Patterning-based nucleic acid detection method

Abstract

The invention discloses a nucleic acid detection method based on patterning, and belongs to the technical field of molecular diagnosis and biosensing. According to the method, a spatial position coded DNA framework is used as a detection carrier, specific capture probes are fixed at a plurality of pre-designed independent sites, a compound formed by target nucleic acid and a biotinylation report chain is subjected to specific hybridization capture, and then a recognizable signal is formed through combination of streptavidin; and the synchronous digital detection of multiple target nucleic acids is realized by reading patterned signals through an atomic force microscope. The position programmability and the single molecule detection sensitivity of the DNA nanotechnology are combined, the multiple detection capacity is only limited by the number of independent sites of a DNA frame, the expandability is extremely high, and the method is suitable for high-specificity detection of multiple nucleic acids in complex biological samples.

Description

Technical Field

[0001] This invention relates to the fields of molecular diagnostics and biosensing technology, specifically to a nucleic acid detection method based on DNA nanoframework patterning. This method utilizes the spatial addressability of DNA frameworks to convert nucleic acid detection signals into predefined spatial patterns, enabling multiplexed digital nucleic acid detection. Background Technology

[0002] Nucleic acid testing, as a core means of decoding life information, plays a cornerstone role in fields such as genetic disease diagnosis, pathogen tracing, early tumor screening, and basic biological research. In recent years, digital detection technologies, such as digital PCR (dPCR) and digital ELISA (dELISA), have achieved absolute quantification at the single-molecule level by dividing the reaction system into numerous micro-units, significantly improving the sensitivity and accuracy of detection. However, these technologies are limited by the types of signal molecules, and their upper limit for multiplexing has not been fundamentally improved.

[0003] The emergence of DNA origami technology has provided a revolutionary tool for precisely constructing programmable structures at the nanoscale. Using this technology, two-dimensional DNA frameworks with defined shapes, such as triangles and rectangles, can be self-assembled, and functional nucleic acid probes can be positioned at predetermined locations within the framework with nanometer-level precision. This inherent spatial programmability lays the foundation for developing a novel detection paradigm: position-encoded detection methods. However, effectively transforming this spatial programming capability of DNA frameworks into a stable, sensitive, and highly information-rich practical detection platform, particularly achieving integrated multiplexing, high sensitivity, high specificity, and addressable operation, remains a significant technological bottleneck in this field.

[0004] This invention designs a patterned nucleic acid detection system based on a triangular DNA origami framework structure. This invention successfully establishes a novel nucleic acid detection paradigm, transforming molecular recognition events into an easily interpretable patterned "molecular location barcode." Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a nucleic acid detection method based on DNA framework patterning, thereby overcoming the limitation of existing digital detection technologies in multiple detection capabilities, achieving simultaneous, highly specific, and digital detection of multiple target nucleic acids, and meeting the practical needs of complex biological sample detection.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A pattern-based nucleic acid detection method includes the following steps: S1. Prepare a DNA framework with a preset spatial site. The DNA framework is formed by PCR reaction of an M13mp18 backbone strand and several staple strands. The preset spatial site includes at least one labeling site M and at least two detection sites. The labeling site M is a staple strand modified with a labeling group. The detection sites are specific capture probes modified on the DNA framework for different target nucleic acids. S2. Mix the target nucleic acid to be tested with biotinylated reporter strand in an equimolar ratio, and anneal to form a target-reporter probe complex; S3. Incubate the target-reporter probe complex with the DNA framework obtained in step S1 in a hybridization buffer, so that the target-reporter probe complex is captured by the DNA framework through specific hybridization; S4. Add streptavidin SA solution, incubate, and it will bind to biotin at the end of the reporter chain to form a signal site; S5. Read the patterned signals on the DNA framework to achieve simultaneous detection of multiple target nucleic acids.

[0007] In some embodiments, the DNA framework has a triangular structure, the staple strands total 208, and the molar ratio of the M13mp18 backbone strand to each staple strand is 1:1.

[0008] In some embodiments, the preset spatial site includes one marker site M and six detection sites: P1-P6, wherein the marker site M is a staple chain modified with biotin.

[0009] In some embodiments, the PCR reaction conditions in step S1 are as follows: starting at 95°C, slowly cooling to 4°C at a rate of -1°C / 1min per cycle, and the reaction system is a 1×TAE buffer containing 12.5mM MgCl2: 40mM Tris, 20mM acetic acid, 2mM EDTA, 12.5mM MgCl2, pH 8.0.

[0010] In some embodiments, step S1 further includes: after the DNA framework is formed by PCR reaction, it is concentrated and purified by 100kDa ultrafiltration tube under the concentration conditions of centrifugation at 3000rcf for 5min.

[0011] In some embodiments, the annealing conditions in step S2 are: heating at 95°C for 10 minutes, then rapidly cooling to 4°C and holding for 20 minutes.

[0012] In some embodiments, the incubation conditions in step S3 are 37°C for 2 hours, and the incubation conditions in step S4 are 37°C for 30 minutes, with a streptavidin solution concentration of 0.1 mg / mL.

[0013] In some embodiments, step S4 further includes, after incubation, purifying the reaction product three times by ultrafiltration using 1×TAE buffer containing 12.5 mM MgCl2.

[0014] A DNA framework for the patterned detection method described above is formed by a PCR reaction of an M13mp18 backbone strand and several staple strands. The preset spatial sites include at least one labeling site M and at least two detection sites. The labeling site M is a staple strand modified with a labeling group, and the detection sites are specific capture probes modified on the DNA framework for different target nucleic acids.

[0015] In some embodiments, the DNA framework is a triangular structure, and the number of detection sites is 6, each corresponding to a capture probe for a different target nucleic acid.

[0016] The present invention also provides the application of the detection method described above in nucleic acid detection of complex biological samples.

[0017] The beneficial effects of this invention are as follows: 1) Breakthrough in multiple detection capabilities: Based on spatially encoded DNA frameworks, it avoids the problem of overlapping spectra of signal molecules, has extremely high scalability, and can achieve simultaneous detection of more targets by increasing the number of detection sites in the DNA framework.

[0018] 2) High specificity: It relies on specific hybridization based on complementary DNA base pairing, combined with the precise spatial positioning of the DNA framework, effectively reducing non-specific binding interference.

[0019] 3) Integrated: It integrates molecular recognition, signal formation and readout, eliminating the need for complex system segmentation steps and simplifying operation.

[0020] 4) Wide range of applications: By replacing nucleic acid probes with different recognition functions, it can achieve sensitive detection and multiplexing analysis of various types of targets (including but not limited to nucleic acids, small molecules, metal ions, etc.). It can be used for the detection of complex biological samples such as bacterial nucleic acid mixtures and has important application value in clinical diagnosis, environmental monitoring and other fields.

[0021] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, some of the drawings in the following description are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram illustrating the principle of the DNA framework patterning detection method described in this invention.

[0024] Figure 2 The spatial localization characterization map of the detection site shown in Embodiment 2 of the present invention includes a preset marker site (M) and detection sites (P1-P6) precisely located in the DNA framework. Detailed Implementation

[0025] To better describe the present invention, specific embodiments are provided below for further explanation. Unless otherwise specified, the methods in the following embodiments are conventional methods.

[0026] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0027] The following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental conditions not specifically stated in the examples are generally performed under conventional conditions or as recommended by the reagent company; reagents, consumables, etc., used in the following examples are commercially available unless otherwise specified.

[0028] Example 1: Preparation of a triangular DNA framework with specific recognition sites 1.1 Experimental Materials and Instruments Reagents: M13mp18 backbone strand, 208 staple strands, 6 specific capture probes, 6 biotinylated reporter strands (the 208 staple strands are A1~A65, B1~B65, C1~C65, Link-A1C~A4C, Link-B1A~B4C, Link-C1B~C4B, Loop, etc.; the sequences of the M13mp18 backbone strand and the 208 staple strands are as shown in reference 1). [1]As shown, the sequence of the M13mp18 backbone strand is identical to that of the M13mp18 strand in Reference 1, and the sequences A1~A65, B1~B65, C1~C65, Link-A1C~A4C, Link-B1A~B4C, Link-C1B~C4B, and Loop are identical to those of the DNA strands with the same names in Reference 1. The six specific capture probes are Tar-1~6, and their sequences are shown in SEQ ID No. 1~6. The six reporter strands are Report-1~6, and their sequences are shown in SEQ ID No. 7~12. All of the above DNA strands were synthesized by Sangon Biotech (Shanghai) Co., Ltd. 1×TAE-Mg 2+ Buffer solution (40mM Tris, 20mM acetic acid, 2mM EDTA, 12.5mM MgCl2, pH 8.0); streptavidin (SA, concentration 0.1mg / mL); hybridization buffer (1×TAE buffer containing 12.5mM MgCl2).

[0029] Instruments: UV-Vis spectrophotometer (Carry-100, Agilent); PCR instrument C100 (Bio-Rad); 100kDa ultrafiltration tubes (Millipore); centrifuge (Eppendorf); Nanodrop (Thermo); gel electrophoresis system PowerPac (Bio-Rad); atomic force microscopy imaging system (AFM, Bruker, MultiMode 8-HR).

[0030] 1.2 Preparation and Validation of DNA Origami Frames See Figure 1 The M13mp18 backbone chain and 208 staple chains were mixed in an equimolar ratio in 1×TAE-Mg 2+ The total volume was 50 μL in a buffer solution (40 mM Tris, 20 mM acetic acid, 2 mM EDTA, 12.5 mM MgCl2, pH 8.0), in which the B18 staple chain was biotin-modified as a marker M to distinguish the position.

[0031] The above solution was subjected to a temperature gradient annealing program in a PCR instrument: starting from 95 °C, the temperature was slowly reduced to 4 °C at a rate of -1 °C / 1 min per cycle to obtain a structurally complete triangular DNA frame.

[0032] The obtained DNA framework solution was transferred to a 0.5 mL 100 kDa ultrafiltration tube, centrifuged at 3000 rcf for 5 min, and the concentrate was collected.

[0033] Take 1 μL of the concentrate and determine the concentration using Nanodrop.

[0034] Example 2: Nucleic acid detection and AFM readout based on patterned DNA framework 2.1 Nucleic acid detection experiment 1) Capture probe modification: Modifying the DNA origami framework structure to target specific targets. Stenotrophomonas, Haemophilus, Klebsiella, Staphylococcus, Lactobacillus, Acinetobacter Capture probes (Tar1-Tar6) of six bacterial DNA sequences were used to capture the target DNA of the above bacteria, corresponding to the P1-P6 sites of the origami frame structure, respectively.

[0035] 2) Preparation of target-reporter probe complex: Six target bacteria were used... Stenotrophomonas, Haemophilus, Klebsiella, Staphylococcus, Lactobacillus, Acinetobacter Bacterial DNA was obtained through centrifugation, RNA extraction, reverse transcription, and asymmetric PCR. The obtained DNA was quantified using nanodrop and diluted to obtain the desired target DNA. 10 μL of 100 nM target DNA and 10 μL of 100 nM biotinylated reporter strand were mixed in hybridization buffer (1× TAE, containing 12.5 mM MgCl2), heated at 95 °C for 10 minutes, and then rapidly cooled to 4 °C to anneal and form a "target-reporter probe complex." The above annealing reaction was performed on the six target DNAs, and each target DNA was mixed with Report-1 to Report-6 respectively.

[0036] 3) Hybridization capture: The pre-hybridized "target-reporter probe complex" solution was added to a centrifuge tube containing a triangular DNA origami frame and incubated at 37 °C for 2 hours, so that the complex was captured by specific hybridization between the target DNA and the capture probe at a specific site on the origami frame.

[0037] 4) Signal labeling: Add streptavidin (SA, 0.1 mg / mL) solution to the system and incubate at 37°C for 30 minutes to allow SA to bind to biotin at the end of the reporter chain, forming a signal site detectable under AFM.

[0038] 4) Purification: Transfer the reaction solution to a 100 kDa ultrafiltration tube, centrifuge at 3000 rcf for 5 min, and then purify with 1×TAE-Mg. 2+ Wash three times with buffer to remove free SA and unbound complexes.

[0039] 5) AFM detection: Take 1 μL of purified DNA framework solution (final concentration 1 nM) and drop it onto freshly peeled mica sheet. Let it stand at room temperature for 2 min, dry it with nitrogen, and then perform AFM imaging. The scanning range is 500 nm × 500 nm. Record the signal distribution of the marker site (M) and the detection site (P1-P6).

[0040] 2.2 Result Determination like Figure 2 As shown, the AFM image clearly displays seven distinct bright spots: one located at the pre-defined marker site (M), and the other six precisely located at detection sites within the DNA framework. This result confirms that targets T1-T6 are specifically captured at specific sites and generate readable signals via SA, successfully achieving digital detection of multiplex nucleic acid targets based on spatial location encoding.

[0041] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0042] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

[0043] [1] Pal S , Deng Z , Wang H ,et al.DNA Directed Self-Assembly ofAnisotropic Plasmonic Nanostructures[J].Journal of the American ChemicalSociety, 2011, 133(44):17606-17609.DOI:10.1021 / ja207898r.

Claims

1. A pattern-based nucleic acid detection method, characterized in that, Includes the following steps: S1. Prepare a DNA framework with a preset spatial site. The DNA framework is formed by PCR reaction of an M13mp18 backbone strand and several staple strands. The preset spatial site includes at least one labeling site M and at least two detection sites. The labeling site M is a staple strand modified with a labeling group. The detection sites are specific capture probes modified on the DNA framework for different target nucleic acids. S2. Mix the target nucleic acid to be tested with biotinylated reporter strand in an equimolar ratio, and anneal to form a target-reporter probe complex; S3. Incubate the target-reporter probe complex with the DNA framework obtained in step S1 in a hybridization buffer, so that the target-reporter probe complex is captured by the DNA framework through specific hybridization; S4. Add streptavidin SA solution, incubate, and it will bind to biotin at the end of the reporter chain to form a signal site; S5. Read the patterned signals on the DNA framework to achieve simultaneous detection of multiple target nucleic acids.

2. The pattern-based nucleic acid detection method as described in claim 1, characterized in that, The DNA framework has a triangular structure, and there are 208 staple strands in total. The molar ratio of the M13mp18 backbone strand to each staple strand is 1:

1.

3. The pattern-based nucleic acid detection method as described in claim 1, characterized in that, The preset spatial sites include one marker site M and six detection sites: P1-P6, wherein the marker site M is a staple chain modified with biotin.

4. The pattern-based nucleic acid detection method as described in claim 1, characterized in that, The PCR reaction conditions in step S1 are as follows: starting at 95℃, slowly cooling to 4℃ at a rate of -1℃ / 1min per cycle, and the reaction system is 1×TAE buffer containing 12.5mM MgCl2: 40mM Tris, 20mM acetic acid, 2mM EDTA, 12.5mM MgCl2, pH 8.

0.

5. The pattern-based nucleic acid detection method as described in claim 1, characterized in that, Step S1 also includes: after the DNA framework is formed by PCR reaction, it is concentrated and purified by 100kDa ultrafiltration tube under the concentration conditions of centrifugation at 3000rcf for 5min.

6. The pattern-based nucleic acid detection method as described in claim 1, characterized in that, The annealing conditions in step S2 are: heating at 95°C for 10 minutes, then rapidly cooling to 4°C and holding for 20 minutes.

7. The pattern-based nucleic acid detection method as described in claim 1, characterized in that, In step S3, the incubation conditions were 37°C for 2 hours, and in step S4, the incubation conditions were 37°C for 30 minutes, with a streptavidin solution concentration of 0.1 mg / mL.

8. The pattern-based nucleic acid detection method as described in claim 1, characterized in that, Step S4 also includes purifying the reaction product three times by ultrafiltration using 1×TAE buffer containing 12.5 mM MgCl2 after incubation.

9. The application of the detection method as described in any one of claims 1-8 in nucleic acid detection of complex biological samples.

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