A method for sorting protoplasts using DNA tetrahedral framework nucleic acid specific anchoring

Abstract

The application relates to the field of DNA nanotechnology, and discloses a method for sorting protoplasts by using DNA tetrahedral framework nucleic acid specific anchoring, as follows: cholesterol functionalized DNA tetrahedral framework nucleic acid is synthesized through a PCR reaction; high-activity protoplasts are obtained through enzymolysis; the DNA tetrahedral framework nucleic acid is uniformly mixed with an MMG buffer solution and then added to a protoplast resuspension solution for incubation; and the DNA tetrahedral framework nucleic acid is specifically anchored on the surface of the protoplasts to realize the sorting of the protoplasts. The DNA tetrahedral framework nucleic acid is functionalized with a cholesterol group by using the above method, the DNA tetrahedral framework nucleic acid is specifically anchored in protoplasts with stronger division ability, and the sorting of the protoplasts is successfully realized; the method is simple in operation, remarkable in effect, and low in cost, and successfully breaks through the lack of a sorting method in the current protoplast research, thereby providing a new direction and thought for protoplast research.

Description

Technical Field

[0001] This invention relates to the field of DNA nanotechnology, and in particular to a method for specific anchoring and sorting of protoplasts using a DNA tetrahedral framework for nucleic acids. Background Technology

[0002] The DNA tetrahedron is a classic representative structure of engineered nucleic acids. Its simple, robust, rigid, hollow three-dimensional model exhibits good and stable mechanical properties, as well as excellent characteristics such as low immunogenicity and nuclease stability. The DNA tetrahedron has four tunable sites, and modifying it with different groups, either individually or in combination, can create biomaterials with multiple functionalities. Currently, the DNA tetrahedron is widely used in animal cell membrane research. A key research strategy involves precisely modifying DNA nanomaterials to enable specific recognition and chemical reactions with functional groups on the cell membrane surface.

[0003] Currently, the main method for protoplast sorting involves staining protoplasts with fluorescein diacetate (FDA) to distinguish between viable and non-viable protoplasts, followed by flow cytometry to obtain highly active cell populations. However, a sorting mechanism for protoplasts with varying meristematic capacity has not yet been developed. Meristematic capacity is a crucial factor determining whether protoplasts can further differentiate and divide, and it is key to protoplast regeneration and plant reconstruction. It allows protoplasts to be reprogrammed into stem cells after cell wall removal and to form new plant tissues through division and differentiation, making protoplasts invaluable in plant genetic improvement and cell engineering. Therefore, developing a method for sorting protoplasts with varying meristematic capacity is one of the key focuses and challenges in protoplast research. Summary of the Invention

[0004] The purpose of this invention is to provide a method for sorting protoplasts by specifically anchoring DNA tetrahedral framework nucleic acids. This method involves functionalizing DNA tetrahedral framework nucleic acids with cholesterol groups, and then using these nucleic acids to specifically anchor them onto protoplasts with strong meristematic capacity, successfully achieving protoplast sorting. The method is simple to operate, highly effective, and low in cost. It also successfully overcomes the current lack of sorting methods in protoplast research, providing a new direction and approach for protoplast studies.

[0005] To achieve the above objectives, the present invention provides a method for nucleic acid-specific anchoring and sorting of protoplasts using a DNA tetrahedral framework, comprising the following steps:

[0006] Step 1: Dissolve the DNA powder in enzyme-free water and dilute it to a 50 μM single-stranded DNA solution.

[0007] Step 2: Take the single-stranded DNA solution required for synthesizing the DNA tetrahedral framework nucleic acid and mix it thoroughly with 10×TM buffer.

[0008] Step 3: Place the mixed solution obtained in Step 2 into a PCR instrument and synthesize cholesterol-functionalized DNA tetrahedral framework nucleic acid according to the program of 95℃ for 5 min; 4℃ for 30 min.

[0009] Step 4: Prepare the enzymatic hydrolysate for extracting protoplasts. Place the plant tissue in the enzymatic hydrolysate for digestion in the dark. After enzymatic hydrolysis, centrifuge to obtain highly active protoplasts.

[0010] Step 5: Resuspend the protoplast in MMG buffer;

[0011] Step 6: Mix the DNA tetrahedral framework nucleic acid from Step 3 with MMG buffer, then add it to the protoplast resuspension solution and incubate at room temperature in the dark for 2-6 hours. The DNA tetrahedral framework nucleic acid will be specifically anchored on the surface of the protoplast, achieving high meristematic sorting of the protoplast.

[0012] Furthermore, the DNA dry powder described in step 1 includes three specific sequences modified with cholesterol molecules at the 5' end and one specific sequence modified with a Cy3 fluorescent group at the 3' end.

[0013] Furthermore, the nucleotide sequences of the three specific sequences and the one specific sequence are shown in SEQ ID NO.1-4.

[0014] It should be understood that the method of the present invention is applicable to DNA strands of any length and sequence capable of synthesizing DNA tetrahedral framework nucleic acids, and the number of cholesterol modifications can be adjusted. As an example and not a limitation, preferably, the sequence length is 46-63 nt.

[0015] Furthermore, in step 2, the size of the DNA tetrahedral framework nucleic acid is 2.4nm-12.6nm, the sequence length is 7bp-37bp, and the concentration of the DNA tetrahedral framework nucleic acid is 1-5μM. If the concentration is too high, too many aggregates will be generated, and the synthesis efficiency will decrease.

[0016] Further, in step 2, Mg in the 10×TM buffer 2+ The concentration is 50mM; too high a concentration can cause cholesterol buildup.

[0017] Furthermore, in step 4, the plant tissue is tobacco leaf, apple callus, or cauliflower meristem; the enzyme hydrolysate for tobacco leaf is prepared with a ratio of 12.5 g / L cellulase and 3 g / L pectinase; the enzyme hydrolysate for apple callus is prepared with a ratio of 20 g / L cellulase, 5 g / L pectinase, and 3 g / L ionase; and the enzyme hydrolysate for cauliflower meristem is prepared with a ratio of 20 g / L cellulase, 1 g / L pectinase, and 5 g / L ionase.

[0018] Furthermore, the enzymatic hydrolysis time for tobacco leaves was 2.5 hours, for apple callus tissue it was 1 hour, and for cauliflower meristem tissue it was 8 hours.

[0019] Furthermore, in step 4, the centrifugal force is ≤100g.

[0020] Furthermore, in step 6, the concentration of the DNA tetrahedral framework nucleic acid is 500 nM, and the protoplast density is 10n. 5 -10 6 The incubation rate is 1 / mL, and the incubation time is preferably 4 hours.

[0021] To achieve the above objectives, the present invention provides the application of the above method in protoplast sorting.

[0022] The advantages and positive effects of the method for nucleic acid-specific anchoring and sorting of protoplasts using the DNA tetrahedral framework described in this invention are as follows:

[0023] 1. This invention achieves successful sorting of protoplasts by functionalizing the DNA tetrahedral framework nucleic acid with cholesterol groups and utilizing the specific anchoring of the DNA tetrahedral framework nucleic acid to protoplasts with strong meristematic ability. The operation is simple, the effect is significant, the cost is low, and it successfully overcomes the lack of sorting methods in current protoplast research, providing a new direction and idea for protoplast research.

[0024] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the method for rapidly sorting protoplasts with high meristematic capacity using DNA tetrahedra in this invention;

[0026] Figure 2 The above are the characterization results of cholesterol-functionalized DNA tetrahedra using 10% non-denaturing polyacrylamide gel electrophoresis in Example 1 of this invention.

[0027] Figure 3 The above describes the characterization of protoplasts using an optical microscope in Example 2 of this invention, where A is a tobacco leaf and its protoplasts, B is an apple callus and its protoplasts, and C is a cauliflower meristem and its protoplasts.

[0028] Figure 4 This is the characterization of DNA tetrahedral anchored protoplasts using laser confocal microscopy in Example 3 of the present invention. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0030] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0031] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0032] The DNA sequences used in this invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequences used in this embodiment are shown in Table 1 below:

[0033] Table 1 ssDNA Sequence

[0034]

[0035] In Table 1, the bases marked in bold and underlined are complementary.

[0036] A method for nucleic acid-specific sorting of protoplasts using a DNA tetrahedral framework includes the following steps (e.g.) Figure 1 As shown):

[0037] Step 1: Dissolve the provided DNA powder in enzyme-free water and dilute it into a 50 μM single-stranded DNA (ssDNA) solution. The DNA powder includes three specific sequences designed and modified with cholesterol molecules at the 5' end, and one specific sequence designed and modified with a Cy3 fluorescent group at the 3' end, as shown in SEQ ID NO.1-4.

[0038] It should be understood that the method of the present invention is applicable to DNA strands of any length and any sequence capable of synthesizing DNA tetrahedral framework nucleic acids, and the number of cholesterol modifications can be adjusted. By way of example and not limitation, preferably, the sequence length is 46-63 nt;

[0039] Step 2: Take the above-mentioned ssDNA solution required for synthesizing DNA tetrahedral framework nucleic acid and mix it evenly with 10×TM buffer.

[0040] The optimal concentration for synthesizing DNA tetrahedral framework nucleic acids is 1-5 μM. Excessive concentration will produce too many aggregates, which will reduce the synthesis efficiency.

[0041] Among them, the 10×TM buffer used contains Mg 2+ The concentration is 50mM; too high a concentration can cause cholesterol to accumulate.

[0042] Step 3: Place the obtained mixed solution into a PCR instrument and synthesize cholesterol-functionalized DNA tetrahedral framework nucleic acid according to the program of 95℃ for 5 min; 4℃ for 30 min.

[0043] Step 4: Prepare an enzymatic hydrolysate for protoplast extraction. Place the plant tissue in the enzymatic hydrolysate for digestion in the dark to obtain highly active protoplasts.

[0044] The concentration and enzyme composition ratio of the enzymatic hydrolysate extracted from protoplasts of different species and tissues need to be adjusted appropriately. For example, the enzymatic hydrolysate ratio for tobacco leaves is 12.5 g / L cellulase and 3 g / L pectinase; the ratio for apple callus tissue is 20 g / L cellulase, 5 g / L pectinase, and 3 g / L isocyanate; and the ratio for cauliflower meristem tissue is 20 g / L cellulase, 1 g / L pectinase, and 5 g / L isocyanate.

[0045] The protoplast enzymatic hydrolysis time needs to be adjusted appropriately according to the plant tissue conditions. Too short a time will result in a small number of protoplasts, while too long a time will result in low protoplast quality.

[0046] In the protoplast processing steps, such as centrifugation, the centrifugal force is no greater than 100g.

[0047] Step 5: Resuspend the protoplast in MMG buffer;

[0048] Step 6: Mix the previously synthesized DNA tetrahedral framework nucleic acid with MMG and add it to the protoplast resuspension. Incubate at room temperature in the dark for 2-6 hours. The DNA tetrahedral framework nucleic acid can then be specifically anchored to the surface of protoplasts with strong meristematic ability, thus achieving high meristematic sorting of protoplasts.

[0049] The ratio of DNA tetrahedral framework nucleic acid to protoplasts can be adjusted. Preferably, the concentration of DNA tetrahedral framework nucleic acid is 500 nM, and the protoplast density is 10n. 5 -10 6 cells / mL;

[0050] Preferably, the incubation time should not be too long (2-6h), as too long may cause the protoplasts to rupture; 4h is preferred.

[0051] This invention utilizes cholesterol-functionalized DNA tetrahedral framework nucleic acids to specifically anchor onto the surface of some protoplasts, which are then distinguished by fluorescence. Meristematic tissues are a type of cell with highly proliferative capacity, commonly distributed at the apical region of plants, including shoot apical meristems and root apical meristems. In this state, the cell wall is newly formed, lacking intercellular spaces and tightly packed. Therefore, the cell membrane is also in its early stages of formation and not yet fully developed, requiring further differentiation and division. The lipids on the membrane are not tightly packed, resulting in high membrane fluidity. Consequently, cholesterol, due to hydrophobic interactions, readily inserts into the intercellular spaces, leading to anchoring. Furthermore, trace amounts of cholesterol are present on the plasma membrane in the early stages of plant cell growth, contributing to subsequent cell growth and being internalized into the endoplasmic reticulum as the cells develop. Therefore, this is also one of the important factors helping cholesterol anchor to the protoplast.

[0052] The following examples provide a detailed description.

[0053] Example 1: Characterization of cholesterol-functionalized DNA tetrahedral framework nucleic acids using 10% non-denaturing polyacrylamide gel electrophoresis.

[0054] A 10% polyacrylamide gel was prepared and electrophoresed for 100 minutes at 80 V using a Bio-Rad electrophoresis apparatus in 1×TAEM buffer (40 mM Tris-acetate, 1 mM EDTA, 12.5 mM Magnesium acetate, pH 8.0–8.6). After electrophoresis, the gel was stained with 1×Gel Red nucleic acid dye for 10–15 minutes. The gel was imaged using a Tanon 2500B gel imaging system to characterize the formation of DNA nanostructures.

[0055] The results are as follows Figure 2 As shown, lane 1 is the marker band, indicating the molecular weight position from 25 to 500 bp. Lanes 2-4 indicate the positions of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and trimer DNA (triDNA), respectively. As the number of synthesized tetrahedrons increases, the complementary sites increase, and the bands shift significantly upward. Lane 4 indicates the correct position of the DNA tetrahedral framework nucleic acid.

[0056] Example 2: Characterization of protoplasts using an optical microscope

[0057] The freshly extracted protoplasts were resuspended in MMG buffer. A certain volume of the protoplast suspension was dropped onto a glass plate and observed under an optical microscope.

[0058] The results are as follows Figure 3 As shown, Figure 3A represents tobacco leaves and their protoplasts. It can be seen that the protoplasts extracted from the leaves are rich in chloroplasts, with a size of about 50 μm. Figure 3 B is apple callus and its protoplast. It can be found that it does not contain chloroplasts, is transparent, contains a lot of vacuoles, and its overall morphology is consistent with that of plant stem cells, with a size of 20-25μm. Figure 3 C represents the inflorescence of the cauliflower and its protoplast. Figure 3 The protoplasts in the mesoporous tissue were all round and plump, indicating that the extraction state and activity were both good.

[0059] Example 3: Characterization of DNA tetrahedral framework nucleic acid anchored protoplasts using laser confocal microscopy

[0060] FDA live cell dye prepared with MMG buffer was added 10 minutes before the end of incubation, and the mixture was incubated for the required time. The incubated DNA tetrahedral framework nucleic acid and protoplast mixture was placed in a confocal microarray and incubated for 3-5 minutes, followed by characterization using CLSM to observe the effect. Dual-channel scanning was used, with excitation wavelengths set at 488 nm and 561 nm, corresponding to emission spectra collected at 500-550 nm and 570-620 nm. The fluorescence of the FDA live cell dye was excited using a 488 nm laser; the tFNA material modified with the Cy3 fluorophore was excited using a 561 nm laser.

[0061] The results are as follows Figure 4 As shown, somatic cells are protoplasts extracted from tobacco leaves, and DNA tetrahedral framework nucleic acids do not interact with them. However, for protoplasts derived from callus tissue, a large amount of DNA tetrahedral framework nucleic acid fluorescence signal can be detected on the plasma membrane surface. This indicates that DNA tetrahedral framework nucleic acids can specifically anchor to protoplasts with strong meristematic ability. By detecting and screening Cy3 signals, the protoplasts can be successfully sorted.

[0062] Therefore, this invention employs the aforementioned method of sorting protoplasts by specifically anchoring DNA tetrahedral framework nucleic acids. This method functionalizes DNA tetrahedral framework nucleic acids with cholesterol groups, allowing them to specifically anchor onto protoplasts with strong meristematic capacity, thus successfully achieving protoplast sorting. The method is simple to operate, highly effective, and low in cost. Furthermore, it successfully overcomes the current lack of sorting methods in protoplast research, providing a new direction and approach for protoplast studies.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. 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 still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for sorting protoplasts using a DNA tetrahedral framework with specific nucleic acid anchoring, characterized in that, Includes the following steps: Step 1: Dissolve the DNA powder in enzyme-free water and dilute it to a 50 μM single-stranded DNA solution. The DNA dry powder comprises three specific sequences modified with cholesterol molecules at the 5' end and one specific sequence modified with a Cy3 fluorescent group at the 3' end; the nucleotide sequences of the three specific sequences and the one specific sequence are shown in SEQ ID NO.1-4; Step 2: Take the single-stranded DNA solution required for synthesizing the DNA tetrahedral framework nucleic acid and mix it thoroughly with 10×TM buffer. Step 3: Place the mixed solution obtained in Step 2 into a PCR instrument and synthesize cholesterol-functionalized DNA tetrahedral framework nucleic acid according to the program of 95℃ for 5 min; 4℃ for 30 min. Step 4: Prepare an enzymatic hydrolysate for protoplast extraction. Place the plant tissue in the enzymatic hydrolysate for digestion in the dark. After enzymatic hydrolysis, centrifuge to obtain highly active protoplasts. The highly active protoplasts are derived from tobacco leaves, apple callus tissue, and cauliflower meristem. Step 5: Resuspend the protoplast in MMG buffer; Step 6: Mix the DNA tetrahedral framework nucleic acid from Step 3 with MMG buffer and add it to the protoplast resuspension. Incubate at room temperature in the dark for 2-6 hours. The DNA tetrahedral framework nucleic acid will be specifically anchored on the surface of the protoplast, achieving high meristematic sorting of the protoplast. The concentration of DNA tetrahedral framework nucleic acid was 500 nM, and the protoplast density was 10n. 5 -10 6 per mL.

2. The method according to claim 1, characterized in that: In step 2, Mg in 10×TM buffer 2+ The concentration is 50 mM.

3. The method according to claim 1, characterized in that: In step 4, the enzyme hydrolysate for tobacco leaves is prepared with a ratio of 12.5 g / L cellulase and 3 g / L pectinase; the enzyme hydrolysate for apple callus is prepared with a ratio of 20 g / L cellulase, 5 g / L pectinase, and 3 g / L ionase; and the enzyme hydrolysate for cauliflower meristem is prepared with a ratio of 20 g / L cellulase, 1 g / L pectinase, and 5 g / L ionase.

4. The method according to claim 3, characterized in that: The enzymatic hydrolysis time for tobacco leaves was 2.5 hours, for apple callus tissue it was 1 hour, and for cauliflower meristem tissue it was 8 hours.

5. The method according to claim 1, characterized in that: In step 4, the centrifugal force is ≤100g.

6. The application of the method according to any one of claims 1-5 in protoplast sorting, characterized in that: Protoplasts originate from tobacco leaves, apple callus, and cauliflower meristem.

Images