CN114660160A

CN114660160A - Method for determining minimum loading binding ratio in polypeptide and siRNA (small interfering ribonucleic acid) co-assembly body

Info

Publication number: CN114660160A
Application number: CN202011527460.1A
Authority: CN
Inventors: 孙婷婷; 舒智愚; 牟泉萌; 陈璞
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2022-06-24

Abstract

The invention belongs to the field of bioengineering, and particularly relates to a method for determining the minimum loading binding ratio in a polypeptide and siRNA co-assembly. The invention discloses a method for determining the minimum loading binding ratio in a polypeptide and siRNA co-assembly body, which comprises the following steps: s1: preparing 2% agarose gel; s2: preparing CPP-siRNA nanoparticles; s3: adding target polypeptide, performing electrophoresis experiment, and imaging with a gel imager; and (3) visually observing a map in the imaging result, selecting a group without trailing of the protein electrophoresis track in the image, wherein the minimum binding ratio in the group without trailing is the minimum packet load binding ratio. The invention finds a method for determining the minimum entrapment binding ratio of self-assembly of polypeptide and siRNA into positively charged nanoparticles, finds several polypeptide sequences suitable for the method, and fills the blank of the prior art. The method is simple to operate, and the judgment method is very visual and easy to operate.

Description

Method for determining minimum loading binding ratio in polypeptide and siRNA (small interfering ribonucleic acid) co-assembly body

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a method for determining the minimum loading binding ratio in a polypeptide and siRNA co-assembly.

Technical Field

RNAi (RNA interference) is a homologous RNA degradation process mediated by dsRNA, found during the study of C.elegans antisense RNA (antisense RNA). RNA interference refers to a highly conserved, double-stranded RNA (dsRNA) -induced, highly efficient and specific degradation of homologous mrnas during evolution. The gene silencing mainly comprises two Types of Gene Silencing (TGS) at a pre-transcription level and gene silencing (PTGS) at a post-transcription level, wherein the TGS refers to the condition that a gene cannot be normally transcribed due to DNA modification or chromosome heterochromatosis and the like; PTGS is a degradation mechanism that initiates sequence specificity of target mRNA within the cytoplasm. Sometimes transgenes will result in both TGS and PTGS.

Since the RNA interference technology can silence the disease-related gene efficiently and specifically, it has been developed into a novel gene therapy approach for treating hereditary or acquired diseases including viral infection and cancer, and so on, so far, a large number of animal experiments and clinical experiments using siRNA for disease treatment have been developed.

Although siRNA shows good silencing effect in vitro application research and at the cellular level, due to its huge molecular weight and the large amount of negative charges carried by siRNA itself, siRNA cannot pass through cell membrane to enter cells to play a role, and is liable to cause nonspecific off-target effect and immune response during systemic transportation, and is also subject to the obstacles such as nuclease degradation, etc., thus posing a huge challenge in vivo systemic transportation of siRNA for the purpose of treating human diseases. The transfection problem of siRNA becomes a major bottleneck limiting its application.

How to enhance the ability of siRNA to penetrate cell membrane and improve the stability and targeting property of the siRNA in vivo is a problem which needs to be solved urgently by siRNA drug carriers. Therefore, designing and synthesizing safe and effective siRNA drug carriers has become an important direction for the development of siRNA drugs at present.

Compared to chemically modified nucleic acids, vector-mediated methods formulate nucleic acid therapies and protect them from degradation, and appear to be simple and rapid. These vectors, including viral and non-viral vectors, are co-assembled or covalently bound to the siRNA. These carriers can enhance cell targeting, prolong drug flux time, and improve membrane penetration.

Common delivery vehicles for non-viral gene drugs are often positively charged cationic compounds, including polycations, positively charged phospholipids, chitosan, albumin, dendrimers, polypeptides, etc., which neutralize the negatively charged phosphate groups on DNA/RNA by positively charged groups, thereby compressing and protecting the gene into assembly particles, allowing the gene to successfully pass through various barriers, such as escape of the immune system, penetration of cell membranes, release from inclusion bodies, etc., to accomplish gene delivery.

The polypeptide as one of the biomolecules has many advantages on the gene carrier which are not possessed by the synthetic macromolecule: the polypeptide has 20 amino acids with different properties, and can form a primary sequence with rich properties; the secondary structure such as beta folding or alpha spiral can be obtained by design according to experimental records; molecules with high purity, single distribution and definite structure can be obtained through solid phase synthesis; can be conveniently modified or be connected with a target sequence with a biological recognizable function to improve the specificity.

Polypeptides have been used for the delivery of synthetic drugs, small molecules, bioactive peptides, therapeutic proteins, but the nucleic acid mechanism is not completely understood. These peptides may include cell-penetrating peptides (CPPs) derived from proteins, cationic peptides, designed polypeptide compounds, fusion peptides, cell-targeting peptides (CTPs), and peptides containing Nuclear Localization Signals (NLS), and the like.

Nucleic acid drugs consisting of polypeptides and siRNA have become a focus of research, and some drugs have already entered clinical research. The polypeptide and siRNA can affect the in vitro and in vivo activity under the condition of not completely encapsulating, so the newly designed polypeptide carrier firstly determines the minimum encapsulating and binding ratio of the polypeptide carrier and siRNA self-assembled into the positively charged nanoparticles before the in vitro experiment, and the activity test experiment is carried out above the minimum binding ratio (including). However, there has been no experimental method described to date how to determine the minimum entrapment binding ratio of a polypeptide to an siRNA, thereby limiting the method of siRNA.

Disclosure of Invention

The invention discovers an experimental method for determining the minimum entrapment binding ratio of the polypeptide and the siRNA, and fills the blank of the prior art.

Specifically, the technical scheme of the invention is as follows:

in a first aspect, the present invention discloses a method for determining the minimum loading binding ratio in a polypeptide and siRNA co-assembly, comprising:

s1: preparing 2% agarose gel;

s2: preparing CPP-siRNA nanoparticles;

s3: adding target polypeptide, performing electrophoresis experiment, and imaging with a gel imager; and (3) visually observing a map in the imaging result, selecting a group without trailing of the protein electrophoresis track in the image, wherein the minimum binding ratio in the group without trailing is the minimum packet load binding ratio.

It should be understood that other additional steps may be performed before step S1, directly after steps S1 and S2, and after step S3, and are all within the scope of the present invention.

Preferably, the amino acid sequence of the target polypeptide is selected from SEQ ID NO: 1. the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 or SEQ ID NO: 5.

It is to be understood that the amino acid sequence of the polypeptide of interest of the present invention is not limited to SEQ ID NO: 1. the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 or SEQ ID NO: 5, those skilled in the art can design different polypeptide sequences to accomplish the present invention according to the needs, and all fall within the scope of the present invention.

In some preferred embodiments of the present invention, in S1, the 2% agarose gel is formulated as follows: a. weighing 3.2g agarose powder, placing into a beaker, adding 1 XTAE to a constant volume of 160mL, slightly shaking and mixing uniformly, placing in a microwave oven to heat for 4-7min until completely dissolving, cooling for a while, adding GelRed Nucleic Acid Gel Stain (BIOTIUM; Cat:41003) dye to make the working concentration 1X; b. pouring the dissolved agarose into a mold inserted with a comb, standing at room temperature for 30min, and pulling out the comb; c. and putting the prepared gel into an electrophoresis tank, and carrying out an electrophoresis experiment after adding the target polypeptide.

Preferably, in S2, the CPP mother liquor and the siRNA mother liquor are prepared separately, and the CPP solution and the siRNA solution with different molar ratios are mixed to obtain the CPP-siRNA nanoparticles.

Preferably, in S3, the electrophoresis test is carried out at a voltage of 80-100V for 20-40 min.

In some embodiments of the present invention, in S3, the electrophoresis is performed at 90V for 30 min.

Preferably, before the target polypeptide is added, glycerol is added into the CPP-siRNA nanoparticles and then the CPP-siRNA nanoparticles are mixed evenly.

Preferably, 40% glycerol is added into the CPP-siRNA nanoparticles.

Preferably, the nucleotide sequences of the sense strand and the antisense strand of the siRNA are respectively as shown in SEQ ID NO: 6 and SEQ ID NO: shown at 7.

Preferably, the co-assembly is a co-assembled positively charged nanoparticle.

The second aspect of the invention discloses the application of the method in the field of medicine preparation; preferably, the drug is a nucleic acid drug.

The polypeptide is positively charged, the siRNA is negatively charged, and the polypeptide and the siRNA can self-assemble into nanoparticles in an aqueous solution due to charge interaction, as shown in FIG. 8, when the polypeptide is excessive, the polypeptide can be dissociated, and the polypeptide cannot move to the positive electrode due to electric property. The positively charged nanoparticles cannot be separated due to size and chargeability, and the negatively charged nanoparticles move to the positive electrode less due to the polypeptide bound by siRNA, but the efficiency of entering cells is greatly reduced, so in order to determine the minimum entrapment binding ratio of the polypeptide and the siRNA self-assembled into the positively charged nanoparticles, an agarose gel electrophoresis with the maximum exclusion of nucleic acid dyes and loading Buffer components can be selected for carrying out a binding ratio confirmation experiment.

Compared with the prior art, the invention has at least the following distinguishing technical characteristics:

the invention finds a method for determining the minimum entrapment binding ratio of self-assembly of polypeptide and siRNA into positively charged nanoparticles, finds several polypeptide sequences suitable for the method, and fills the blank of the prior art. The method is simple to operate, and the judgment method is very visual and easy to operate. By determining the minimum packet loading binding ratio, a basis is provided for in vitro binding ratio experiments, and the in vivo and in vitro experiment progress of polypeptide and siRNA nucleic acid is greatly promoted.

Drawings

FIG. 1 is a graph of gel images of Carrier 1 and Carrier 2 with siRNA at different ratios of binding ratios;

FIG. 2 is an image of a gel of carrier 3 and siRNA at different ratios of binding;

FIG. 3 is an image of a gel of Carrier 3 and siRNA at different ratios of binding;

FIG. 4 is an image of a gel of carrier 6 and siRNA at different ratios of binding;

FIG. 5 is a graph of gel images of carrier 3 and siRNA at different ratios of binding;

FIG. 6 is a graph of gel images of carriers 4 and 5 and siRNA at different ratios of binding;

FIG. 7 is an image of a gel of vector 6 and siRNA at different ratios of binding;

fig. 8 is a three-dimensional schematic diagram of assembly of a polypeptide and siRNA into a nanoparticle.

Detailed Description

The present application is further illustrated by the following detailed examples, which should be construed to be merely illustrative and not limitative of the remainder of the disclosure.

The instruments, apparatuses, and reagents used in the examples are available from various sources, for example, commercially available, or may be prepared.

The following embodiments relate to vector 1, vector 2, vector 3, vector 4, vector 5 and vector 6, the amino acid sequences of which are shown below:

carrier 1:

LysLysLys-(TrpTrpArgHisHisHisHisArgHisHisHisHisArgHisHisHisHisArgHisHisHisHisArgHisHisHisHis)4(SEQ ID NO：1)；

carrier 2:

LysLysLys-(TrpProLysHisHisHisHisLysHisHisHisHisLysHisHisHisHisLysHisHisHisHisLys)4(SEQ ID NO：2)；

carrier 3: ArgLeuTrpArgLeuTrpArgLeuTrpArgLeuTrpArgTrArg (SEQ ID NO: 8);

carrier 4:

LysLysLys-(ProLysHisHisHisHisLysHisHisHisHisLysHisHisHisHisLysHisHisHisHisLys)4(SEQ ID NO：3)；

carrier 5:

GlnGlnGln-(ArgHisHisHisHisArgHisHisHisHisArgHisHisHisHisArgHisHisHisHisArg)4(SEQ ID NO：4)；

carrier 6:

LysLysLys-(TrpTrpGlnHisHisHisHisGlnHisHisHisHisGlnHisHisHisHisGlnHisHisHisHisGlnHisHisHisHis)4(SEQ ID NO：5)。

except for vector 3, which consists of a backbone and branches, "-" is preceded by a backbone and followed by branches.

The vector 1, the vector 2, the vector 3, the vector 4, the vector 5 and the vector 6 belong to cell-penetrating peptide (CPP).

Sense strand of siRNA related in the examples of the present invention: 5 '- (uucuccgaacgugucacgutt) -3' (SEQ ID NO: 6), antisense strand: 5 '- (acgugacacguucggagaatt) -3' (SEQ ID NO: 7).

Example 1

The embodiment discloses an experimental method for detecting the binding ratio of carrier polypeptide and siRNA, which specifically comprises the following steps:

(1) 2% agarose gel is prepared in advance, and the preparation method is as follows: a. weighing 3.2g agarose powder, placing into a beaker, adding 1 XTAE to a constant volume of 160mL, slightly shaking and mixing uniformly, placing in a microwave oven to heat for 4-7min until completely dissolving, cooling for a while, adding GelRed Nucleic Acid Gel Stain (BIOTIUM; Cat:41003) dye to make the working concentration 1X; b. pouring the dissolved agarose into a mold inserted with a comb, standing at room temperature for 30min, and pulling out the comb; c. and putting the prepared gel into an electrophoresis tank, and carrying out an electrophoresis experiment after a sample is added.

(2) The sample preparation method comprises the following steps:

CPP and siRNA were formulated in a range of ratios as shown in Table 1 below. Polypeptide mother liquor concentration: 500 mu M; concentration of siRNA mother liquor: 50 μ M solution.

TABLE 1

CPP and siRNA were formulated in a range of ratios as shown in Table 2 below. Polypeptide mother liquor concentration: 100 mu M; concentration of siRNA mother liquor: 50 μ M solution.

TABLE 2

(3) Mixing the prepared CPP and the siRNA solution, carrying out vortex oscillation for 10s, and standing for 20min to form the CPP-siRNA nano-particles.

(4) After 20min, 3.4. mu.l of 6 XDNA Loading Buffer (Solambio; Cat. No.: D1010) was added to each tube and mixed well by vortexing.

(5) Adding 15 mul of sample into a sample groove, carrying out electrophoresis experiment, carrying out voltage of 90V for 30min, imaging by a gel imager and storing.

The CPP in this example is vector 3 (described in patent CN 104487450A) or vector 6.

As shown in FIG. 2, the carrier 3 and siRNA were completely encapsulated at 20: 1. Further refinement of the binding ratio for vector 3, as shown in FIG. 3, determined that the minimum binding ratio of vector 3 to siRNA was 16: 1.

As can be seen from fig. 4, the minimum binding ratio of the carrier 6 is 5: 1.

Example 2

(1) 2% agarose gel is prepared in advance, and the preparation method is as follows: a. weighing 3.2g of agarose powder, putting into a beaker, adding 1 XTAE to a constant volume of 160ml, slightly shaking and uniformly mixing, and placing in a microwave oven to heat for 4-7min until the agarose powder is completely dissolved; b. pouring the dissolved agarose into a mold inserted with a comb, standing at room temperature for 30min, and pulling out the comb; c. and putting the prepared gel into an electrophoresis tank, and carrying out an electrophoresis experiment after a sample is added.

(2) The sample preparation method comprises the following steps:

CPP and siRNA were formulated in a range of ratios as shown in Table 3 below. Polypeptide mother liquor concentration: 500 mu M; concentration of siRNA mother liquor: 50 μ M solution.

TABLE 3

CPP and siRNA were formulated in a series of ratios as shown in Table 4 below. Polypeptide mother liquor concentration: 100 mu M; concentration of siRNA mother liquor: 50 μ M solution.

TABLE 4

(4) After 20min, 4. mu.l of 40% glycerol was added to each tube and mixed well by vortexing.

(5) Add 15. mu.l sample into sample tank, and perform electrophoresis experiment at 120V for 30 min.

(6) Finally, the Gel was stained in 1 XGelRed Nucleic Acid Gel Stain (BIOTIUM; Cat:41003) dye for 30min, imaged and stored in a Gel imager using siRNA as scramble siRNA, sense strand: 5 '- (UUCUCCGAACGUGUCACGUTT) -3', antisense strand: 5 '- (ACGUGACACGUUCGGAGAATT) -3'. The CPP in this example is vector 1, vector 2, vector 3, vector 4, vector 5, or vector 6.

By gel imager, according to fig. 1, the minimum binding ratio of carrier 1, carrier 2 to siRNA can be determined to be 5: 1.

the entrapment binding ratio of vector 3 to siRNA was determined in fig. 5. Vector 3 with siRNA at 20:1, the sample Loading hole of the example 2 is brighter than the polypeptide nucleic acid nanoparticles in the example 1 under the condition of higher binding ratio, the analysis reason is probably that components in GelRed and 6 multiplied DNA Loading Buffer (the main components are glycerol, EDTA, bromophenol blue, xylene cyan and the like) are combined with the polypeptide nanoparticles and are tightly wrapped, and the phenomenon is obvious when the combination ratio is higher. Therefore, the method of determining the minimum pack ratio disclosed in example 2 is more preferable.

FIG. 6 shows that the minimum binding ratio of carriers 4, 5 to siRNA is 5: 1. Fig. 7 shows that the minimum binding ratio of carrier 6 to siRNA is 5: 1.

as can be seen from the above examples, the method disclosed in the present invention for determining the minimum entrapment binding ratio of a polypeptide to siRNA is feasible.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A method for determining the minimum loading binding ratio in a polypeptide and siRNA co-assembly, comprising:

s1: preparing 2% agarose gel;

s2: preparing CPP-siRNA nanoparticles;

s3: adding target polypeptide, performing electrophoresis experiment, and imaging with a gel imager; and (3) observing a map in an imaging result by naked eyes, selecting a group without trailing of the protein electrophoresis track in the image, wherein the minimum binding ratio in the group without trailing is the minimum packet load binding ratio.

2. The method of claim 1, wherein the amino acid sequence of the target polypeptide is selected from the group consisting of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 or SEQ ID NO: 5.

3. The method of claim 1, wherein in S2, the CPP mother liquor and the siRNA mother liquor are prepared separately, and the CPP solution and the siRNA solution with different molar ratios are mixed to obtain the CPP-siRNA nanoparticles.

4. The method of claim 1, wherein in S3, the electrophoresis is performed at a voltage of 80-100V for a time of 20-40 min.

5. The method of claim 4, wherein in S3, the electrophoresis is performed at 90V for 30 min.

6. The method of claim 1, wherein glycerol is added to the CPP-siRNA nanoparticles prior to adding the target polypeptide and then the CPP-siRNA nanoparticles are mixed.

7. The method of claim 6, wherein 40% glycerol is added to the CPP-siRNA nanoparticles.

8. The method of claim 1, wherein the nucleotide sequences of the sense and antisense strands of the siRNA are set forth in SEQ ID NO: 6 and SEQ ID NO: shown at 7.

9. The method of claim 1, wherein the co-assembly is a co-assembly of positively charged nanoparticles.

10. Use of a process according to any one of claims 1 to 9 in the field of pharmaceutical preparation; preferably, the drug is a nucleic acid drug.