A nucleic acid composition for mRNA delivery and expression and its applications

Abstract

The present invention discloses a nucleic acid composition for mRNA delivery and expression and its application, which relates to the field of biomedical technology. The nucleic acid composition includes mRNA, nucleic acid composition I and nucleic acid composition II; the nucleic acid composition I and the nucleic acid composition II are complementary to and bind with the mRNA. The present invention combines the mRNA with the nucleic acid composition I and the nucleic acid composition II in a complementary manner to form a nucleic acid composition with uniform physical characteristics. In the nucleic acid composition, the mRNA is connected by the nucleic acid composition I and the nucleic acid composition II in a preset manner, and a large number of mRNA active regions are still exposed in the connected nucleic acid composition, thus facilitating the efficient delivery and expression of mRNA in cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a nucleic acid composition for mRNA delivery and expression and its applications. Background Art

[0002] In recent years, mRNA has emerged as a promising strategy for treating various diseases as an alternative to protein-based drugs. However, native mRNA faces numerous obstacles such as electrostatic repulsion and nuclease degradation when entering cells without carrier assistance. Solving these problems is the core solution to promoting the application of mRNA. Nanopreparations can interact with cells using their unique delivery methods for drugs, proteins, nucleic acid molecules, etc., and achieve controlled release, which is also the main way to solve the problems of intracellular delivery efficiency and stability of mRNA. Specifically, common solutions include encapsulating mRNA into nanosized carriers (such as lipo8000, LNP, etc.), loading mRNA onto nanomaterials, and nanosizing mRNA with metal ions, etc.

[0003] The above mRNA delivery methods usually require auxiliary substances far greater in mass than the mRNA itself for construction, and there are also problems such as relatively complex operations and difficulty in ensuring uniformity. Here, the present invention hopes to develop a simplified operation strategy to achieve efficient delivery and expression of mRNA to cultured cells and living cells. Summary of the Invention

[0004] The object of the present invention is to provide a nucleic acid composition for mRNA delivery and expression and its applications to solve the problems existing in the above prior art. This nucleic acid composition can achieve efficient delivery and expression of mRNA in cells.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a nucleic acid composition for mRNA delivery and expression, which comprises mRNA, nucleic acid composition I, and nucleic acid composition II; nucleic acid composition I and nucleic acid composition II are complementary to and bind with the mRNA;

[0007] The mRNA is divided into N mRNA结合区 mRNA binding regions and N mRNA结合区 -1 mRNA active regions; the mRNA binding regions and the mRNA active regions are alternately distributed in sequence; the N mRNA结合区 mRNA binding regions are sequentially numbered as binding region 1 to binding region N mRNA结合区 ; the length L mRNA结合区 of the mRNA binding region is 5 - 20 nt; the length L mRNA活性区 of the mRNA active region is 10 - 150 nt;

[0008] The length L of the mRNA mRNA = L mRNA结合区 × N mRNA结合区 + L mRNA活性区 × (N mRNA结合区 - 1);

[0009] The nucleic acid sequence I is divided into N 核酸组合物I结合区 nucleic acid composition I binding regions and N 核酸组合物I间隔区 nucleic acid composition I spacer regions; the nucleic acid composition I binding regions and the nucleic acid composition I spacer regions are alternately distributed in sequence; they are sequentially numbered as nucleic acid composition I binding region 1 to nucleic acid composition I binding region N 核酸组合物I结合区 ;

[0010] The length of the nucleic acid composition I spacer region is 1 - 10 nt;

[0011] N 核酸组合物I结合区 = N 核酸组合物I间隔区 ;

[0012] The nucleic acid sequence II is divided into N 核酸组合物II结合区 nucleic acid composition II binding regions and N 核酸组合物II间隔区 nucleic acid composition II spacer regions; the nucleic acid composition II binding regions and the nucleic acid composition II spacer regions are alternately distributed in sequence; the N 核酸组合物II结合区 nucleic acid composition II binding regions are sequentially numbered as nucleic acid composition II binding region 1 to nucleic acid composition II binding region N 核酸组合物II结合区 ;

[0013] The length of the nucleic acid composition II spacer region is 1 - 10 nt;

[0014] N 核酸组合物II结合区 = N 核酸组合物II间隔区 ;

[0015] N 核酸组合物I结合区 + N 核酸组合物II结合区 = N mRNA结合区 ;

[0016] The nucleic acid composition I binding region 1 to nucleic acid composition I binding region N 核酸组合物I结合区 are sequentially complementary to the mRNA binding regions with odd numbers; the nucleic acid composition II binding region 1 to nucleic acid composition II binding region N 核酸组合物II结合区 are sequentially complementary to the mRNA binding regions with even numbers.

[0017] Furthermore, both the nucleic acid composition I and the nucleic acid composition II are modified with a cell-penetrating peptide.

[0018] Furthermore, the cell-penetrating peptide is TAT.

[0019] Furthermore, the nucleic acid sequence I is circular DNA, circular RNA, single-stranded DNA or single-stranded RNA; the nucleic acid sequence II is circular DNA, circular RNA, single-stranded DNA or single-stranded RNA.

[0020] Furthermore, the nucleotide sequence of the spacer region of the nucleic acid composition I is CCCCCC; the nucleotide sequence of the spacer region of the nucleic acid composition II is CCCCCC.

[0021] The present invention also provides a method for improving the delivery efficiency of mRNA, including the step of forming a nucleic acid composition by complementary binding of the nucleic acid composition I and the nucleic acid composition II with the mRNA to improve the delivery efficiency of the mRNA;

[0022] The mRNA is divided into N mRNA结合区 mRNA binding regions and N mRNA结合区 -1 mRNA active regions; the mRNA binding regions and the mRNA active regions are alternately distributed in sequence; the N mRNA结合区 mRNA binding regions are sequentially numbered as binding region 1 to binding region N mRNA结合区 ; the length L mRNA结合区 of the mRNA binding region is 5-20 nt; the length L mRNA活性区 of the mRNA active region is 10-150 nt;

[0023] The length L mRNA of the mRNA = L mRNA结合区 ×N mRNA结合区 +L mRNA活性区 ×(N mRNA结合区 -1);

[0024] The nucleic acid sequence I is divided into N 核酸组合物I结合区 nucleic acid composition I binding regions and N 核酸组合物I间隔区 nucleic acid composition I spacer regions; the nucleic acid composition I binding regions and the nucleic acid composition I spacer regions are alternately distributed in sequence; sequentially numbered as nucleic acid composition I binding region 1 to nucleic acid composition I binding region N 核酸组合物I结合区 ;

[0025] The length of the nucleic acid composition I spacer region is 1-10 nt;

[0026] N 核酸组合物I结合区 = N 核酸组合物I间隔区 ;

[0027] The nucleic acid sequence II is divided into N 核酸组合物II结合区 nucleic acid composition II binding regions and N 核酸组合物II间隔区 nucleic acid composition II spacer regions; the nucleic acid composition II binding regions and the nucleic acid composition II spacer regions are alternately distributed in sequence; the N 核酸组合物II结合区The nucleic acid composition II binding regions are sequentially numbered as nucleic acid composition II binding region 1 to nucleic acid composition II binding region N 核酸组合物II结合区 ;

[0028] The length of the spacer region of the nucleic acid composition II is 1 - 10 nt;

[0029] N 核酸组合物II结合区 = N 核酸组合物II间隔区 ;

[0030] N 核酸组合物I结合区 + N 核酸组合物II结合区 = N mRNA结合区 ;

[0031] The nucleic acid composition I binding regions 1 to nucleic acid composition I binding region N 核酸组合物I结合区 are sequentially complementary to the mRNA binding regions with odd numbers; the nucleic acid composition II binding regions 1 to nucleic acid composition II binding region N 核酸组合物II结合区 are sequentially complementary to the mRNA binding regions with even numbers.

[0032] Furthermore, both the nucleic acid composition I and the nucleic acid composition II are modified with a cell-penetrating peptide.

[0033] Furthermore, the method for modifying the nucleic acid composition I with a cell-penetrating peptide includes: subjecting the nucleic acid sequence I to azide modification to obtain an azide-functionalized nucleic acid composition I, and then reacting it with a DBCO-modified cell-penetrating peptide to obtain a cell-penetrating peptide-modified nucleic acid composition I;

[0034] The method for modifying the nucleic acid composition II with a cell-penetrating peptide includes: subjecting the nucleic acid sequence II to azide modification to obtain an azide-functionalized nucleic acid composition II, and then reacting it with a DBCO-modified cell-penetrating peptide to obtain a cell-penetrating peptide-modified nucleic acid composition II.

[0035] Furthermore, the nucleotide sequence of the spacer region of the nucleic acid composition I is CCCCC; the nucleotide sequence of the spacer region of the nucleic acid composition II is CCCCC.

[0036] The present invention also provides the use of the above nucleic acid composition for mRNA delivery and expression in the preparation of mRNA drugs.

[0037] The present invention discloses the following technical effects:

[0038] The present invention provides a nucleic acid composition for mRNA delivery and expression. Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention combines mRNA with nucleic acid composition I and nucleic acid composition II in a complementary manner to form a nucleic acid composition with uniform physical characteristics. Among them, nucleic acid composition I and nucleic acid composition II contain multiple mRNA binding regions, which can accurately pair and bind with the corresponding regions on mRNA; there are spacer regions between the binding regions to reduce the interference between the binding regions. After co-incubating mRNA with nucleic acid composition I and nucleic acid composition II, they can form a nucleic acid composition with nanoscale size. In the nucleic acid composition, mRNA is connected by nucleic acid composition I and nucleic acid composition II in a preset manner, and a large number of mRNA active regions are still exposed in the connected nucleic acid composition, which is beneficial to the efficient delivery and expression of mRNA in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0040] Figure 1 is a schematic structural diagram of the nucleic acid composition;

[0041] Figure 2 is an atomic force microscope characterization diagram of the nucleic acid composition;

[0042] Figure 3 is the detection result of the delivery and expression of the nucleic acid composition into cultured cells;

[0043] Figure 4 is the detection result of the delivery and expression of the nucleic acid composition into living cells;

[0044] Figure 5 is the detection result of the delivery and expression of the nucleic acid composition without penetratin modification into cultured cells and living cells. DETAILED DESCRIPTION OF THE INVENTION

[0045] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0046] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0048] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0049] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0050] The present invention provides a nucleic acid composition for intracellular delivery and expression of mRNA. Using mRNA as the core backbone, the nucleic acid composition I and nucleic acid composition II are used to connect mRNA to form a nucleic acid composition of nanoscale size. Specifically, the nucleic acid composition I and nucleic acid composition II contain multiple mRNA binding regions, which can accurately pair and bind with the corresponding regions on mRNA; the mRNA in the nucleic acid composition is connected by the nucleic acid composition I and nucleic acid composition II in a preset manner, and a large number of mRNA active regions are still exposed in the connected nucleic acid composition, so as to facilitate the intracellular delivery of the nucleic acid composition and the expression of mRNA in cells ( Figure 1 ). Among them, except for the 5'-end capping region and 3'-end polyA tail region of mRNA, the middle is all functional regions (i.e., exons), which can be further translated into proteins in cells later. The functional regions of mRNA are divided into binding regions and active regions according to the binding characteristics of the nucleic acid composition. Among them, the binding regions bind to the corresponding nucleic acid composition I and nucleic acid composition II. The present invention limits the length of the binding region (L mRNA结合区 ) and the length of the active region (L mRNA活性区 ), so, the number of binding regions (NmRNA结合区 ) and the number of active regions (N mRNA活性区 ) will vary with the length of the mRNA (L mRNA , excluding the 5'-capped region and the 3'-polyA tail region). Nucleic acid composition I and nucleic acid composition II also include two characteristic regions: a binding region and a spacer region. The present invention limits the length of the binding region (L 结合区 ) and the length of the spacer region (L 间隔区 ), so the number of binding regions (N 核酸组合物I结合区 , N 核酸组合物II结合区 ) and the number of spacer regions (N 核酸组合物I间隔区 , N 核酸组合物II间隔区 ) will also change with the mRNA length. Based on the fact that the nucleic acid composition is a symmetric model, its design rules are as follows:

[0051] i. L mRNA结合区 × N mRNA结合区 + L mRNA活性区 × N mRNA活性区 = L mRNA ;

[0052] ii. N mRNA结合区 = N mRNA活性区 + 1;

[0053] iii. N 核酸组合物I结合区 + N 核酸组合物II结合区 = N mRNA结合区 ;

[0054] iv. N 核酸组合物I间隔区 = N 核酸组合物I结合区 ;

[0055] v. N 核酸组合物II间隔区 = N 核酸组合物II结合区 ;

[0056] vi. N = int[N];

[0057] Wherein, N represents the number; L represents the length; int[] is the floor function.

[0058] The following takes the tool mRNA - mcherry mRNA (which is translated into red fluorescent protein with a length of 861 nt) as an example to illustrate the method of the present invention.

[0059] Example 1 Construction and Characterization of Nucleic Acid Composition

[0060] The 5' end of mcherry mRNA is capped with Cap1AG, contains N1-me-pseudoU modified bases in the middle (to reduce immunogenicity), and has a polyA tail at the 3' end. The length of the active region is set to 90 nt, the length of the binding region is set to 17 nt, and the number of binding regions is calculated to be 5. By modifying and conjugating the nucleic acid composition I and nucleic acid composition II with the cell-penetrating peptide (TAT), and then binding them to mRNA, the feasibility of constructing the nucleic acid composition was observed. It includes the following steps:

[0061] Step a: Modify the nucleic acid sequence I or II with the cell-penetrating peptide TAT. Use click chemistry reaction to conjugate the cell-penetrating peptide with the nucleic acid sequence I or II. The specific operation is as follows: In a buffer with a total volume of 100 μL, the nucleic acid sequence I (SEQ ID NO.1 in Table 1) and the nucleic acid sequence II (SEQ ID NO.2 in Table 1) are respectively combined with NHS-PEG 4 -N 3 crosslinker at a molar ratio of 30 μM: 1.5 mM and magnetically stirred at room temperature for 2 h (1000 rpm). The azide-modified nucleic acid composition I and azide-modified nucleic acid composition II are obtained by ultrafiltration centrifugation purification; the azide-modified nucleic acid composition I and azide-modified nucleic acid composition II are respectively incubated with the DBCO-modified cell-penetrating peptide (TAT) at a molar ratio of 10 μM: 100 μM at 4 °C for 1 day while magnetically stirring (1000 rpm), and then ultrafiltration centrifugation is carried out to obtain the cell-penetrating peptide-modified nucleic acid composition I and cell-penetrating peptide-modified nucleic acid composition II.

[0062] Table 1 Nucleic acid sequences

[0063]

[0064]

[0065] Note: Both the nucleic acid sequence I and nucleic acid sequence II are circular RNAs connected head to tail; the underlined part is the spacer region.

[0066] Step b: Mix mcherry mRNA, the cell-penetrating peptide-modified nucleic acid composition I, and the cell-penetrating peptide-modified nucleic acid composition II at a molar ratio of 1:1:1 in 1×TAE / Mg 2+ buffer, react at 65 °C for 10 min, and cool to room temperature within 2 h to obtain the nucleic acid composition containing mRNA. The morphology was characterized by atomic force microscopy, and the results are as Figure 2 shown. Figure 2 The morphology characterization results shown in the control group in [reference] are the results of mixing the nucleic acid sequence I and nucleic acid sequence II at a molar ratio of 1:1.

[0067] From Figure 2It can be seen that the nucleic acid composition I / II modified with cell-penetrating peptides shows a uniform dot-like appearance under atomic force microscopy. At the same time, after the reaction of the two, the nucleic acid composition containing mRNA shows a nano-scale morphological feature, indicating the successful construction of the nucleic acid composition, which is beneficial for subsequent verification at the cellular level.

[0068] Example 2 Characterization of the Delivery Effect of Nucleic Acid Composition Assisting mRNA to Cultured Cells in Vitro and Living Cells

[0069] In vitro cell experiment:

[0070] Seed 1×10 4 HeLa cells in a confocal dish and incubate at 37 °C for 24 h. Set three groups: blank control, mRNA (mcherry mRNA), and the nucleic acid composition containing mRNA (prepared in Example 1). The mRNA and nucleic acid composition containing mRNA groups co-incubate mcherry mRNA or the nucleic acid composition containing mRNA with the cells under the same conditions (mRNA concentration, incubation time, and buffer). After 2 h, add Hoechst to stain the living cell nuclei for cell localization. The blank control group does not add mcherry mRNA or the nucleic acid composition containing mRNA, directly incubate the cells, and after 2 h, add Hoechst to stain the living cell nuclei for cell localization. Take confocal images of the above groups of cells. Use a filter (ex: 587 nm, em: 610 nm) to image mcherry mRNA and a filter (ex: 350 nm, em: 460 nm) to image the Hoechst dye, so as to obtain the intracellular fluorescence intensity and conduct comparative analysis. The results are as Figure 3 shown.

[0071] Tissue (living cell) experiment:

[0072] Inject mRNA or the nucleic acid composition into the subcutaneous tumor of nude mice (MCF-7 breast cancer). Similarly, set three groups: blank control, mRNA (mcherry mRNA), and the nucleic acid composition containing mRNA (prepared in Example 1). The mRNA and nucleic acid composition containing mRNA groups inject mcherry mRNA or the nucleic acid composition containing mRNA under the same conditions (mRNA concentration, incubation time, buffer), and then take out the tumor tissue to make tissue sections. The blank control is to inject an equal amount of PBS. Take confocal images of the above groups of tumor sections. Use a filter (ex: 587 nm, em: 610 nm) to image mcherry mRNA and a filter (ex: 350 nm, em: 460 nm) to image the DAPI dye, so as to obtain the intracellular fluorescence intensity and conduct comparative analysis. The results are as Figure 4 shown.

[0073] From Figure 3 it can be seen that when mRNA is incubated with cells alone, it is almost impossible to enter the cells and the fluorescence is weak; while when the nucleic acid composition is formed, due to the special nature of its nanostructure, it can be delivered into the cells and present fluorescence. From Figure 4 it can be seen that under the same conditions, the mcherry mRNA forming the nucleic acid composition can still be expressed in tissues (living cells) and present fluorescence, which means that the nucleic acid composition can effectively deliver mRNA into living cells and achieve intracellular expression.

[0074] Example 3

[0075] Method for constructing nucleic acid composition:

[0076] Mix mcherry mRNA, nucleic acid sequence I (SEQ ID NO.1) and nucleic acid sequence II (SEQ ID NO.2) in a molar ratio of 1:1:1 in 1×TAE / Mg 2+ buffer, react at 65°C for 10 min, and cool to room temperature within 2 h to obtain a nucleic acid composition containing mRNA.

[0077] Characterization of the delivery effect of the nucleic acid composition without penetratin modification on mRNA to in vitro cultured cells and living cells

[0078] In vitro cell experiment:

[0079] Seed 1×10 4 HeLa cells in a confocal dish and incubate at 37°C for 24 h. Set two groups: blank control and nucleic acid composition group containing mRNA. The nucleic acid composition group containing mRNA is co-incubated with the cells under the same conditions as in Example 2 with the nucleic acid composition containing mRNA prepared in Example 3. After 2 h, add Hoechst to stain the living cell nuclei for cell localization. The blank control group is to incubate the cells directly without adding the nucleic acid composition containing mRNA, and add Hoechst to stain the living cell nuclei for cell localization after 2 h. Image the above groups of cells by confocal microscopy, image mcherry mRNA using a filter (ex: 587 nm, em: 610 nm), and image the Hoechst dye using a filter (ex: 350 nm, em: 460 nm), so as to obtain the intracellular fluorescence intensity and conduct comparative analysis. The results are as Figure 5 shown.

[0080] Tissue (living cell) experiment:

[0081] Inject the nucleic acid composition into subcutaneous tumors (MCF-7 breast cancer) in nude mice. Similarly, two groups are set: a blank control group and a nucleic acid composition group containing mRNA. The nucleic acid composition group containing mRNA is injected with the nucleic acid composition containing mRNA prepared in Example 3 under the same conditions as in Example 2, and then the tumor tissues are taken out to make tissue sections. The blank control is injected with an equal amount of PBS. The above-mentioned tumor sections of each group are photographed by confocal microscopy. The mcherry mRNA is imaged using a filter (ex: 587 nm, em: 610 nm), and the DAPI dye is imaged using a filter (ex: 350 nm, em: 460 nm), so as to obtain the intracellular fluorescence intensity and conduct comparative analysis. The results are as Figure 5 shown.

[0082] As can be seen from Figure 5 , the nucleic acid composition without cell-penetrating peptide modification can still maintain its performance, and based on the special nature of its nanostructure, the mRNA is delivered into cells and tissues (living cells), showing fluorescence.

[0083] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A nucleic acid composition for mRNA delivery and expression, characterized in that: The nucleic acid composition comprises mRNA, nucleic acid composition I and nucleic acid composition II; the nucleic acid composition I and the nucleic acid composition II are complementary to the mRNA; The mRNA is divided into N mRNA结合区 mRNA binding region and N mRNA结合区 -1 mRNA active region; the mRNA binding region and the mRNA active region are alternately distributed in sequence; the N mRNA结合区 The mRNA binding regions are numbered from binding region 1 to binding region N. mRNA结合区 ; The length L of the mRNA binding region mRNA结合区 is 5-20nt; the length L of the mRNA active region mRNA活性区 10-150 nt; The length of mRNA L mRNA =L mRNA结合区 ×N mRNA结合区 +L mRNA活性区 ×(N mRNA结合区 -1); The nucleic acid composition I is divided into N 核酸组合物I结合区 A nucleic acid composition I binding region and N 核酸组合物I间隔区 The nucleic acid composition I spacer region; the nucleic acid composition I binding region and the nucleic acid composition I spacer region are alternately distributed in sequence; and are numbered as nucleic acid composition I binding region 1 to nucleic acid composition I binding region N in sequence. 核酸组合物I结合区 ; The length of the spacer region of the nucleic acid composition I is 1-10 nt; N 核酸组合物I结合区 =N 核酸组合物I间隔区 ; The nucleic acid composition II is divided into N 核酸组合物II结合区 Nucleic acid composition II binding region and N 核酸组合物II间隔区 The nucleic acid composition II spacer region; the nucleic acid composition II binding region and the nucleic acid composition II spacer region are alternately distributed in sequence; the N 核酸组合物II结合区 The nucleic acid composition II binding regions are numbered sequentially as nucleic acid composition II binding region 1 to nucleic acid composition II binding region N. II Binding area; The length of the spacer region of the nucleic acid composition II is 1-10 nt; N 核酸组合物II结合区 =N 核酸组合物II间隔区 ; N 核酸组合物I结合区 +N 核酸组合物II结合区 =N mRNA结合区 ; The nucleic acid composition I binding region 1 to the nucleic acid composition I binding region N 核酸组合物I结合区 Complementary to the odd-numbered mRNA binding regions in sequence; the nucleic acid composition II binding region 1 to the nucleic acid composition II binding region N 核酸组合物II结合区 They are complementary to the even-numbered mRNA binding regions in sequence; The mRNA is mcherry mRNA; The nucleic acid composition I and the nucleic acid composition II are both circular RNAs connected end to end; The nucleotide sequence of the nucleic acid composition I is shown in SEQ ID NO.1; The nucleotide sequence of the nucleic acid composition II is shown in SEQ ID NO.

2.

2. The nucleic acid composition for mRNA delivery and expression according to claim 1, characterized in that: The nucleic acid composition I and the nucleic acid composition II are both modified with a cell-penetrating peptide.

3. The nucleic acid composition for mRNA delivery and expression according to claim 2, characterized in that: The cell-penetrating peptide is TAT.

4. A method for improving mRNA delivery efficiency, characterized in that: The method comprises the steps of complementary combining the nucleic acid composition I and the nucleic acid composition II with the mRNA to form a nucleic acid composition, so as to improve the delivery efficiency of the mRNA; The mRNA is divided into N mRNA结合区 mRNA binding region and N mRNA结合区 -1 mRNA active region; the mRNA binding region and the mRNA active region are alternately distributed in sequence; the N mRNA结合区 The mRNA binding regions are numbered from binding region 1 to binding region N. mRNA结合区 ; The length L of the mRNA binding region mRNA结合区 is 5-20nt; the length L of the mRNA active region mRNA活性区 10-150 nt; The length of mRNA L mRNA =L mRNA结合区 ×N mRNA结合区 +L mRNA活性区 ×(N mRNA结合区 -1); The nucleic acid composition I and the nucleic acid composition II are both circular RNAs connected end to end; The nucleic acid composition I is divided into N 核酸组合物I结合区 A nucleic acid composition I binding region and N 核酸组合物I间隔区 The nucleic acid composition I spacer region; the nucleic acid composition I binding region and the nucleic acid composition I spacer region are alternately distributed in sequence; and are numbered as nucleic acid composition I binding region 1 to nucleic acid composition I binding region N in sequence. 核酸组合物I结合区 ; The nucleotide sequence of the spacer region of the nucleic acid composition I is CCCCCC; N 核酸组合物I结合区 =N 核酸组合物I间隔区 ; The nucleic acid composition II is divided into N 核酸组合物II结合区 Nucleic acid composition II binding region and N 核酸组合物II间隔区 The nucleic acid composition II spacer region; the nucleic acid composition II binding region and the nucleic acid composition II spacer region are alternately distributed in sequence; the N 核酸组合物II结合区 The nucleic acid composition II binding regions are numbered sequentially as nucleic acid composition II binding region 1 to nucleic acid composition II binding region N. II Binding area; The nucleotide sequence of the spacer region of the nucleic acid composition II is CCCCCC; N 核酸组合物II结合区 =N 核酸组合物II间隔区 ; N 核酸组合物I结合区 +N 核酸组合物II结合区 =N mRNA结合区 ; The nucleic acid composition I binding region 1 to the nucleic acid composition I binding region N 核酸组合物I结合区 Complementary to the odd-numbered mRNA binding regions in sequence; the nucleic acid composition II binding region 1 to the nucleic acid composition II binding region N 核酸组合物II结合区 They are complementary to the even-numbered mRNA binding regions in sequence.

5. The method according to claim 4, characterized in that The nucleic acid composition I and the nucleic acid composition II are both modified with a cell-penetrating peptide.

6. The method according to claim 5, characterized in that The method for modifying a cell-penetrating peptide with the nucleic acid composition I comprises: performing azide modification on the nucleic acid composition I to obtain an azide-functionalized nucleic acid composition I, and then reacting the nucleic acid composition I with a DBCO-modified cell-penetrating peptide to obtain a cell-penetrating peptide-modified nucleic acid composition I; The method for modifying the cell-penetrating peptide with the nucleic acid composition II comprises: subjecting the nucleic acid composition II to azide modification to obtain an azide-functionalized nucleic acid composition II, and then reacting the nucleic acid composition II with a DBCO-modified cell-penetrating peptide to obtain a cell-penetrating peptide-modified nucleic acid composition II.

7. Use of the nucleic acid composition for mRNA delivery and expression as claimed in any one of claims 1 to 3 in the preparation of mRNA drugs.

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