Method for identifying free nucleic acid and nucleic acid wrapped by carrier based on nuclear magnetic resonance phosphorus spectrum

Abstract

The invention relates to the technical field of analysis and detection, in particular to a method for identifying free nucleic acid and nucleic acid wrapped by a carrier based on nuclear magnetic resonance phosphorus spectrums.The method comprises the following steps that 1, the free nucleic acid serves as a contrast, nuclear magnetic resonance phosphorus spectrum detection is conducted, and a nuclear magnetic phosphorus spectrogram of the free nucleic acid is obtained; (2) taking the nucleic acid combined with the carrier as an experimental group, and performing nuclear magnetic resonance phosphorus spectrum detection under the same condition to obtain a phosphorus spectrogram of the experimental group; and (3) comparing the characteristic changes of peak positions, peak shapes, peak heights, peak areas and the like of the experimental group and the control group to identify the free nucleic acid and the nucleic acid wrapped by the carrier. The method has good accuracy, repeatability and stability, can be used for nondestructive testing, and is suitable for analyzing sensitive samples. The method can be used for detecting the binding condition of a carrier and nucleic acid in a nucleic acid drug, predicting the encapsulation efficiency and release rate of the nucleic acid drug, and providing an important analysis tool for opening, production and quality control of the nucleic acid drug.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for identifying free nucleic acid and nucleic acid wrapped by a carrier based on nuclear magnetic resonance phosphorus spectrum. Background Art

[0002] Nucleic acids play an extremely critical role in the life activities of organisms, and are of vital importance to heredity, variation, protein synthesis, gene expression regulation, etc. However, when exogenous nucleic acids enter the body, the widely existing nucleases in the body will degrade them, making it difficult to exert their biological effects (K. Paunovska, D. Loughrey and J. E. Dahlman, Nat. Rev. Genet., 2022, 23, 265–280.). In addition, nucleic acid molecules are hydrophilic and oleophobic, and have a strong negative charge on the surface, which makes it difficult for them to cross the cell membrane with a double-layer phospholipid membrane structure.

[0003] Therefore, nucleic acid needs a carrier to protect it and transport it to the cell to play a role. In early gene therapy research, adenovirus, lentivirus, herpes virus and other viruses are often used as carriers to deliver nucleic acids. Although this type of carrier has a high transfection efficiency, there are problems such as potential carcinogenicity, limited carrier capacity, complex preparation process and high cost, which limit its clinical application. After this, nanomaterials have gradually become the main research carriers for nucleic acid delivery. For example, the main components of lipid nanoparticles include cationic lipids, neutral auxiliary phospholipids, cholesterol and polyethylene glycol lipids, which have good biocompatibility (L.Juncheng, N.Elena Atochina-Vasserman, S.Devendra, J.Am.Chem.Soc.2023,145,18760-18766). At this stage, dendritic molecules, polymers, ferritin and other materials have been experimentally proven to have the characteristics of high efficiency, safety, targeting and multifunctionality in nucleic acid delivery. Although more and more nucleic acid carriers are constantly discovered and prepared, there is currently no clear method to detect nucleic acids wrapped by carriers. Summary of the invention

[0004] Based on the above situation, the object of the present invention is to provide a method for distinguishing free nucleic acid from nucleic acid wrapped by a carrier based on nuclear magnetic resonance phosphorus spectrum, by comparing the peak position, peak shape, peak height and peak area of ​​the target peak of the experimental group and the target of the control group, it can be used to distinguish free nucleic acid from nucleic acid wrapped by a carrier, and analyze the binding of carrier to nucleic acid. This method can simply, quickly and accurately detect nucleic acid wrapped by a carrier, and realize non-destructive testing, and the experimental sample after recovery can continue to be used. On this basis, the peak area of ​​nucleic acid and nucleic acid wrapped by a carrier is integrated, and the encapsulation efficiency and release rate of nucleic acid can be predicted by the ratio of the two peak areas.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for distinguishing free nucleic acid from nucleic acid encapsulated by a carrier based on nuclear magnetic resonance phosphorus spectroscopy, the method comprising the following steps:

[0007] (1) dissolving the free nucleic acid to be identified and the nucleic acid encapsulated by the carrier in a solvent that does not contain phosphorus to obtain a test solution, and performing nuclear magnetic resonance phosphorus spectrum detection on the two test solutions;

[0008] (2) By comparing the NMR phosphorus spectra of the two substances to be identified obtained in step (1), the two substances can be identified based on the peak height, peak area, and peak position of phosphorus, and the binding between the carrier and the nucleic acid can be analyzed: in the NMR phosphorus spectrum, the sample with lower peak height, smaller peak area, and greater peak position deviation is the nucleic acid wrapped by the carrier.

[0009] In (1), when the two test solutions are subjected to nuclear magnetic resonance phosphorus spectrum detection, the sampling parameters are as follows: a zgig pulse sequence is used, the test temperature is 284K, the pulse width is 9.9μs, D1 is 1s, the spectrum width is 503.2ppm, the RF center frequency is 161.993176Hz, LB=100Hz, the number of acquisition points is 65536, the number of sampling accumulation times is 8192 times, the number of empty scans is 0 times, and the gain is 2050;

[0010] The solvent is water, anhydrous ethanol or citrate buffer.

[0011] Furthermore, the solvent is a citrate buffer solution with a concentration of 0.05 M and a pH of 3.

[0012] Further, the nucleic acid is DNA, mRNA, siRNA or miRNA;

[0013] Furthermore, when the nuclear magnetic resonance phosphorus spectrum is detected, the frequency of the nuclear magnetic resonance instrument used is 400-600 MHz, preferably 400 MHz.

[0014] A method for predicting the encapsulation rate of nucleic acid in nucleic acid wrapped by a carrier based on the above method, after obtaining the nuclear magnetic phosphorus spectra of free nucleic acid and nucleic acid wrapped by the carrier, the peak areas of each are integrated to obtain the peak area ratio of the two, which is used to predict the encapsulation rate of nucleic acid. The peak areas of nucleic acid wrapped by the carrier and nucleic acid released from the carrier can also be integrated to obtain the peak area ratio of the two, which is used to predict the release rate of nucleic acid.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0016] The method of the present invention can achieve non-destructive detection of nucleic acids and nucleic acids bound to carriers, without the need for irreversible treatments such as enzyme cutting, dissociation, and precipitation, and the results are intuitive, without the need for complex processing of experimental spectra or large amounts of data calculations, and the binding of carriers and nucleic acids in samples can be analyzed by directly comparing the peaks of the two. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a cryo-electron microscopy image of lipid nanoparticles encapsulating pEGFP plasmid;

[0018] Figure 2 This is the gel electrophoresis picture of lipid nanoparticles and pEGFP;

[0019] Figure 3 is the NMR phosphor spectrum of the plasmid containing free pEGFP;

[0020] Figure 4 This is the NMR phosphor spectrum of the pEGFP plasmid encapsulated by lipid nanoparticles. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is described in detail below in conjunction with specific embodiments and drawings.

[0022] The main reagents and materials used in the following examples are described as follows: When nuclear magnetic resonance phosphorus spectrum detection, a 400MHz nuclear magnetic resonance instrument (Bruker, Switzerland) was used, and a 10mm nuclear magnetic tube (purchased from Wilmad, USA) was used. Heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate) was purchased from Xiamen Sinobond Biotechnology Co., Ltd., distearoyl phosphatidylcholine and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 were purchased from Ruixi Biotechnology, and cholesterol was purchased from Sigma. 0.5M citrate buffer was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., pH=3. High-purity plasmid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. DP116. RNase-free ddH 2O Purchase from Shengong Biotechnology Co., Ltd.

[0023] Preparation of 50 mM citrate buffer: Take 300 μL of pH 3 0.5 M citrate buffer and add 2.7 mL RNase-free ddH 2 D, mix well, and dilute to obtain 50 mM citrate buffer.

[0024] Example 1 Preparation of free pEGFP plasmid solution, the specific steps are as follows:

[0025] Preparation of pEGFP plasmid solution: Use a high-purity plasmid extraction kit to extract the EGFP-expressing plasmid pEGFP from E. coli, and then dilute it to 1 mg / mL with 50 mM citrate buffer for subsequent experiments.

[0026] Take 750 μL of the above 1 mg / mL pEGFP plasmid solution, add 50 mM citrate buffer to make up to 1.5 mL, then add 0.5 mL of anhydrous ethanol, mix well to obtain a free pEGFP plasmid detection solution as a control group.

[0027] Example 2 Preparation of pEGFP plasmid (LNP-pEGFP) solution encapsulated by lipid nanoparticles, the specific steps are as follows:

[0028] (1) Heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate) (abbreviated as SM102), distearoylphosphatidylcholine (abbreviated as DSPC), cholesterol (abbreviated as Chol) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (abbreviated as DMG-PEG2000) were prepared in a molar ratio of SM102:DSPC:Chol:DMG-PEG2000=50:10:8.5:1.5 (the amount of DMG-PEG2000 was 0.08 mg, the amount of substance was 3×10 -8 mol) was measured and dissolved in 2 mL of anhydrous ethanol to complete the preparation of the ethanol phase.

[0029] (2) Take 750 μL of the 1 mg / mL pEGFP plasmid solution prepared in Example 1 and add it to 5250 μL of 50 mM citrate buffer to complete the preparation of the aqueous phase.

[0030] (3) The ethanol phase was mixed with the aqueous phase containing the pEGFP plasmid, and after mixing evenly, the mixture was passed through a 100 kDa ultrafiltration tube and centrifuged at 4000 rpm to concentrate the sample to 2 mL, thereby completing the preparation of the experimental group test solution LNP-pEGFP.

[0031] Example 3 Cryo-electron microscopy characterization of LNP-pEGFP, the specific steps are as follows:

[0032] The LNP-pEGFP was dropped onto a copper mesh covered with a carbon film to prepare the sample to be observed. The sample was quickly frozen using liquid nitrogen. After freezing, the sample on the copper mesh was characterized by cryo-electron microscopy ( Figure 1 ).

[0033] Result analysis: After lipid nanoparticles LNP encapsulate pEGFP plasmid, they can form spheres with a particle size of 50-80nm.

[0034] Example 4 Agarose gel electrophoresis experiment characterizes the packaging of nucleic acids, and the specific steps are as follows:

[0035] After LNP-pEGFP was digested with DNase I, agarose gel electrophoresis was performed to characterize the encapsulation of nucleic acid by LNP. Free pEGFP plasmid was used as the control group. The gel electrophoresis experiment was performed for 1 hour at a voltage of 80 V ( Figure 2 ).

[0036] The results of agarose gel electrophoresis experiments show that the free pEGFP plasmid with a chain length of about 3000-5000bp is concentrated in the middle and upper part of the agarose gel. DNase I can degrade the pEGFP plasmid into shorter fragments, which move to the bottom of the agarose gel under the action of an applied voltage and show a tailing phenomenon. The bands of the two groups of LNP-pEGFP are at the top of the agarose gel and there is no tailing phenomenon, which indicates that the pEGFP plasmid is wrapped inside the LNP and can prevent it from being degraded by DNase I.

[0037] Example 5 Detecting the nucleic acid encapsulated by the carrier, the specific steps are as follows:

[0038] The free pEGFP plasmid test solution of the control group prepared in Example 1 and the pEGFP plasmid solution encapsulated by lipid nanoparticles of the experimental group prepared in Example 2 were subjected to nuclear magnetic resonance phosphorus spectrum detection to obtain the nuclear magnetic spectra of the two ( Figure 3 , Figure 4 ).

[0039] The sampling parameters of NMR phosphorus spectrum of the control group and the experimental group are as follows: using zgig pulse sequence, the test temperature is 284K, the pulse width is 9.9μs, D1 is 1s, the spectrum width is 503.2ppm, the RF center frequency is 161.993176Hz, LB=100Hz, the number of acquisition points is 65536, the number of sampling accumulation times is 8192 times, the number of empty scans is 0 times, and the gain is 2050.

[0040] Results: After the free pEGFP plasmid in the control group and the pEGFP plasmid encapsulated by lipid nanoparticles in the experimental group were detected by nuclear magnetic resonance phosphorus spectrum, the peak positions of both were -3--5ppm, but the peak area of ​​the target peak of the pEGFP plasmid encapsulated by lipid nanoparticles was greatly reduced, the peak height was greatly reduced, the peak width was widened, and there was also a nuclear magnetic signal at -16.4ppm (it may be because the LNP used contains cationic lipids, which will carry electric and magnetic fields. After binding with nucleic acids, it affects the chemical environment of phosphorus atoms in nucleic acids, resulting in changes in the peak position). This shows that the self-rotation of phosphorus atoms in the pEGFP plasmid is restricted after being encapsulated by lipid nanoparticles, and it cannot produce a good nuclear magnetic signal. Moreover, on this basis, the peak area of ​​free nucleic acid and its peak area after being encapsulated by the carrier can be integrated to obtain the peak area ratio of the two, which is used to predict the encapsulation rate of nucleic acid. The peak area of ​​nucleic acid encapsulated by the carrier and the peak area of ​​nucleic acid released by the carrier can also be integrated to obtain the peak area ratio of the two, which is used to predict the release rate of nucleic acid.

Claims

1. A method for identifying free nucleic acid and nucleic acid encapsulated by a carrier based on nuclear magnetic resonance phosphorus spectroscopy, the method comprising the following steps: (1) Dissolving the free nucleic acid to be identified and the nucleic acid encapsulated by the carrier in a solvent that does not contain phosphorus to obtain a test solution, and performing nuclear magnetic resonance phosphorus spectrum detection on the two test solutions; (2) By comparing the NMR phosphorus spectra of the two substances to be identified obtained in step (1), the two substances can be identified based on the peak height, peak area, and peak position of phosphorus, and the binding between the carrier and the nucleic acid can be analyzed: in the NMR phosphorus spectrum, the sample with lower peak height, smaller peak area, and greater peak position deviation is the nucleic acid wrapped by the carrier; In (1), when the two test solutions were subjected to nuclear magnetic resonance phosphorus spectrum detection, the sampling parameters were as follows: a zgig pulse sequence was used, the test temperature was 284 K, the pulse width was 9.9 μs, D1 was 1 s, the spectrum width was 503.2 ppm, the RF center frequency was 161.993176 Hz, LB = 100 Hz, the number of acquisition points was 65536, the number of sampling accumulation times was 8192 times, the number of empty scans was 0 times, and the gain was 2050; The solvent is water, anhydrous ethanol or citrate buffer.

2. The method according to claim 1, characterized in that The nucleic acid is DNA, mRNA, siRNA or miRNA.

3. The method according to claim 1, characterized in that When the nuclear magnetic resonance phosphorus spectrum is detected, the frequency of the nuclear magnetic resonance instrument used is 400-600 MHz.

4. A method for predicting the encapsulation efficiency of nucleic acid in a carrier-encapsulated nucleic acid based on the method described in any one of claims 1 to 3, integrating the respective peak areas based on the nuclear magnetic phosphorus spectra of free nucleic acid and carrier-encapsulated nucleic acid obtained by the method described in any one of claims 1 to 3 to obtain the peak area ratio of the two for predicting the encapsulation efficiency of nucleic acid.

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