DNA tetrahedral fluorescent probe based on multivalent spatial pattern recognition, and use thereof

Abstract

The present invention relates to the technical field of biological detection, in particular to a DNA tetrahedral fluorescent probe based on multivalent spatial pattern recognition and the use thereof. The fluorescent probe is of a tetrahedral structure, and comprises five DNA single strands, three DNA long strands and three DNA short strands complementary to the DNA long strands, wherein the three DNA long strands comprise a CY5 dye modifying the 3' end, and the three DNA short strands comprise a BHQ2 dye modifying the 3' end. The DNA tetrahedral fluorescent probe of the present invention can achieve multivalent recognition of a circular RNA and structurally matches same, and thus has enhanced specificity and sensitivity of circular RNA detection and is suitable for imaging and detection of circular RNAs.

Description

A DNA tetrahedron fluorescent probe based on multivalent spatial pattern recognition and its application Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a DNA tetrahedron fluorescent probe based on multivalent spatial pattern recognition and its application. Background Art

[0002] Circular RNA (circRNA) is a novel endogenous noncoding RNA with a covalently closed circular structure. Recent studies have shown that circRNA is closely associated with the development and progression of various diseases, including tumors, cardiovascular diseases, and neurological diseases. Therefore, the development of detection and imaging methods for circRNA is of great significance for the early diagnosis of related diseases. However, due to the complex secondary structure and spatial geometry of circRNA molecules and the wide variety of interfering RNA molecules, their specific detection and imaging in the complex system of living cells remains a significant challenge.

[0003] Currently, the most widely used method for detecting circular RNA is quantitative PCR (qPCR). While qPCR offers good sensitivity, it is also associated with time-consuming, cumbersome procedures, and high costs. Furthermore, the commonly used imaging method, fluorescence in situ hybridization (FISH), has drawbacks such as low sensitivity and the need to fix cells.

[0004] In living systems, biomolecules can significantly enhance interactions through allosteric effects of spatial geometric structure matching and multivalent effects of multiple recognition sites. DNA nanotechnology, with its excellent properties such as sequence programmability, precise and controllable spatial structure, biorecognition and stimulus-responsive properties, and biocompatibility, is an excellent tool for constructing molecular probes. Among them, framework nucleic acid tetrahedron nanostructures have the advantages of ease of synthesis, structural controllability, and ease of cellular internalization. Inspired by the above, the present invention proposes a DNA tetrahedron fluorescent probe that enhances recognition capabilities through multivalent recognition of circular RNA molecules and spatial structure matching, and is used for specific detection of circular RNA and cell imaging.

[0005] Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention proposes a DNA tetrahedron fluorescent probe based on multivalent spatial pattern recognition and its application.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] A DNA tetrahedral fluorescent probe based on multivalent spatial pattern recognition, wherein the fluorescent probe has a tetrahedral structure and comprises five single DNA strands, three long DNA strands, and three short DNA strands complementary to the long DNA strands; three of the long DNA strands comprise CY5 dyes modified at their 3' ends, and three of the short DNA strands comprise BHQ2 dyes modified at their 3' ends.

[0009] Preferably, the nucleotide sequences of the five single-stranded DNAs are respectively shown as SEQIDNO.1-5, the sequence of A37-1 is shown as SEQIDNO.1, the sequence of B37-1 is shown as SEQIDNO.2, the sequence of C37-1 is shown as SEQIDNO.3, the sequence of D37-1 is shown as SEQIDNO.4, and the sequence of D37-2 is shown as SEQIDNO.5.

[0010] Preferably, the nucleotide sequences of the three DNA long chains are shown as SEQ ID NO.6-8, respectively, the sequence of A37-2 is shown as SEQ ID NO.6, the sequence of B37-2 is shown as SEQ ID NO.7, and the sequence of C37-2 is shown as SEQ ID NO.8.

[0011] Preferably, the nucleotide sequences of the three short DNA chains are shown as SEQIDNO.9-11, the sequence of Q1 is shown as SEQIDNO.9, the sequence of Q2 is shown as SEQIDNO.10, and the sequence of Q3 is shown as SEQIDNO.11; wherein the short DNA chain Q1 is complementary to the long DNA chain A37-2, the short DNA chain Q2 is complementary to the long DNA chain B37-2, and the short DNA chain Q3 is complementary to the long DNA chain C37-2.

[0012] Preferably, the length of the five single-stranded DNAs is 58 nt; the length of the three long DNA chains is 77 nt; and the length of the three short DNA chains is 12 nt.

[0013] The above-mentioned application of a DNA tetrahedron fluorescent probe based on multivalent spatial pattern recognition in circular RNA imaging and non-diagnostic detection.

[0014] Preferably, the applications specifically include: dynamic real-time monitoring of circular RNA in cultured cells in vitro; sensitive and rapid in situ imaging of circular RNA in mouse brain slices; and ultra-rapid detection of circular RNA in unamplified RNA in human plasma samples.

[0015] Beneficial effects of the present invention:

[0016] 1. The DNA tetrahedral structure is biocompatible; thus, it solves the problems of probe toxicity in existing technologies, greatly improving the possibility of live cell imaging, and has the advantages of being non-toxic and easy to prepare.

[0017] 2. The DNA tetrahedral fluorescent probe provided by the present invention can be actively taken up by cells and stably exist in the cells for more than 48 hours. When the target is not present, there is almost no fluorescence leakage, which greatly increases the fluorescence concentration and improves the resolution.

[0018] 3. The DNA tetrahedral fluorescent probe provided by the present invention has great programmability and can be modified with different types and numbers of fluorescent dye groups on different vertex extension chains; the operation is simple, fast and low-cost.

[0019] 4. The spatial geometric structure of the DNA tetrahedral fluorescent probe provided by the present invention matches the circular SCMH1 RNA and has multiple sites for recognizing the circular SCMH1 RNA, which can enhance the interaction between the probe and the circular SCMH1 RNA and significantly improve the sensing performance and cell imaging effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0021] FIG1 is a three-dimensional structural diagram of a DNA tetrahedron fluorescent probe proposed in the present invention;

[0022] FIG2 is a plan view of the DNA tetrahedron fluorescent probe proposed by the present invention;

[0023] FIG3 is a schematic diagram showing the principle of the method for identifying SCHM1 RNA based on spatial recognition pattern using tetrahedral fluorescent probes according to the present invention;

[0024] FIG4 is an agarose gel electrophoresis run image of the present invention, demonstrating the successful preparation of the probe;

[0025] FIG5 is a fluorescence image of the present invention verifying the tetrahedral fluorescent probe multivalent recognition strategy by detecting circular RNA;

[0026] FIG6 shows the fluorescence intensity of the probe of the present invention for detecting 0-500 nM SCHM1 RNA, demonstrating that the probe has good detection sensitivity;

[0027] FIG7 shows the fluorescence intensity of the probe of the present invention for detecting 0-20 nM SCHM1 RNA, demonstrating that the probe has good detection sensitivity;

[0028] FIG8 is a diagram showing that the tetrahedral fluorescent probe of the present invention has good selectivity by detecting different RNAs;

[0029] FIG9 shows the dynamic real-time monitoring of circular RNA in cultured cells in vitro according to the present invention;

[0030] FIG10 shows the sensitive and rapid in situ imaging of circular RNA in mouse brain slices according to the present invention;

[0031] FIG11 shows the ultra-rapid detection of circular RNA in unamplified RNA of human plasma samples according to the present invention. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0033] Example 1

[0034] Design and preparation of DNA tetrahedral fluorescent probe based on multivalent spatial recognition mode

[0035] 1. Design of DNA tetrahedral fluorescent probe

[0036] The fluorescent probe has a tetrahedral structure and includes 5 single DNA strands, 3 long DNA strands and 3 short DNA strands complementary to the long DNA strands; the 3 long DNA strands include CY5 dye modified at the 3' end, and the 3 short DNA strands include BHQ2 dye modified at the 3' end.

[0037] The length of the five DNA single chains in the main structure of the tetrahedral fluorescent probe is 58 nt; the length of the three DNA long chains is 77 nt; and the length of the three DNA short chains is 12 nt.

[0038] 2. Preparation of Tetrahedron Probe

[0039] (1) Materials and preparation

[0040] PB buffer (10mM PB, 50mM MgCl2, pH 7.0) and other reagents were prepared by our own laboratory. All reagents were prepared with ultrapure water. DNA oligo was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and purified by HPLC. The sequences are shown in SEQID NO.1-11. The nucleotide sequences of the five single-stranded DNAs are shown in SEQIDNO.1-5, the sequence of A37-1 is shown in SEQIDNO.1, the sequence of B37-1 is shown in SEQIDNO.2, the sequence of C37-1 is shown in SEQIDNO.3, the sequence of D37-1 is shown in SEQIDNO.4, and the sequence of D37-2 is shown in SEQIDNO.5; the nucleotide sequences of the three long-chain DNAs are shown in SEQIDNO.6-8, the sequence of A37-2 is shown in SEQIDNO.6, and the sequence of B37-1 is shown in SEQIDNO.2. The sequence of 7-2 is shown as SEQIDNO.7, and the sequence of C37-2 is shown as SEQIDNO.8; the nucleotide sequences of the three short DNA chains are shown as SEQIDNO.9-11, respectively, the sequence of Q1 is shown as SEQIDNO.9, the sequence of Q2 is shown as SEQIDNO.10, and the sequence of Q3 is shown as SEQIDNO.11; wherein the short DNA chain Q1 is complementary to the long DNA chain A37-2, the short DNA chain Q2 is complementary to the long DNA chain B37-2, and the short DNA chain Q3 is complementary to the long DNA chain C37-2.

[0041] The equipment used in this example includes: a refrigerated centrifuge (Eppendorf), a gel developer (Tanon, 1600), a vortex shaker (DLAB), a polyacrylamide gel electrophoresis apparatus (Tanon, EPS 300), a PCR instrument (Eppendorf, Mastercycler nexus GX2), an analytical balance (Sartorius, BSA224S), a shaking table (IKA KS 260 basic), and a fluorescence confocal microscope (OLYMPUS);

[0042] (2) Preparation and characterization of DNA tetrahedral probes

[0043] First, the single-stranded DNA was dissolved, and the eleven strands forming the tetrahedral nanostructures were mixed in equal proportions in PB buffer to a final concentration of 1 μM. The prepared sample was then placed in a PCR instrument and incubated at 95°C for 5 minutes, followed by rapid cooling to 4°C over 1 minute and stabilization at 4°C for 10 minutes to obtain the tetrahedral DNA nanostructures. Agarose gel electrophoresis was used to initially characterize the yield and size of the resulting DNA tetrahedral structures. As shown in Figure 4, the agarose gel electrophoresis results indicate that the DNA tetrahedrons were successfully synthesized, with a yield exceeding 90%.

[0044] Example 2

[0045] Performance test of tetrahedral fluorescent probe

[0046] In this example, the RNA sequences used are the nucleotide sequences shown in SEQ ID NOs. 12-14.

[0047] In this embodiment, the synthesized tetrahedral probe is used to detect circular RNA in vitro, comprising the following steps:

[0048] S1. Synthesize 1 μM tetrahedral probe according to the preparation method of Example 1;

[0049] S2, incubate the circular SCMH1 RNA and tetrahedral probe at 25°C;

[0050] S3. Fluorescence measurement: As shown in Figure 5, the feasibility of the tetrahedral probe multivalent recognition strategy was verified by fluorescence measurement.

[0051] The sensitivity of the probe was verified by varying the concentration of circular RNA. As shown in Figures 6 and 7 , the tetrahedral probe of the present invention has good sensitivity, ranging from 0.01 to 20 nM.

[0052] The selectivity of the tetrahedral probe was verified by mixing it with 50 nM of different RNAs. As shown in Figure 8, the tetrahedral probe of the present invention has good selectivity.

[0053] Example 3

[0054] Cell culture and real-time monitoring of circular RNA dynamics

[0055] (1) The culture steps of HT-22 cells are as follows: freeze the frozen HT-22 cells in a water bath at 37°C for 1 to 2 minutes, centrifuge after complete dissolution, and remove the supernatant; resuspend the cells in fresh DMEM medium containing 10% fetal bovine serum and 1% double antibody, pipette the cells with a sterile pipette and inject them into 10 ml of fresh DMEM medium containing 10% fetal bovine serum and 1% double antibody, and shake clockwise; place the cells in an incubator maintained at 37°C and 5% carbon dioxide; subculture after one to two days of full growth.

[0056] (2) The HT-22 cell culture procedure is as follows: discard the supernatant of the cultured cells, wash the residual liquid with sterile PBS, add 1 ml of trypsin and digest at 37°C for 1 min, add an equal volume of DMEM medium containing 10% fetal bovine serum and 1% double antibody to terminate the digestion, and aspirate the cells into a centrifuge tube; centrifuge at 1000r for 5 min, remove the supernatant; resuspend in DMEM medium containing 10% fetal bovine serum and 1% double antibody, distribute to two culture flasks containing fresh culture medium, shake gently and place in the incubator.

[0057] (3) Dynamic imaging of cells using probes:

[0058] The cells were digested and transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the supernatant removed. The cells were resuspended in DMEM medium containing 10% fetal bovine serum and 1% double-antibody, and then transferred to a culture dish for culture. The culture medium was pre-added with the DNA tetrahedron fluorescent probe prepared in Example 1, gently shaken, and incubated in an incubator at 37°C and 5% carbon dioxide for 3-12 hours. Imaging analysis was performed using a live cell workstation. As shown in Figure 9, the present invention demonstrated excellent live-cell imaging capabilities for circSCMH1 RNA.

[0059] Example 4

[0060] Sensitive and rapid in situ imaging of circular RNA in mouse brain slices

[0061] Preparation of mouse brain frozen sections, specifically the following steps: anesthetize the mouse in a closed anesthesia chamber containing 3% isoflurane, 30% oxygen and 70% carbon dioxide; cut the mouse chest cavity, insert a needle connected to a constant flow pump into the mouse left ventricle, and cut open the right atrial appendage to drain the fluid; first perfuse with 250mL 0.01mol / L PBS, and then fix the brain tissue with 40mL 4% PFA; remove the mouse brain, place it in 4% PFA solution at 4°C overnight; transfer it to 30% sucrose solution and settle at 4°C; after the brain tissue is completely settled, place it in a -20°C freezing microtome for equilibration for 2 hours; embed the brain tissue with OCT embedding solution and equilibrate again for 30 minutes; maintain -20°C for sectioning, and the section thickness is 30μm / slice.

[0062] (2) The DNA tetrahedral fluorescent probe prepared in Example 1 was incubated with frozen cell sections for 12 hours, and then imaged and analyzed using a laser confocal microscope. The analysis results are shown in Figure 10, demonstrating that the present invention has excellent live cell imaging capabilities for circSCMH1 RNA.

[0063] Example 5

[0064] Ultra-rapid detection of circular RNA in unamplified RNA from human plasma samples

[0065] Blood was collected from 30 healthy controls and 30 AIS patients, and plasma was separated and total RNA was extracted. The RNA samples were incubated with the DNA tetrahedron fluorescent probe prepared in Example 1, and the results were measured using a fluorescence detector. The detection results are shown in Figure 11. The data in Figure 11 show that the probe can still quantify the copy number of circSCMH1 in plasma samples without nucleic acid amplification, demonstrating that the present invention has excellent ultra-rapid detection capabilities for circular RNA in unamplified RNA from human plasma samples.

[0066] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0067] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A DNA tetrahedron fluorescent probe based on multivalent spatial pattern recognition, characterized in that: The fluorescent probe has a tetrahedral structure and includes five DNA single chains, three DNA long chains and three DNA short chains complementary to the DNA long chains; three of the DNA long chains include CY5 dye modified at the 3' end, and three of the DNA short chains include BHQ2 dye modified at the 3' end.

2. The DNA tetrahedron fluorescent probe according to claim 1, characterized in that The nucleotide sequences of the five single-stranded DNAs are shown in SEQ ID NOs. 1-5, respectively. The sequence of A37-1 is shown in SEQ ID NO. 1, the sequence of B37-1 is shown in SEQ ID NO. 2, the sequence of C37-1 is shown in SEQ ID NO. 3, the sequence of D37-1 is shown in SEQ ID NO. 4, and the sequence of D37-2 is shown in SEQ ID NO.

5.

3. The DNA tetrahedron fluorescent probe according to claim 1, characterized in that The nucleotide sequences of the three DNA long chains are shown in SEQ ID NOs. 6-8, respectively. The sequence of A37-2 is shown in SEQ ID NO. 6, the sequence of B37-2 is shown in SEQ ID NO. 7, and the sequence of C37-2 is shown in SEQ ID NO.

8.

4. The DNA tetrahedron fluorescent probe according to claim 1, characterized in that The nucleotide sequences of the three DNA short chains are shown in SEQ ID NOs.9-11, respectively, the sequence of Q1 is shown in SEQ ID NO.9, the sequence of Q2 is shown in SEQ ID NO.10, and the sequence of Q3 is shown in SEQ ID NO.11; wherein the DNA short chain Q1 is complementary to the DNA long chain A37-2, the DNA short chain Q2 is complementary to the DNA long chain B37-2, and the DNA short chain Q3 is complementary to the DNA long chain C37-2.

5. The DNA tetrahedral fluorescent probe according to claim 1, characterized in that The length of the five single-stranded DNAs is 58 nt; the length of the three long DNA chains is 77 nt; and the length of the three short DNA chains is 12 nt.

6. Use of a DNA tetrahedron fluorescent probe based on multivalent spatial pattern recognition as described in any one of claims 1 to 5 in circular RNA imaging and non-diagnostic detection.

7. The use according to claim 6, characterized in that The applications specifically include: dynamic real-time monitoring of circular RNA in cultured cells in vitro; sensitive and rapid in situ imaging of circular RNA in mouse brain slices; and ultra-rapid detection of circular RNA in unamplified RNA in human plasma samples.

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