A method for distinguishing free nucleic acid from nucleic acid encapsulated by a carrier based on nuclear magnetic resonance phosphorus spectroscopy

Abstract

The present invention relates to the field of analytical detection technology, and specifically to 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: (1) using free nucleic acid as a control, performing nuclear magnetic resonance phosphorus spectroscopy detection to obtain its nuclear magnetic phosphorus spectrum; (2) using nucleic acid bound to a carrier as an experimental group, performing nuclear magnetic resonance phosphorus spectroscopy detection under the same conditions to obtain the phosphorus spectrum of the experimental group; (3) distinguishing free nucleic acid from nucleic acid encapsulated by a carrier by comparing characteristic changes such as peak position, peak shape, peak height and peak area between the experimental group and the control group. The method has good accuracy, repeatability and stability, can perform non-destructive testing, and is suitable for analyzing sensitive samples. It can be used to detect the binding of carriers and nucleic acids in nucleic acid drugs, predict the encapsulation rate and release rate of nucleic acid drugs, and provide an important analytical tool for the development, production and quality control of nucleic acid drugs.

Description

Technical Field

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

[0002] Nucleic acids play a crucial role in the life processes of organisms, playing a crucial role in inheritance, mutation, protein synthesis, and gene expression regulation. However, when exogenous nucleic acids enter an organism, they are degraded by the widespread presence of nucleases, making their biological effects difficult to achieve (K. Paunovska, D. Loughrey and J. E. Dahlman, Nat. Rev. Genet., 2022, 23, 265–280). Furthermore, nucleic acids are hydrophilic and oleophobic, and their surface carries a strong negative charge, making them difficult to cross the cell membrane, which has a double-layer phospholipid membrane structure.

[0003] Therefore, nucleic acids need carriers to protect them and transport them to cells to play a role. In early gene therapy research, viruses such as adenovirus, lentivirus, and herpes virus are commonly used as carriers to deliver nucleic acids. Although such carriers have high transfection efficiency, they have potential carcinogenicity, limited carrier capacity, complex preparation process, and high cost, which limit their 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, various materials such as dendrimers, polymers, and ferritin 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 being 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 kind of method for differentiating free nucleic acid and the nucleic acid wrapped by carrier based on nuclear magnetic resonance phosphorus spectrum, by contrasting the peak of experimental group target peak and control group target, go out changes such as peak position, peak shape, peak height and peak area, can be used for differentiating free nucleic acid and the nucleic acid wrapped by carrier, analyze the binding situation of carrier and nucleic acid.This method can simply, quickly and accurately detect the nucleic acid wrapped by carrier, and realize nondestructive testing, and the experimental sample after recovery can continue to use.On this basis, nucleic acid and the peak area of ​​the nucleic acid wrapped by carrier are integrated, and the encapsulation efficiency and the 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 free of phosphorus to obtain a test solution, and performing nuclear magnetic resonance phosphorus spectroscopy on the two test solutions;

[0008] (2) By comparing the nuclear magnetic resonance 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 nuclear magnetic resonance phosphorus spectrum, the sample with lower peak height, smaller peak area, and larger peak position offset is the nucleic acid wrapped by the carrier.

[0009] 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 284K, the pulse width was 9.9μs, D1 was 1s, the spectrum width was 503.2ppm, the radio frequency center frequency was 161.993176Hz, LB=100Hz, 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;

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

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

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

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

[0014] A method for predicting the encapsulation efficiency of nucleic acid in a vector-encapsulated nucleic acid based on the above method comprises obtaining nuclear magnetic resonance phosphorus spectra of free nucleic acid and nucleic acid encapsulated by the vector, integrating the respective peak areas, and obtaining a peak area ratio for use in predicting the encapsulation efficiency of the nucleic acid. Alternatively, the peak areas of nucleic acid encapsulated by the vector and nucleic acid released from the vector can be integrated to obtain a peak area ratio for use in predicting the release rate of the 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 methods such as enzyme digestion, dissociation, and precipitation. The results are intuitive, and there is no need for complex processing of experimental spectra or large-scale calculations of data. The binding status of the carrier and nucleic acid in the sample 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 image of lipid nanoparticles and pEGFP;

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

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

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

[0022] The main reagents and materials used in the following examples are described as follows: Nuclear magnetic resonance (NMR) phosphorus spectrometry was performed using a 400 MHz NMR spectrometer (Bruker, Switzerland) with 10 mm NMR tubes (purchased from Wilmad, USA). Heptadecane-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate was purchased from Xiamen Sinobank Biotechnology Co., Ltd., distearoylphosphatidylcholine and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 were purchased from Ruixi Biotechnology, and cholesterol was purchased from Sigma. 0.5 M citrate buffer (pH 3) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. A high-purity plasmid extraction kit (DP116) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. RNase-free ddH2O was purchased from Shenggong Biotechnology Co., Ltd.

[0023] Preparation of 50 mM citrate buffer: Add 300 μL of 0.5 M citrate buffer at pH 3 to 2.7 mL of RNase-free ddH2O, 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] Prepare 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, which is used as the 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-((undedecyloxy)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 mixed 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) 750 μL of the 1 mg / mL pEGFP plasmid solution prepared in Example 1 was added 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 sample to be observed was prepared by dropping LNP-pEGFP onto a copper mesh covered with a carbon film. 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: Lipid nanoparticles LNP can form spherical particles after encapsulating pEGFP plasmid, with a particle size of 50-80nm.

[0034] Example 4 Agarose gel electrophoresis experiment characterizes the encapsulation of nucleic acids. 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] Agarose gel electrophoresis results showed that free pEGFP plasmids with a chain length of approximately 3000-5000 bp were concentrated in the upper middle portion of the agarose gel. DNase I degraded the pEGFP plasmid into shorter fragments, which, under the action of an applied voltage, migrated to the bottom of the agarose gel and exhibited a tailing phenomenon. In contrast, the bands of both LNP-pEGFP groups were located at the top of the agarose gel and showed no tailing phenomenon, indicating that the pEGFP plasmid was encapsulated within the LNPs and protected from DNase I degradation.

[0037] Example 5 Detection of nucleic acid encapsulated by a 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 the nuclear magnetic resonance phosphorus spectrum of the control group and the experimental group are as follows: a zgig pulse sequence was used, the test temperature was 284K, the pulse width was 9.9μs, D1 was 1s, the spectrum width was 503.2ppm, the RF center frequency was 161.993176Hz, LB=100Hz, 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.

[0040] Results: Nuclear magnetic resonance (NMR) phosphorus spectroscopy revealed peaks at -3 to -5 ppm for both the free pEGFP plasmid in the control group and the lipid nanoparticle-encapsulated pEGFP plasmid in the experimental group. However, the target peak for the lipid nanoparticle-encapsulated pEGFP plasmid exhibited a significantly reduced peak area, height, and width, and also generated an NMR signal at -16.4 ppm. This is likely due to the cationic lipids in the LNPs, which carry electric and magnetic fields. After binding to nucleic acids, these interactions affect the chemical environment of the phosphorus atoms in the nucleic acids, leading to a shift in peak position. This suggests that lipid nanoparticle encapsulation restricts the spin rotation of the phosphorus atoms in the pEGFP plasmid, preventing it from generating a strong NMR signal. Furthermore, the peak area ratio of the free nucleic acid and the peak area of ​​the vector-encapsulated nucleic acid can be integrated to estimate the encapsulation efficiency. Alternatively, the peak area ratio of the vector-encapsulated nucleic acid and the peak area of ​​the nucleic acid released from the vector can be integrated to estimate the release rate.

Claims

1. 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: (1) Dissolving the free nucleic acid to be identified and the nucleic acid encapsulated by the carrier in a solvent free of phosphorus to obtain a test solution, and performing nuclear magnetic resonance phosphorus spectroscopy on the two test solutions; (2) By comparing the nuclear magnetic resonance 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 nuclear magnetic resonance phosphorus spectrum, the sample with lower peak height, smaller peak area, and greater peak position shift 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 During the nuclear magnetic resonance phosphorus spectrum detection, 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 according to any one of claims 1 to 3, wherein the nuclear magnetic resonance phosphorus spectra of free nucleic acid and carrier-encapsulated nucleic acid obtained by the method according to any one of claims 1 to 3 are integrated to obtain the peak area ratio of the two for predicting the encapsulation efficiency of nucleic acid.

Images