DNA tetrahedral nano-element and preparation method and imaging application thereof

Abstract

The invention relates to a DNA tetrahedral nano-element as well as a preparation method and imaging application thereof, and belongs to the technical field of biological detection. The technical problems that in the prior art, the miRNA detection sensitivity is low, traditional tetrahedral DNA is difficult to enter cells, and the living body imaging specificity is insufficient are solved. According to the invention, a nano-element composed of a tetrahedral DNA frame, a polyguanine modified chain and a catalytic hairpin chain (H1 / H2) is constructed, and cycle signal amplification of to-be-detected miRNA is realized through a chain displacement reaction; after in-vitro verification, the probe is applied to macrophage targeted detection and ARDS mouse model lung imaging. According to the invention, efficient targeting of macrophages can be realized without a transfection reagent, excellent specificity is shown in vivo and in vitro, and a reliable tool is provided for early diagnosis and mechanism research of ARDS.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, and relates to DNA tetrahedral nanoelements, their preparation methods, and imaging applications. Background Technology

[0002] Acute respiratory distress syndrome (ARDS) is a life-threatening clinical condition characterized by acute hypoxic respiratory failure, with a mortality rate as high as 35%-45%. The main causes include severe infection, trauma, and shock. Although supportive care strategies have improved the clinical prognosis of ARDS patients, the mortality rate remains high. Therefore, there is an urgent need to explore more reliable diagnostic methods. The pathophysiology of ARDS is closely related to an excessive inflammatory cascade, in which alveolar macrophages (AMs) play a key regulatory role, promoting the secretion of pro-inflammatory cytokines and chemokines, ultimately leading to alveolar epithelial damage and impaired gas exchange. ARDS is a high-mortality disease whose pathophysiology is closely related to the inflammatory response. However, existing detection methods (such as free hairpin strand assays) have low detection limits for miRNAs, making early or trace detection difficult and limiting their application in clinical diagnosis.

[0003] Tetrahedral DNA (TET) is a tunable nanomaterial formed by four DNA strands linked by complementary base pairing. Compared with other nanomaterials, tetrahedral DNA has advantages such as good stability, high controllability, and good biocompatibility. Due to these properties, it has been widely studied and applied in recent years in fields such as biosensors, drug delivery, and bioimaging. However, traditional tetrahedral DNA has difficulty entering cells, which poses a challenge to its application in intracellular imaging or drug delivery. Summary of the Invention

[0004] In view of this, the present invention enables tetrahedral DNA modified with polyguanine (poly G) to be efficiently internalized by macrophages without additional transfection reagents, thereby increasing the ability of tetrahedral DNA to enter macrophages. Simultaneously, by synthesizing various multifunctional tetrahedral DNAs, highly sensitive imaging detection of microRNA-155 (miR-155) in macrophages is achieved. One objective of this invention is to provide a DNA tetrahedral nanoelement; another objective is to provide a method for preparing a DNA tetrahedral nanoelement; and a third objective is to provide an imaging application for the DNA tetrahedral nanoelement.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a DNA tetrahedral nanoelement, which consists of a DNA tetrahedron, two H hairpin strands, and two identical polyG strands. The DNA tetrahedron is self-assembled from TET1, TET2, TET3, and TET4 single nucleotides. The H hairpin strands include H1 and H2 strands. The H1 strand contains a fluorescent group and a quenching group. The H1 strand initiates a strand displacement reaction with the target miRNA. The two polyG strands are used to target macrophages. The hairpin strand H1 is used to undergo a strand displacement reaction with the target miRNA, releasing fluorescence. The H2 strand is used to undergo a further strand displacement reaction with the reaction product of H1+ miRNA, displacing the miRNA and amplifying the signal. Preferably, the nucleic acid sequences of TET1-TET4 are as shown in SEQ ID NO:1-SEQ ID NO:4; the nucleic acid sequence of the H1 chain is as shown in SEQ ID NO:5 or SEQ ID NO:6; the H1 chain is modified with a fluorescent group and a quenching group; the fluorescent group is 6FAM or Cy5; the quenching group is preferably BHQ1 or BHQ2; the nucleic acid sequence of the H2 chain is as shown in SEQ ID NO:7; and the nucleic acid sequence of the poly G chain is as shown in SEQ ID NO:8. Furthermore, the preparation method of DNA tetrahedral nanodevices includes the following steps: S1: Mix TET-1, TET-2, TET-3 and TET-4, each with a concentration of 50 µM, at a molar ratio, then add 10×TAE. The annealing program is: 95℃ for 10 min, followed by 4℃ for 30 min. S2: Mix the annealed H1 and H2 with TET at a molar concentration ratio and incubate at 37°C for 30 min; S3: Add polyG and incubate at 37°C for 30 min; Preferably, the molar ratio of TET-1, TET-2, TET-3 and TET-4 is 1:1:1:1, and the molar concentration ratio of H1, H2 and TET is 1:1. Furthermore, the application of the aforementioned DNA tetrahedral nanoelement in imaging detection; Preferably, the application of the DNA tetrahedral nanoelement in imaging detection of miRNA; Preferably, the miRNA is macrophage miRNA-155.

[0006] The beneficial effects of this invention are as follows: High-sensitivity detection: The DNA nanoelement, through a CHA cycle amplification mechanism, achieves ultrasensitive detection of miRNA-155, with a detection limit of 250 fM (femtomolar), approximately 10 times more sensitive than free hairpin strands (detection limit 2.5 nM). This facilitates early diagnosis and in-depth research into the molecular mechanisms of ARDS.

[0007] High specificity: By designing a specific sequence, this element is highly specific for miRNA-155 and has no significant cross-reactivity with other miRNAs, reducing the risk of false positives and improving detection reliability.

[0008] Highly efficient cell targeting: polyG modification enables DNA nanoelements to effectively target macrophages (especially alveolar macrophages), allowing them to be intracellularly endocytosed without additional transfection reagents, thus reducing experimental complexity and toxicity.

[0009] Enhanced in vivo imaging capabilities: In an ARDS mouse model, this element successfully enabled real-time imaging of lung miRNA-155, with fluorescence signal intensity significantly increasing in the disease state and exhibiting time-dependent peak intensity (peak at 3 hours). This provides a new tool for in vivo diagnosis and treatment assessment of ARDS.

[0010] Biocompatibility and stability: Tetrahedral DNA frameworks have good biocompatibility and stability, reducing the potential risks of in vivo application and making them suitable for preclinical studies.

[0011] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0012] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1The assembly process and application model of DNA nanoelement are shown in the diagram: DNA nanoelement (TET-H1H22G (14)) can enter macrophages without the assistance of transfection reagents; after entering the cell, in the presence of miRNA-155, the hairpin H1 chain will unfold, and the fluorescence of 6-FAM will no longer be quenched by BHQ1, thus generating a fluorescence signal; the product formed by the reaction of miRNA-155 and H1 will further undergo a chain displacement reaction with hairpin H2, so that the miRNA-155 chain is released. The released miRNA-155 can participate in the reaction again, realize the amplification of the fluorescence signal, and finally realize the ultrasensitive imaging of miRNA-155 in macrophages.

[0013] Figure 2 Figure for in vitro detection of miRNA-155: 2a: Tetrahedral DNA (TET) can be successfully synthesized from four DNA strands, and two polyguanine nucleotide chains (polyG) can be modified onto TET.

[0014] 2b: The reaction of TET-H1H2 with miRNA-155 showed that the migration rate of the TET-H1H2 and miRNA-155 reactants decreased after the addition of miRNA-155, suggesting the formation of a larger complex.

[0015] 2c: Similar conclusions were also reached regarding the reaction of hairpin structure H1H2 with miRNA-155.

[0016] 2d: Compared with TET-H1H2, TET-H1H22G (14) can polymerize to form a larger complex regardless of the presence of miRNA-155; however, it is more likely to form a larger polymer in the presence of miRNA-155.

[0017] 2e-2f: Transmission electron microscopy (TEM) images and dynamic light scattering (DLS) data show that both TET-H1H2 and TET-H1H22G (14) can polymerize in the presence of miRNA-155; however, compared to TET-H1H2, TET-H1H22G (14) generates a larger polymer when reacting with miRNA-155.

[0018] 2g-2h: The performance of free hairpins H1 and H2, and those immobilized on TET or TET-H1H22G (14), in in vitro detection of miRNA-155 was compared. Under miRNA-155 induction, free H1 and H2 can undergo a catalytic hairpin assembly reaction (CHA) to achieve cyclic amplification and detection of the target miRNA-155; when these two hairpins are immobilized on TET-H1H2 or TET-H1H22G (14), CHA can also occur in the reaction with miRNA-155.

[0019] 2i: Compared with free H1 and H2, H1 and H2 immobilized on TET-H1H2 or TET-H1H22G (14) exhibited higher cyclic amplification efficiency, with H1 and H2 immobilized on TET-H1H22G (14) showing the strongest efficiency. The detection limit of free H1 and H2 for miRNA-155 was 2.5 nM, while the detection limit of H1 and H2 immobilized on TET-H1H22G (14) for miRNA-155 reached 250 fM, with a sensitivity approximately 10 times that of the former.

[0020] The 2j: TET-H1H22G (14) detection system is specific for the detection of miRNA-155 and does not show significant amplification compared to other miRNAs.

[0021] Figure 3 Figure for imaging detection of miRNA-155: 3a: Laser confocal scanning microscopy comparison of the imaging effects of four systems—H1+H2, TET-H1H2, TET-H12G (14), and TET-H1H22G (14)—on intracellular miRNA-155. The H1+H2 and TET-H1H2 groups showed almost no fluorescence, indicating that free hairpins (H1+H2) and TET-H1H2 had difficulty entering macrophages without transfection reagents. Both the TET-H12G (14) and TET-H1H22G (14) groups showed fluorescence, with the TET-H1H22G (14) group exhibiting significantly higher fluorescence intensity than the TET-H12G (14) group. The fluorescence intensity of the LPS-treated group was significantly higher than the control group, because LPS induction increases the expression level of miRNA-155 in RAW264.7 macrophages.

[0022] 3b: The same results were obtained by semi-quantitative detection using flow cytometry.

[0023] 3c-3d: As the co-incubation time continued to increase, the fluorescence intensity gradually increased, and the fluorescence intensity of the LPS-treated group was always significantly higher than that of the control group.

[0024] Figure 4 Here is a graph showing the results of the flow cytometry analysis: Flow cytometry analysis showed that tetrahedrons carrying polyA (TET-2A(14)), polyT (TET-2T(14)), polyC (TET-2C(14)), and polyG (TET-2G(14)) and co-incubated with RAW264.7 cells resulted in an increase in fluorescence intensity only for TET-2G(14). This indicates that polyG has good specificity for macrophages.

[0025] Figure 5 Diagram of an in vivo detection experiment in animals: 5a: The imaging capability of DNA nanoelement (TET-H1H22G (14)) for miRNA-155 was investigated in an ARDS mouse model. As shown in Figure 5a, different materials or PBS were injected into the model mice via endotracheal infusion. 5b: The imaging intensity of each group showed a trend of TET-H1H22G (14) > TET-H12G (14) > TET-H1H2 > H1+H2.

[0026] 5c: In the ARDS group, the imaging intensity of TET-H1H22G (14) was significantly higher than that in the PBS group, which was due to the abnormally high level of miRNA-155 in the ARDS mice.

[0027] 5d-5e: When TET-H1H22G (14) was instilled into mice 3 hours later, the lung imaging intensity reached its peak; as time went on, the lung imaging intensity gradually decreased, which may be due to the fluorescence decay of the fluorescent group. Detailed Implementation

[0028] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0029] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0030] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0031] Example 1: Preparation of DNA nanoelement (TET-H1H22G (14)) TET-1, TET-2, TET-3, and TET-4, each at a concentration of 50 µM, were mixed in a molar ratio of 1:1:1:1, resulting in a total volume of 30 µL. 3 µL of 10×TAE was added, and the mixture was annealed at 95 °C for 10 min, followed by 4 °C for 30 min. The annealed H1 and H2 were then mixed with TET in a 1:1:1 molar ratio and incubated at 37 °C for 30 min. PolyG was then added, and the mixture was incubated at 37 °C for another 30 min. All DNA sequences are as follows:

[0032] Example 2: In vitro detection of miRNA-155 using DNA nanoelements In a 20 μL system containing 1×TE buffer, the final concentrations of H1, H1+H2, TET-H1H2, or TEE-H1H22G (14) were all controlled to 1 μM. Different concentrations of miRNA-155 were added to the experimental groups, while the control group was replaced with an equal volume of double-distilled water (ddH2O). After adding miRNA-155 or double-distilled water, the system was incubated at 37℃ for 60 min, and then the fluorescence intensity emitted by the system was detected using an F-7000 fluorescence spectrophotometer.

[0033] like Figure 2As shown: 2a: Tetrahedral DNA (TET) can be successfully synthesized from four DNA strands, and two polyguanine nucleotide chains (polyG) can be modified onto TET. 2b: The reaction of TET-H1H2 with miRNA-155 showed that the migration rate of the TET-H1H2 reactant with miRNA-155 decreased after the addition of miRNA-155, suggesting the formation of a larger complex. 2c: The reaction of hairpin structure H1H2 with miRNA-155 also yielded similar conclusions. 2d: Compared with TET-H1H2, TET-H1H22G (14) can polymerize to form a larger complex regardless of the presence or absence of miRNA-155; however, it is more likely to form a larger polymer in the presence of miRNA-155. 2e-2f: Transmission electron microscopy (TEM) images and dynamic light scattering (DLS) data show that both TET-H1H2 and TET-H1H22G (14) can polymerize in the presence of miRNA-155; however, TET-H1H22G (14) generates larger polymers under miRNA-155 induction compared to TET-H1H2. 2g-2h: The performance of free hairpins H1 and H2, and H1 and H2 immobilized on TET or TET-H1H22G (14), in in vitro detection of miRNA-155 was compared. Under miRNA-155 induction, free H1 and H2 can undergo a catalytic hairpin assembly reaction (CHA) to achieve cyclic amplification and detection of the target miRNA-155; when these two hairpins are immobilized on TET-H1H2 or TET-H1H22G (14), CHA can also occur under miRNA-155 induction. 2i: Compared with free H1 and H2, H1 and H2 immobilized on TET-H1H2 or TET-H1H22G (14) exhibit higher cyclic amplification efficiency, with H1 and H2 immobilized on TET-H1H22G (14) showing the strongest efficiency. The detection limit of free H1 and H2 for miRNA-155 is 2.5 nM, while the detection limit of H1 and H2 immobilized on TET-H1H22G (14) for miRNA-155 can reach 250 fM, with a sensitivity approximately 10 times that of the former. The 2j: TET-H1H22G (14) detection system is specific for the detection of miRNA-155 and does not show significant amplification compared to other miRNAs.

[0034] Example 3: DNA nanoelement successfully targeted macrophages and miRNA-155 was detected within macrophages. RAW264.7 cells were seeded in 6-well plates to detect the targeting of DNA nanomaterials to macrophages. Then, the various nanomaterials were added to the cells at a final concentration of 200 nM and incubated together. Intracellular fluorescence intensity was detected using flow cytometry and laser confocal microscopy.

[0035] like Figure 3 As shown in Figure 3a: Laser confocal scanning microscopy comparison of the imaging effects of four systems—H1+H2, TET-H1H2, TET-H12G (14), and TET-H1H22G (14)—on intracellular miRNA-155. The H1+H2 and TET-H1H2 groups showed almost no fluorescence, indicating that free hairpins (H1+H2) and TET-H1H2 were difficult to enter macrophages without transfection reagents. Both the TET-H12G (14) and TET-H1H22G (14) groups showed fluorescence, with the TET-H1H22G (14) group exhibiting significantly higher fluorescence intensity than the TET-H12G (14) group. The fluorescence intensity of the LPS-treated group was significantly higher than that of the control group, because LPS induction increased the expression level of miRNA-155 in RAW264.7 macrophages. 3b: The same results were obtained by semi-quantitative flow cytometry. 3c-3d: With the continuous extension of co-incubation time, the fluorescence intensity gradually increased, and the fluorescence intensity of the LPS-treated group was consistently significantly higher than that of the control group.

[0036] like Figure 4 As shown, flow cytometry analysis revealed that only TET-2G(14) showed increased fluorescence intensity after co-incubation with tetrahedrons carrying polyA (TET-2A(14)), polyT (TET-2T(14)), polyC (TET-2C(14)), and polyG (TET-2G(14)). This indicates that polyG has good targeting ability for macrophages.

[0037] Example 4: Detection of miRNA-155 in ADRS mice using DNA nanoelements Male C57BL / 6 mice aged 6-8 weeks were randomly divided into four groups: control group (Ctrl group), H1+H2 group, TET-H1H2 group, TET-H12G (14) group, and TET-H1H22G (14) group, with 6 mice in each group. Each group was further divided into two subgroups: one group was stimulated with lipopolysaccharide, and the other group was treated with an equal volume of PBS. After anesthesia, mice were instilled with 50 μL of PBS (5 mg / kg) or an equal volume of PBS via the airway. Two hours later, 50 μL of H1+H2, TET-H1H2, TET-H12G (14), or TET-H1H22G (14) was instilled via the same airway, with each material being 30 μg. The mice were then observed at room temperature for 6 hours, after which tissue samples (lung, heart, liver, spleen, and kidney) were collected. In the experiment investigating the effect of observation time on the imaging effect of miRNA-155 in the mice, all other conditions were kept constant, and only the observation time of the mice was changed. Finally, the acquired tissue samples were subjected to imaging analysis. The results are analyzed as follows. Figure 5 As shown, 5a: The imaging ability of DNA nanoelement (TET-H1H22G (14)) on miRNA-155 was investigated in an ARDS mouse model. Researchers injected different materials or PBS into the model mice via tracheal instillation; 5b: The imaging intensity of each group showed a trend of TET-H1H22G (14)>TET-H12G (14)>TET-H1H2>H1+H2; 5c: In the ARDS group, the imaging intensity of TET-H1H22G (14) was significantly higher than that of the PBS group, which was due to the abnormally high level of miRNA-155 in the ARDS mice; 5d-5e: The lung imaging intensity reached its peak 3 hours after TET-H1H22G (14) was instilled into the mice; as time went on, the lung imaging intensity gradually decreased, which may be caused by the fluorescence decay of the fluorescent group.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A DNA tetrahedral nanodevice, characterized in that: The DNA tetrahedral nanoelement consists of a DNA tetrahedron, two H hairpin strands, and two identical poly G strands. The DNA tetrahedron is self-assembled from TET1, TET2, TET3, and TET4 single nucleotides. The H hairpin strands include H1 and H2 strands. The H1 strand contains a fluorescent group and a quenching group. The H1 strand undergoes a displacement reaction with the sending strand of the target miRNA.

2. The DNA tetrahedral nanodevice according to claim 1, characterized in that: The nucleic acid sequences of TET1-TET4 are shown in SEQ ID NO:1-SEQ ID NO:4; the nucleic acid sequence of the H1 chain is shown in SEQ ID NO:5 or SEQ ID NO:6, and the H1 chain is modified with a fluorescent group and a quenching group; the fluorescent group is 6-FAM or Cy5, and the quenching group is preferably BHQ1 or BHQ2; the nucleic acid sequence of the H2 chain is shown in SEQ ID NO:7; and the nucleic acid sequence of the poly G chain is shown in SEQ ID NO:

8.

3. A method for preparing DNA tetrahedral nanodevices, characterized in that, The steps are as follows: S1: Mix TET-1, TET-2, TET-3 and TET-4, each with a concentration of 50 µM, at a molar ratio, then add 10×TAE. The annealing program is: 95℃ for 10 min, followed by 4℃ for 30 min. S2: Mix the annealed H1 and H2 with TET at a molar concentration ratio and incubate at 37°C for 30 min; S3: Add polyG and incubate at 37°C for 30 min.

4. The preparation method according to claim 3, characterized in that: The molar ratio of TET-1, TET-2, TET-3 and TET-4 is 1:1:1:1, and the molar concentration ratio of H1, H2 and TET is 1:

1.

5. The application of the DNA tetrahedral nanoelement according to claim 1 or 2 in imaging detection.

6. The application of the DNA tetrahedral nanoelement according to claim 1 or 2 in imaging detection of miRNA.

7. The application according to claim 6, characterized in that: The miRNA in question is miRNA-155, which is found in macrophages.

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