[0075] The nucleotide sequences of the five DNA single strands of the synthesized OR logic gate tetrahedral framework nucleic acid are shown in Table 1 as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

[0076] 2. Tetrahedral framework nucleic acids based on AND logic gate operations

[0077] In the AND logic gate tetrahedral framework nucleic acid: a first single-stranded probe DNA is attached to one end of the first single-stranded DNA, a second single-stranded probe DNA is attached to one end of the second single-stranded DNA, and a third single-stranded probe DNA is attached to one end of the fourth single-stranded DNA. A portion of the first single-stranded probe DNA extends into the tetrahedral framework nucleic acid. The second single-stranded probe DNA sequentially pairs complementaryly with portions of the first, third, and fourth single-stranded DNA, respectively. A portion of the third single-stranded probe DNA extends into the tetrahedral framework nucleic acid, and a portion of the third single-stranded probe DNA sequentially pairs complementaryly with portions of the first and second single-stranded DNA, respectively. The starting position of the second single-stranded probe DNA in the second single-stranded DNA is denoted as point A, and the starting position of the third single-stranded probe DNA in the fourth single-stranded DNA is denoted as point B. Points A and B are modified with a fluorescent group (Cy5) and a quencher group (BHQ2), respectively. The first single-stranded probe DNA is used to recognize input strand 1 (as shown in SEQ ID NO. 24), and the third single-stranded probe DNA is used to recognize input strand 3 (as shown in SEQ ID NO. 24). As shown in NO.26), the second single-stranded probe DNA is used to recognize input strand 2 (as shown in SEQ ID NO.25); when the three single-stranded probe DNAs recognize the three input strands at the same time, the fluorescent group and quencher group are separated through the strand substitution reaction, releasing a fluorescent signal, and the AND logic gate outputs 1;

[0078] The nucleotide sequences of the four DNA single strands of the synthesized AND logic gate tetrahedral framework nucleic acid are shown in Table 1 as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9.

[0079] 3. Tetrahedral framework nucleic acids based on MAJ logic gate operations

[0080] In the MAJ logic gate tetrahedral framework nucleic acid: A first single-stranded DNA probe (STP) is attached to one end of each of the first, second, and third single-stranded DNA strands. A portion of each STP extends to a second, third, and fourth vertex, respectively. The remaining portions of each STP are located between the first and third vertices. Each of the three single-stranded DNA strands at the first vertex has a thymine-containing deoxyribonucleotide (called a spacer) that does not form complementary hydrogen bonds with other bases. There are three spacers in total. Two spacers are modified with a fluorescent group (Cy5) and a quencher group (BHQ2), respectively. The remaining spacer is not modified with a fluorescent group but is located on the DNA strand a short distance from the remaining spacer. A fluorescent group is modified on the thymine at a distance of 2 bases. This is because if two fluorescent groups are too close together, they will quench each other's fluorescence, resulting in decreased fluorescence. Therefore, each fluorescent group needs to be about 2 bases away from the spacer to mitigate the fluorescence attenuation. (This fluorescent group needs to be the same as the fluorescent group modified on the spacer, i.e., Cy5). When three single-stranded probe DNAs are used to specifically recognize three single-stranded DNA segments (i.e., the input strands are shown in SEQ ID NO.21, SEQ ID NO.22, and SEQ ID NO.23), in this structure, when the single-stranded probe DNA recognizes at least two input strands, a strand displacement reaction causes one or two fluorescent groups to detach from the quenching group, releasing a fluorescent signal, and the MAJ logic gate output is 1.

[0081] The nucleotide sequences of the four DNA single strands of the synthesized MAJ logic gate tetrahedral framework nucleic acid are shown in Table 1 as SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.

[0082] 4. Tetrahedral framework nucleic acids based on OA logic gate operations

[0083] In the OA logic gate tetrahedral framework nucleic acid: A first single-stranded DNA, a second single-stranded DNA, and a third single-stranded DNA are respectively attached to one end of each of the three single-stranded DNAs. A portion of each of these DNAs extends to a second, third, and fourth vertex, respectively. The remaining portions of each DNA are located between the first vertex and another vertex. Each of the three single-stranded DNAs at the first vertex has a thymine-containing deoxyribonucleotide (called a spacer), which does not form complementary hydrogen bonds with other bases. There are three spacers in total; two spacers are modified with a fluorescent group (Cy5) and a quencher group (BHQ2), respectively, while the remaining spacer is not modified with a fluorescent group. The three single-stranded DNA probes are used to specifically recognize three single-stranded DNA segments (i.e., the input strands such as SEQ ID NO.21, SEQ ID NO.22, and SEQ ID NO.23). (As shown in IDNO.23); In this structure, the two input chains recognized by the two toeholds on both sides of the unmodified fluorescent group spacer are both optional inputs, while the other input chain is the prerequisite input. Only when the prerequisite input is recognized and at least one optional input is recognized can the fluorescent group be removed from the quenching group through the chain substitution reaction, thereby releasing a high fluorescence signal. The output of the OA logic gate is 1.

[0084] The nucleotide sequences of the four DNA single strands of the synthesized OA logic gate tetrahedral framework nucleic acid are shown in Table 1 as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.12, and SEQ ID NO.13.

[0085] 5. Tetrahedral framework nucleic acids based on NOR logic gates

[0086] In the described NOR logic gate tetrahedral framework nucleic acid: A first single-stranded probe DNA, a second single-stranded probe DNA, and a third single-stranded probe DNA are respectively attached to one end of the first, second, and third single-stranded DNA. A portion of each of the first, second, and third single-stranded probe DNAs extends to a second, third, and fourth vertex, respectively. The remaining portions of the first, second, and third single-stranded probe DNAs are located between the first vertex and another vertex. One of the single-stranded DNAs at the first vertex has a DNA breakpoint, with both ends of the breakpoint modified with a fluorescent resonance energy transfer (FRET) group pair (Cy3 group and FAM group). The three single-stranded probe DNAs can serve as three input ends, specifically recognizing three single-stranded DNA segments (i.e., the input strands are shown in SEQ ID NO. 21, SEQ ID NO. 22, and SEQ ID NO. 23). The effects of FRET are: group separation – increased distance – energy transition failure – low emission intensity of the acceptor group. When the single-stranded probe DNA recognizes at least one input strand, a fluorescence resonance energy transfer group pair is separated through a strand displacement reaction, resulting in a decrease in fluorescence intensity and a NOR logic gate output of 0.

[0087] The nucleotide sequences of the five DNA single strands that synthesize the NOR logic gate tetrahedral framework nucleic acid are shown in Table 1 as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.14, and SEQ ID NO.15.

[0088] 6. Tetrahedral framework nucleic acids based on NAND logic gate operations

[0089] In the NAND logic gate tetrahedral framework nucleic acid: a first single-stranded probe DNA is attached to one end of the first single-stranded DNA, a second single-stranded probe DNA is attached to one end of the second single-stranded DNA, and a third single-stranded probe DNA is attached to one end of the fourth single-stranded DNA. A portion of the first single-stranded probe DNA extends into the tetrahedral framework nucleic acid. The second single-stranded probe DNA sequentially pairs complementaryly with portions of the first, third, and fourth single-stranded DNA, respectively. A portion of the third single-stranded probe DNA extends into the tetrahedral framework nucleic acid, and a portion of the third single-stranded probe DNA sequentially pairs complementaryly with portions of the first and second single-stranded DNA, respectively. The starting position of the second single-stranded probe DNA in the second single-stranded DNA is denoted as point A, and the starting position of the third single-stranded probe DNA in the fourth single-stranded DNA is denoted as point B. Points A and B are modified with fluorescence resonance energy transfer group pairs (Cy3 group and FAM group), respectively. The first single-stranded probe DNA is used to recognize input strand 1 (as shown in SEQ ID NO. 24), and the third single-stranded probe DNA is used to recognize input strand 3 (as shown in SEQ ID NO. 24). As shown in NO.26, the second single-stranded probe DNA is used to recognize input strand 2 (as shown in SEQ ID NO.25); the effects of fluorescence resonance energy transfer are: group separation - increased distance - energy transition failure - low emission intensity of the acceptor group. Therefore, when three single-stranded probe DNAs simultaneously recognize three input strands, the separation of the group pairs is triggered by the strand substitution reaction, resulting in a decrease in fluorescence intensity and a NAND logic gate output of 0;

[0090] The nucleotide sequences of the four DNA single strands that synthesize the NAND logic gate tetrahedral framework nucleic acid are shown in Table 1, namely SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.9, and SEQ ID NO.16.

[0091] 7. Tetrahedral framework nucleic acids based on XOR logic gate operations

[0092] In a tetrahedral framework nucleic acid using an XOR logic gate: A first single-stranded DNA probe is attached to one end of the first single-stranded DNA, and the other end of the first single-stranded DNA is denoted as endpoint A. A second single-stranded DNA probe is attached to one end of the second single-stranded DNA, and the other end of the second single-stranded DNA is denoted as endpoint B. A third single-stranded DNA probe is attached to one end of the fourth single-stranded DNA, and the other end of the fourth single-stranded DNA is denoted as endpoint C. A portion of the first, second, and third single-stranded DNA probes extends to the first, third, and second vertices, respectively. The remaining portions of the first, second, and third single-stranded DNA probes are located at their respective endpoints. Between the extended vertex and another vertex, endpoints A, B, and C are located at the first, third, and second vertices, respectively. All endpoints A, B, and C are modified with a quenching group (BHQ1). The first and third single-stranded probe DNA at the first vertex are modified with a fluorescent resonance energy transfer group pair (Cy3, FAM). Similarly, the third and first single-stranded probe DNA at the second vertex are modified with the same pair, and the second and first single-stranded probe DNA at the third vertex are also modified with the same pair. These three single-stranded probe DNAs are used to identify the input strand (as shown in SEQ ID NO.21, SEQ ID NO.22, and SEQ ID NO.23). When the single-stranded probe DNA does not recognize any input strand or recognizes three input strands simultaneously, the fluorescence resonance energy transfer group pair is quenched by the quenching group, and the XOR logic gate output is 0; when the single-stranded probe DNA recognizes one or two input strands, the fluorescence resonance energy transfer group pair leaves the quenching group through a strand displacement reaction, releasing a fluorescence signal, and the XOR logic gate output is 1.

[0093] The nucleotide sequences of the four DNA single strands of the synthetic XOR logic gate tetrahedral framework nucleic acid are shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, and SEQ ID NO.20, as shown in Table 1. The structures of the tetrahedral framework nucleic acids for the seven logic gate operations are shown in Table 1. Figure 1 As shown.

[0094] Table 1. Specific DNA sequences for synthesized DNA logic gates.

[0095]

[0096]

[0097] The truth tables for various DNA logic gates (i.e., the output of DNA logic gates under different input states) are shown in Table 2. 0 represents negative input / negative output, and 1 represents positive input / positive output.

[0098] Table 2 Truth Tables for Various DNA Logic Gates

[0099]

[0100] The preparation method of tetrahedral framework nucleic acid based on the above 7 logic gate operations is as follows: The synthetic single strands corresponding to the above 7 logic gates are added at a concentration of 1 μM to TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0) to form a 100 μL synthesis system. The temperature of the PCR thermal cycler is set to be stably maintained at 95℃ for 10 minutes, and then rapidly cooled to 4℃ for 20 minutes to obtain the tetrahedral framework nucleic acid based on DNA logic gate operations.

[0101] The technical solution of the present invention will be further explained through experiments below.

[0102] All samples used in this experiment were prepared according to the method in Example 1.

[0103] Example 1: Characterization and Identification Results of Tetrahedral Framework Nucleic Acids Based on DNA Logic Gate Operations

[0104] I. Experimental Methods

[0105] Polyacrylamide gel electrophoresis (PAGE): Prepare an 8% non-denaturing polyacrylamide gel. Add each single strand mixed with loading buffer and the prepared OR, AND, NOR, NAND, XOR, Majority, and OA logic gate samples to the lanes. Adjust the electrophoresis parameters (~80V, 80 minutes) and run the gel. After completion, incubate with gel red staining solution in the dark for 10 minutes. Use a gel imaging system to expose and capture images in the fluorescence channel and gel red channel to clarify the synthesis and yield of each three-input logic gate.

[0106] II. Experimental Results

[0107] like Figure 2As shown, the specific content of each band is as follows: lane1: S23, lane2: S24, lane3: S25, lane4: S26, lane5: S27, lane6: S23-24, lane7: S23-24-25, lane8: S23-24-25-26, lane9: OR gate, lane 10: S27+in1, lane11: S25+in2, lane12: S24+in3. The bands are clear, confirming the successful synthesis of DNA-based OR logic gate tetrahedral framework nucleic acid.

[0108] like Figure 3 As shown, the specific content of each band is as follows: lane1: S28, lane2: S29, lane3: S30, lane4: S31, lane5: S68, lane6: S69, lane7: S70, lane8: S28-29, lane9: S28-29-30, lane10: ANDgate, lane 11: S31+in1, lane12: S29+in2, lane13: S30+in3. The bands are clear, confirming the successful synthesis of DNA-based AND logic gate tetrahedral framework nucleic acids.

[0109] like Figure 4 As shown, the specific contents of each stripe are as follows: lane1: S23, lane2: S32, lane3: S33, lane4: S34, lane5: S65 (as in1), lane6: S66 (as in2), lane7: S67 (as in3), lane8:

[0110] S23-32, lane 9: S23-32-33, lane 10: MAJ gate, lane 11: S34+in1, lane 12: S33+in2, lane 13: S32+in3. Each band is clear, confirming the successful synthesis of DNA-based MAJ logic gate tetrahedral framework nucleic acid.

[0111] like Figure 5 As shown, the specific content of each band is as follows: lane1: S23, lane2: S25, lane3: S35, lane4: S34, lane5: S23-25, lane6: S23-25-35, lane7: OA gate, lane8: S34+in1, lane9: S35+in2, lane10: S25+in3. The bands are clear, confirming the successful synthesis of DNA-based OA logic gate tetrahedral framework nucleic acid.

[0112] like Figure 6As shown, the specific content of each band is as follows: lane1: S23, lane2: S24, lane3: S25, lane4: S36, lane5: S37, lane6: S23-24, lane7: S23-24-25, lane8: S23-24-25-36, lane9: NOR gate, lane10: lane 11: S34+in1, lane12: S35+in2, lane13: S25+in3. The bands are clear, confirming the successful synthesis of DNA-based NOR logic gate tetrahedral framework nucleic acids.

[0113] like Figure 7 As shown, the specific content of each band is as follows: lane1: S28, lane2: S29, lane3: S38, lane4: S31, lane5: S68 (in1), lane6: S69 (in2), lane7: S70 (in3), lane8: S28-29, lane9: S29-29-38, lane10: NOR gate, lane11: S31+in1, lane12: S38+in2, lane13: S29+in3. The bands are clear, confirming the successful synthesis of DNA-based NAND logic gate tetrahedral framework nucleic acids.

[0114] like Figure 8 As shown, the specific contents of each stripe are as follows: lane1: S46, lane2: S49, lane3: S48, lane4: S47, lane5: S65 (as in1), lane6: S66 (as in2), lane7: S67 (as in3), lane8:

[0115] S46-47, lane 9: S46-47-48, lane 10: XOR gate, lane 11: S46+in1, lane 12: S49+in2, lane 13: S48+in3. Each band is clear, confirming the successful synthesis of DNA-based XOR logic gate tetrahedral framework nucleic acid.

[0116] Experiment 2: Tetrahedral framework nucleic acids based on DNA logic gate operations can achieve logical recognition of three target nucleic acid single strands.

[0117] I. Experimental Methods

[0118] (1) Logical Reaction Incubation: Here, we take the 10 μL reaction system of the OR gate as an example; the operation procedure for other logic gates is the same. Add 1 μL of 1 μM single-stranded DNA (in1, in2, in3) to 5 μL of the prepared 1 μM OR gate solution, and bring the volume to 10 μL with enzyme-free water. Gently swirl the EP tube to mix the solution thoroughly, then place it in a PCR thermal cycler and incubate at 25°C for 60 minutes.

[0119] (2) Polyacrylamide gel electrophoresis (PAGE): Prepare 10 μL of reaction solution according to method (1). After the reaction is completed, mix it with the loading buffer and add it to the 8% PAGE gel lane. Adjust the electrophoresis parameters (~80V, 80 minutes) and run the gel.

[0120] (3) The effect of three-input logic gates on logic operations in cells: The target DNA single strands with eight different signal states (i.e. 000, 100, 010, 001, 110, 101, 011, 111) were transfected into MCF cells using Lipofectamine 3000. After culturing for 1 day, serum-free medium was added to dilute each logic gate to a concentration of 250 nM. After incubation for 24 hours, the fluorescence distribution in the cells was observed using a confocal fluorescence microscope.

[0121] II. Experimental Results

[0122] like Figure 9 As shown, inputting any one of the input chains in1, in2, and in3 will produce a positive fluorescence output, confirming the successful synthesis of DNA-based OR logic gate tetrahedral framework nucleic acid.

[0123] like Figure 10 As shown, positive fluorescence output can only be obtained by inputting all input chains in1, in2, and in3, confirming the successful synthesis of DNA-based AND logic gate tetrahedral framework nucleic acids.

[0124] like Figure 11 As shown, positive fluorescence output can be obtained by inputting any two input chains in1, in2 and in3, confirming the successful synthesis of DNA-based MAJ logic gate tetrahedral framework nucleic acids.

[0125] like Figure 12 As shown, with in1 as the input, inputting either in2 or in3 will produce a positive fluorescence output, confirming the successful synthesis of DNA-based OA logic gate tetrahedral framework nucleic acid.

[0126] like Figure 13As shown, in the absence of an input strand, there is a FRET phenomenon between the FAM-Cy3 group pairs, and the FAM fluorescence is absorbed by Cy3, resulting in no fluorescence output from FAM. The FRET phenomenon can be disrupted by inputting any one of the input strands in1, in2, and in3, resulting in FAM being able to emit fluorescence, thus confirming the successful synthesis of DNA-based NOR logic gate tetrahedral framework nucleic acids.

[0127] like Figure 14 As shown, when there is no input strand, only one input strand, or only two input strands, there is a FRET phenomenon between the FAM-Cy3 group pairs. The FAM fluorescence is absorbed by Cy3, resulting in no fluorescence output from the FAM. Only when all three input strands (in1, in2, and in3) are input can the FRET phenomenon be destroyed, resulting in the FAM being able to emit fluorescence. This confirms the successful synthesis of DNA-based NAND logic gate tetrahedral framework nucleic acids.

[0128] like Figure 15 As shown, the FRET phenomenon between FAM-Cy3 group pairs is suppressed when there is no input strand or when all three input strands are input; the FRET phenomenon can be activated when only one or two input strands are input, confirming the successful synthesis of DNA-based XOR logic gate tetrahedral framework nucleic acids.

[0129] like Figure 16 As shown, the statistical changes in fluorescence intensity are related to Figure 9 The consistency indicates that OR logic gate tetrahedral framework nucleic acids can complete logical recognition and response to the target output chain within MCF living cells.

[0130] like Figure 17 As shown, the statistical changes in fluorescence intensity are related to Figure 10 The consistency indicates that the AND logic gate tetrahedral framework nucleic acid can complete the logical recognition and response to the target output chain within MCF living cells.

[0131] like Figure 18 As shown, the statistical changes in fluorescence intensity are related to Figure 11 The consistency indicates that MAJ logic gate tetrahedral framework nucleic acids can complete logical recognition and response to the target output chain within MCF living cells.

[0132] like Figure 19 As shown, the statistical changes in fluorescence intensity are related to Figure 12 The consistency indicates that OA logic gate tetrahedral framework nucleic acids can complete logical recognition and response to the target output chain within MCF living cells.

[0133] In summary, this invention successfully constructed a tetrahedral framework nucleic acid that conforms to DNA logic gates (OR, AND, MAJ, OA, NOR, NAND, and XOR), enabling the excited fluorescence intensity changes to conform to the logical operation results of three-input logic gates. This achieves logical recognition of three target nucleic acid single strands and has good application prospects.

Claims

1. A reagent with a three-input DNA logic gate function, characterized in that, The reagent is a tetrahedral framework nucleic acid with DNA logic gate operation function; the tetrahedral framework nucleic acid includes three single-stranded probe DNAs for recognizing three input strands respectively; The tetrahedral framework nucleic acid with DNA logic gate operation function is selected from at least one of the following: OR logic gate tetrahedral framework nucleic acid, AND logic gate tetrahedral framework nucleic acid, MAJ logic gate tetrahedral framework nucleic acid, OA logic gate tetrahedral framework nucleic acid, NOR logic gate tetrahedral framework nucleic acid, NAND logic gate tetrahedral framework nucleic acid, and XOR logic gate tetrahedral framework nucleic acid. The OR logic gate tetrahedral framework nucleic acid is modified with fluorescent and quenching groups. When the single-stranded probe DNA recognizes at least one input strand, the fluorescent and quenching groups are separated through a strand displacement reaction, releasing a fluorescent signal, and the OR logic gate outputs 1. The AND logic gate tetrahedral framework nucleic acid is modified with fluorescent groups and quenching groups. When the single-stranded probe DNA recognizes three input strands at the same time, the fluorescent group and quenching group are separated through a strand displacement reaction, releasing a fluorescent signal, and the AND logic gate outputs 1. The MAJ logic gate tetrahedral framework nucleic acid is modified with fluorescent groups and quenching groups. When the single-stranded probe DNA recognizes at least two input strands, a strand displacement reaction is initiated to cause one or two fluorescent groups to detach from the quenching group, releasing a fluorescent signal, and the MAJ logic gate output is 1. The OA logic gate tetrahedral framework nucleic acid is modified with fluorescent groups and quenching groups. When the single-stranded probe DNA recognizes at least one other input strand in addition to one specified input strand, the fluorescent group and quenching group are separated through a strand substitution reaction, releasing a fluorescent signal, and the OA logic gate outputs 1. The NOR logic gate tetrahedral framework nucleic acid is modified with fluorescent resonance energy transfer group pairs. When the single-stranded probe DNA recognizes at least one input strand, the fluorescent resonance energy transfer group pairs are separated through a strand displacement reaction, the fluorescence intensity decreases, and the NOR logic gate output is 0. The NAND logic gate tetrahedral framework nucleic acid is modified with fluorescent resonance energy transfer group pairs. When the single-stranded probe DNA recognizes three input strands at the same time, the fluorescent resonance energy transfer group pairs are separated through a strand displacement reaction, the fluorescence intensity decreases, and the NAND logic gate output is 0. The XOR logic gate's tetrahedral framework nucleic acid is modified with a fluorescent resonance energy transfer group pair and a quenching group. When the single-stranded probe DNA does not recognize any input strand or recognizes three input strands simultaneously, the fluorescent resonance energy transfer group pair is quenched by the quenching group, and the XOR logic gate outputs 0. When the single-stranded probe DNA recognizes one or two input strands, a strand displacement reaction causes the fluorescent resonance energy transfer group pair to leave the quenching group, releasing a fluorescent signal, and the XOR logic gate outputs 1.

2. The reagent with a three-input DNA logic gate function according to claim 1, characterized in that, The tetrahedral framework nucleic acid with DNA logic gate operation function includes a first vertex, a second vertex, a third vertex, and a fourth vertex; wherein, the first vertex is composed of a first single-stranded DNA, a second single-stranded DNA, and a third single-stranded DNA; the second vertex is composed of a first single-stranded DNA, a third single-stranded DNA, and a fourth single-stranded DNA; the third vertex is composed of a first single-stranded DNA, a second single-stranded DNA, and a fourth single-stranded DNA; and the fourth vertex is composed of a second single-stranded DNA, a third single-stranded DNA, and a fourth single-stranded DNA. One end of any three of the first single-stranded DNA, the second single-stranded DNA, the third single-stranded DNA, and the fourth single-stranded DNA includes a first single-stranded probe DNA, a second single-stranded probe DNA, and a third single-stranded probe DNA for recognizing the input strand. In the OR logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA, the second single-stranded DNA, and the third single-stranded DNA respectively includes a first single-stranded probe DNA, a second single-stranded probe DNA, and a third single-stranded probe DNA; a portion of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA extends to a second vertex, a third vertex, and a fourth vertex, respectively; the remaining fragments of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA completely cover the region between the first vertex and another vertex; one of the single-stranded DNA strands at the first vertex has a DNA breakpoint; the two ends of the DNA breakpoint are modified with a fluorescent group and a quenching group, respectively. And / or, in the AND logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA includes a first single-stranded probe DNA, one end of the second single-stranded DNA includes a second single-stranded probe DNA, one end of the fourth single-stranded DNA includes a third single-stranded probe DNA, a portion of the first single-stranded probe DNA extends into a tetrahedral framework nucleic acid, the second single-stranded probe DNA is complementary to a portion of the first single-stranded probe DNA, a portion of the third single-stranded DNA, and a portion of the fourth single-stranded DNA in sequence, respectively, a portion of the third single-stranded probe DNA extends into a tetrahedral framework nucleic acid, a portion of the third single-stranded probe DNA is complementary to a portion of the first single-stranded DNA and a portion of the second single-stranded DNA in sequence, the starting position of the second single-stranded probe DNA in the second single-stranded DNA is denoted as point A, the starting position of the third single-stranded probe DNA in the fourth single-stranded DNA is denoted as point B, and fluorescent groups and quenching groups are modified at points A and B, respectively; And / or, in the MAJ logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA, the second single-stranded DNA, and the third single-stranded DNA respectively includes a first single-stranded probe DNA, a second single-stranded probe DNA, and a third single-stranded probe DNA; a portion of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA extends to a second vertex, a third vertex, and a fourth vertex, respectively; the remaining fragments of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA completely cover the region between the first vertex and another vertex; each of the three single-stranded DNAs at the first vertex is provided with a thymine-containing deoxyribonucleotide, the thymine-containing deoxyribonucleotide is denoted as a spacer, wherein two spacers are modified with a fluorescent group and a quenching group, respectively, and the thymine at a distance of 2 bases from the remaining spacer is modified with a fluorescent group; And / or, in the OA logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA, the second single-stranded DNA, and the third single-stranded DNA respectively includes a first single-stranded probe DNA, a second single-stranded probe DNA, and a third single-stranded probe DNA; a portion of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA extends to a second vertex, a third vertex, and a fourth vertex, respectively; the remaining fragments of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA completely cover the region between the first vertex and another vertex; each of the three single-stranded DNA strands at the first vertex is provided with a spacer, wherein two of the spacers are modified with a fluorescent group and a quenching group, respectively; And / or, in the NOR logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA, the second single-stranded DNA, and the third single-stranded DNA respectively includes a first single-stranded probe DNA, a second single-stranded probe DNA, and a third single-stranded probe DNA; a portion of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA respectively extends to a second vertex, a third vertex, and a fourth vertex; the remaining fragments of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA respectively completely cover the region between the first vertex and another vertex; one of the single-stranded DNAs at the first vertex has a DNA breakpoint; the two ends of the DNA breakpoint are modified with fluorescent resonance energy transfer group pairs. And / or, in the NAND logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA includes a first single-stranded probe DNA, one end of the second single-stranded DNA includes a second single-stranded probe DNA, one end of the fourth single-stranded DNA includes a third single-stranded probe DNA, a portion of the first single-stranded probe DNA extends into the tetrahedral framework nucleic acid, the second single-stranded probe DNA is complementary to a portion of the first single-stranded probe DNA, a portion of the third single-stranded DNA, and a portion of the fourth single-stranded DNA in sequence, a portion of the third single-stranded probe DNA extends into the tetrahedral framework nucleic acid, a portion of the third single-stranded probe DNA is complementary to a portion of the first single-stranded DNA and a portion of the second single-stranded DNA in sequence, the starting position of the second single-stranded probe DNA in the second single-stranded DNA is denoted as point A, the starting position of the third single-stranded probe DNA in the fourth single-stranded DNA is denoted as point B, and fluorescent resonance energy transfer group pairs are modified at points A and B; And / or, in the XOR logic gate tetrahedral framework nucleic acid: one end of the first single-stranded DNA includes a first single-stranded probe DNA, and the other end of the first single-stranded DNA is denoted as endpoint A; one end of the second single-stranded DNA includes a second single-stranded probe DNA, and the other end of the second single-stranded DNA is denoted as endpoint B; one end of the fourth single-stranded DNA includes a third single-stranded probe DNA, and the other end of the fourth single-stranded DNA is denoted as endpoint C; a portion of the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA extends to a first vertex, a third vertex, and a second vertex, respectively; the first single-stranded probe DNA, the second single-stranded probe DNA, and the third single-stranded probe DNA... The remaining fragments of the single-stranded probe DNA completely cover the region between its extended vertex and another vertex. Endpoints A, B, and C are located at the first vertex, the third vertex, and the second vertex, respectively. All three endpoints are modified with quenching groups. The first and third single-stranded probe DNA at the first vertex are modified with fluorescent resonance energy transfer group pairs. The third and first single-stranded probe DNA at the second vertex are modified with fluorescent resonance energy transfer group pairs. The second and first single-stranded probe DNA at the third vertex are modified with fluorescent resonance energy transfer group pairs.

3. The reagent with a three-input DNA logic gate function according to claim 2, characterized in that, The fluorescent group is selected from at least one of Cy5, Cy3, and FAM groups; the quenching group is selected from at least one of BHQ1 and BHQ2 groups. And / or, in MAJ logic gate tetrahedral framework nucleic acids, the thymine-containing deoxyribonucleotide does not form complementary hydrogen bonds with other bases; the fluorescent group modified on the thymine at a distance of 2 bases from the remaining spacer needs to be the same as the fluorescent group modified on the spacer; And / or, the fluorescence resonance energy transfer group pair is selected from Cy5 and Cy3, or the fluorescence resonance energy transfer group pair is selected from Cy5 and FAM; And / or, in the XOR logic gate tetrahedral framework nucleic acid, the fluorescence resonance energy transfer group pair is selected from at least two of Cy5, Cy3, and FAM groups; the quenching group is selected from at least one of BHQ1 and BHQ2 groups.

4. The reagent with a three-input DNA logic gate function according to claim 2, characterized in that: The OR logic gate tetrahedral framework nucleic acid is assembled from 5 DNA single strands through complementary base pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively. And / or, the AND logic gate tetrahedral framework nucleic acid is assembled from four DNA single strands through complementary base pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively; And / or, the Majority logic gate tetrahedral framework nucleic acid is assembled from four DNA single strands through complementary base pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively; And / or, the OA logic gate tetrahedral framework nucleic acid is assembled from four DNA single strands through complementary base pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.12, and SEQ ID NO.13, respectively; And / or, the NOR logic gate tetrahedral framework nucleic acid is assembled from 5 DNA single strands through complementary base pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.14, and SEQ ID NO.15, respectively; And / or, the NAND logic gate tetrahedral framework nucleic acid is assembled from four DNA single strands through complementary base pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.9, and SEQ ID NO.16; And / or, the XOR logic gate tetrahedral framework nucleic acid is assembled from four DNA single strands through complementary base pairing, and the nucleotide sequences of the DNA single strands are shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, and SEQ ID NO.

20.

5. A method for preparing the reagent according to any one of claims 1-4, characterized in that, The process includes the following steps: adding the DNA single strand corresponding to each logic gate tetrahedral framework nucleic acid to a buffer solution, mixing to obtain a logic gate solution, and incubating to obtain the final product; the ratio of the amount of DNA single strands corresponding to each logic gate tetrahedral framework nucleic acid is 1:1:1:1 or 1:1:1:1:

1.

6. Use of the reagent with three-input DNA logic gate function as described in any one of claims 1-4 in multi-target recognition.

7. The use according to claim 6, characterized in that, The reagent is used for logical detection of multiple targets.

8. A reagent kit for target detection, characterized in that, Includes the input strand and the reagent with three-input DNA logic gate function as described in any one of claims 1-4.

9. The kit according to claim 8, characterized in that, The input strand consists of three DNA single strands; when the tetrahedral framework nucleic acid with DNA logic gate operation function is any one of OR logic gate tetrahedral framework nucleic acid, MAJ logic gate tetrahedral framework nucleic acid, OA logic gate tetrahedral framework nucleic acid, NOR logic gate tetrahedral framework nucleic acid, or XOR logic gate tetrahedral framework nucleic acid, the nucleotide sequence of the target DNA single strand is as shown in SEQ ID NO.21, SEQ ID NO.22, and SEQ ID NO.23; And / or, when the tetrahedral framework nucleic acid with DNA logic gate operation function is any one of AND logic gate tetrahedral framework nucleic acid or NAND logic gate tetrahedral framework nucleic acid, the nucleotide sequence of the target DNA single strand is as shown in SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.

26.

10. A method for target detection using the kit according to claim 8 or 9, characterized in that, The process includes the following steps: mixing the logic gate solution with the input chain and incubating to obtain the final product.

Images