Method for recovering nucleic acid

Abstract

The present invention provides a method, with which it is possible to consistently perform the separation of extracellular vesicles from an extracellular vesicle-containing sample and the recovery of a nucleic acid (e.g., RNA) from the extracellular vesicles. More specifically, the present invention provides a method for recovering a nucleic acid, the method including the following: (A) separating extracellular vesicles from an extracellular vesicle-containing sample by using sedimentation particles in which a substance having affinity to an extracellular vesicle membrane is fixed by non-charged binding; (B) eluting the nucleic acid from the extracellular vesicle; and (C) recovering the eluted nucleic acid using nucleic acid recovery particles.

Description

Nucleic acid recovery method

[0001] The present invention relates to a method for recovering nucleic acids.

[0002] Extracellular vesicles (EVs) are tiny vesicles with a membrane structure secreted by various cells and present in body fluids such as blood or cell culture media. Extracellular vesicles secreted outside cells include exosomes, ectosomes, and apoptotic blebs. Because extracellular vesicles are a diverse group containing various substances that perform functions such as intercellular signaling, they are being analyzed for purposes such as diagnosis and drug discovery.

[0003] Analyzing nucleic acids (e.g., RNA) in extracellular vesicles requires a) a process for separating extracellular vesicles from an extracellular vesicle-containing sample (e.g., serum, cell culture supernatant), and b) a process for recovering nucleic acids from the separated extracellular vesicles. For example, Patent Document 1 describes a method including a) a process using immunoprecipitation using magnetic particles with tosyl groups, and b) a process using a nucleic acid purification kit (Example 11).

[0004] International Publication No. 2020 / 080387

[0005] The above processes a) and b) have been developed independently, and no method has yet been developed that can consistently separate extracellular vesicles from an extracellular vesicle-containing sample and recover nucleic acids (e.g., RNA) from the extracellular vesicles. Current methods that include the above processes a) and b) require a step of transferring the separated extracellular vesicles to a nucleic acid recovery system. For example, in the process using a nucleic acid purification kit in the method of Patent Document 1, a step of transferring the separated extracellular vesicles to a nucleic acid recovery system is required, which requires the replacement of containers (e.g., tubes). Furthermore, this step raises concerns about the loss of rare nucleic acids and sample mix-ups.

[0006] Therefore, an object of the present invention is to develop a method that can be consistently performed to separate extracellular vesicles from an extracellular vesicle-containing sample and to recover nucleic acids from the extracellular vesicles.

[0007] As a result of extensive research, the inventors have discovered that the above-mentioned problems can be solved by using sedimentation particles to which a substance having affinity for the extracellular vesicle membrane is fixed by an uncharged bond in the separation of extracellular vesicles from an extracellular vesicle-containing sample, and by using nucleic acid extraction particles in the recovery of nucleic acids (e.g., RNA) from the extracellular vesicles, and have thus completed the present invention.

[0008] That is, the present invention is as follows. [1] A method for recovering nucleic acids, comprising: (A) separating extracellular vesicles from an extracellular vesicle-containing sample using sedimentation particles to which a substance having affinity for extracellular vesicle membranes is immobilized via an uncharged bond; (B) eluting nucleic acids from the extracellular vesicles; and (C) recovering the eluted nucleic acids using nucleic acid extraction particles. [2] The method of [1], wherein the nucleic acid is RNA, and (C) is performed in the presence of a chaotropic agent. [3] The method of [1] or [2], wherein the substance having affinity for extracellular vesicle membranes is an antibody against a surface marker of extracellular vesicles. [4] Any of the methods of [1] to [3], wherein the surface marker of extracellular vesicles is CD9 and / or CD63. [5] Any of the methods of [1] to [4], wherein the uncharged bond is an amide bond. [6] The method of any one of [1] to [5], wherein the sedimentation particles are particles surface-modified with carboxy groups, obtained by reacting an antibody against a surface marker of extracellular vesicles, to which the antibody is immobilized via an amide bond, and the amide bond is a bond formed by reaction between the carboxy group and an amino group in the antibody. [7] The method of any one of [1] to [6], wherein the particles for nucleic acid extraction are particles surface-modified with carboxy groups or silica particles. [8] The method of any one of [1] to [7], wherein the method is carried out in a single container. [9] The method of any one of [1] to [8], wherein the particles for sedimentation are particles different from the particles for nucleic acid extraction.

[10] Any of the methods of [1] to [9], comprising the following steps: (1) mixing a sample containing extracellular vesicles with sedimentation particles having immobilized thereon a substance having affinity for extracellular vesicle membranes to produce a first mixture containing the sample containing extracellular vesicles and the sedimentation particles; (2) incubating the first mixture to produce a complex containing the extracellular vesicles and the sedimentation particles; and (3) separating the first mixture into a supernatant and a precipitate containing the complex; (4) removing the supernatant to obtain a precipitate containing the complex; (5) simultaneously or separately adding (i) a lysis solution of extracellular vesicles and (ii) particles for nucleic acid extraction to the precipitate containing the complex to produce a second mixture containing the complex, the lysis solution, and particles for nucleic acid extraction; (6) incubating the second mixture to adsorb nucleic acids eluted from the extracellular vesicles to the particles for nucleic acid extraction; and (7) recovering the particles for nucleic acid extraction to which nucleic acids are adsorbed from the second mixture.

[11] Any of the methods of [1] to [9], which comprises the following steps: (1) mixing a sample containing extracellular vesicles with sedimentation particles having immobilized thereon a substance having affinity for extracellular vesicle membranes to produce a first mixture containing the sample containing extracellular vesicles and the sedimentation particles; (2) incubating the first mixture to produce a complex containing the extracellular vesicles and the sedimentation particles; (3) separating the first mixture into a supernatant and a precipitate containing the complex; (4) removing the supernatant to obtain a precipitate containing the complex; (5) adding a lysis solution of extracellular vesicles to the precipitate containing the complex to produce a second mixture containing the complex and the lysis solution; (6) incubating the second mixture to elute nucleic acids from the extracellular vesicles; (7) adding particles for nucleic acid extraction to the eluted nucleic acids to produce a third mixture containing the eluted nucleic acids and particles for nucleic acid extraction; (8) incubating the third mixture to adsorb the eluted nucleic acid onto the particles for nucleic acid extraction; and (9) recovering the particles for nucleic acid extraction to which the nucleic acid has been adsorbed from the third mixture.

[12] The method of any of [1] to [8], wherein the particles for sedimentation are the same particles as the particles for nucleic acid extraction.

[13] Any of the methods [1] to [8] and

[12] , which comprises the following steps: (1) mixing a sample containing extracellular vesicles with particles for nucleic acid extraction to which a substance having affinity for the extracellular vesicle membrane has been immobilized to produce a first mixture containing the sample containing extracellular vesicles and the particles for nucleic acid extraction; (2) incubating the first mixture to produce a complex containing the extracellular vesicles and the particles for nucleic acid extraction; and (3) separating the first mixture into a supernatant and a precipitate containing the complex; (4) removing the supernatant to obtain a precipitate containing the complex; (5) adding a solution of lysing extracellular vesicles to the precipitate containing the complex to produce a second mixture containing the complex and the solution of lysing extracellular vesicles; (6) incubating the second mixture to adsorb nucleic acids eluted from the extracellular vesicles to the particles for nucleic acid extraction; and (7) recovering the particles for nucleic acid extraction from the second mixture.

[14] The method of any of [1] to

[13] , wherein either the sedimentation particles or the particles for nucleic acid extraction, or both the sedimentation particles and the particles for nucleic acid extraction, are magnetic particles.

[15] A method for analyzing nucleic acids, comprising: (1) recovering nucleic acids by any of the methods of [1] to

[14] ; and (2) analyzing the recovered nucleic acids.

[16] A reagent for recovering nucleic acids, comprising: (I) (I-1) sedimentation particles to which a substance having affinity for extracellular vesicle membranes is immobilized by an uncharged bond, or (I-2) sedimentation particles capable of immobilizing (a) a substance having affinity for extracellular vesicle membranes and (b) a substance having affinity for extracellular vesicle membranes via an uncharged bond; and (II) particles for nucleic acid extraction.

[17] The reagent of

[16] , wherein the sedimentation particles to which a substance having affinity for extracellular vesicle membranes is immobilized via an uncharged bond are sedimentation particles to which a substance having affinity for extracellular vesicle membranes is immobilized via an amide bond, or the sedimentation particles capable of immobilizing a substance having affinity for extracellular vesicle membranes via an uncharged bond are sedimentation particles surface-modified with carboxy groups.

[18] A reagent for recovering nucleic acids, comprising particles or silica particles surface-modified with carboxy groups to which a substance having affinity for extracellular vesicle membranes is immobilized via an uncharged bond.

[19] The reagent according to any one of

[16] to

[18] , wherein the nucleic acid is RNA and further comprises a chaotropic agent.

[20] A nucleic acid analysis kit comprising: (A) the reagent according to any one of

[16] to

[19] ; ​​and (B) a substance for analyzing nucleic acids; and (C) a chaotropic agent, if necessary.

[0009] According to the present invention, the step of transferring the separated extracellular vesicles to a nucleic acid (e.g., RNA) recovery system can be omitted, and thus nucleic acids in the extracellular vesicles can be recovered from an extracellular vesicle-containing sample in a single container (e.g., tube). Furthermore, according to the present invention, loss of rare nucleic acids and sample mix-up can be avoided.

[0010] Figure 1 shows the effect of the type and properties of magnetic particles used in immunoprecipitation on nucleic acid extraction efficiency. Figure 2 shows the results of nucleic acid extraction from EV immunoprecipitation using one type of magnetic particles. Figure 3 shows the results of a comparison between the present invention and a conventional method (phenol-chloroform extraction method). Figure 4 shows the effect of the size and surface structure of silica magnetic particles on nucleic acid extraction efficiency. Figure 5 shows the effect of the amount of particles for immunoprecipitation on nucleic acid extraction efficiency. Figure 6 shows the effect of the type of particles for nucleic acid extraction on nucleic acid extraction efficiency.

[0011] 1. Method for recovering nucleic acids The present invention provides a method for recovering nucleic acids, comprising: (A) separating extracellular vesicles from an extracellular vesicle-containing sample using sedimentation particles to which a substance having affinity for extracellular vesicle membranes is immobilized by an uncharged bond; (B) eluting nucleic acids from the extracellular vesicles; and (C) recovering the eluted nucleic acids using particles for nucleic acid extraction.

[0012] Examples of nucleic acids include DNA and RNA, and mixtures thereof. Preferably, the nucleic acid may be RNA.

[0013] In the above step (A), extracellular vesicles are separated from a sample containing extracellular vesicles using sedimentation particles to which a substance having affinity for the extracellular vesicle membrane is immobilized by an uncharged bond. The extracellular vesicles can be bound to the sedimentation particles via the substance having affinity for the extracellular vesicle membrane. Therefore, the extracellular vesicle membranes can be separated from the sample containing extracellular vesicles in a form bound to the sedimentation particles (precipitate).

[0014] Extracellular vesicles are tiny vesicles with a membrane structure secreted from various cells. Examples of extracellular vesicles include exosomes, ectosomes, and apoptotic vesicles. Preferably, the extracellular vesicles are exosomes. Extracellular vesicles can also be defined by their size. The size of extracellular vesicles is, for example, 30 to 1000 nm, preferably 50 to 300 nm, and more preferably 80 to 200 nm. The size of extracellular vesicles can be measured by, for example, a method based on the Brownian motion of extracellular vesicles, a light scattering method, an electrical resistance method, or the like. Preferably, the size of extracellular vesicles is measured using NanoSight (Malvern Instruments).

[0015] The sample containing extracellular vesicles can be any sample containing extracellular vesicles. Preferably, the sample containing extracellular vesicles is a biological liquid sample. The sample containing extracellular vesicles can be subjected to other treatments before being used in the method of the present invention. Such treatments include, for example, centrifugation, extraction, filtration, precipitation, heating, freezing, refrigeration, and stirring.

[0016] In one embodiment, the extracellular vesicle-containing sample is a culture supernatant sample. The culture supernatant sample may be a cell culture supernatant sample or a tissue culture supernatant sample. Examples of organisms from which the cultured cells or tissues are derived include mammals (e.g., primates such as humans and monkeys; rodents such as mice, rats, and rabbits; ungulates such as cows, pigs, goats, horses, and sheep; birds (e.g., chickens); insects, microorganisms (e.g., bacteria), plants, and fish. Preferably, the organism is a mammal, preferably a human.

[0017] In another embodiment, the extracellular vesicle-containing sample is an animal-derived liquid sample. An animal-derived liquid sample is a bodily fluid sample derived from an organism as described above. Examples of bodily fluid samples include blood samples (e.g., whole blood, serum, and plasma), urine, saliva, lymph, tissue fluid, cerebrospinal fluid, ascites, sweat, semen, tears, mucus, milk, pleural fluid, bronchoalveolar lavage fluid, and amniotic fluid. Preferably, the bodily fluid is a blood sample, urine, or saliva. Examples of plasma include EDTA plasma, heparin plasma, citrate plasma, sodium fluoride plasma, and plasma containing ACD (acid-citrate-dextrose) or CPD (citrate phosphate dextrose).

[0018] As the sedimentation particles, sedimentation particles (preferably immunoprecipitation particles) to which a substance having affinity for the extracellular vesicle membrane is immobilized by an uncharged bond can be used. The sedimentation particles are not particularly limited as long as they can immobilize a substance having affinity for the extracellular vesicle membrane and can be recovered by a predetermined operation (e.g., magnetic operation or centrifugation). Examples of sedimentation particles include microparticles, nanoparticles, microbeads, nanobeads, microspheres, and nanospheres. Examples of sedimentation particles include inorganic particles (e.g., metal particles, silica particles), organic particles (e.g., polymer particles), and organic-inorganic composite particles. The shape of the sedimentation particles may be spherical or non-spherical (e.g., elliptical).

[0019] The average particle size of the particles for precipitation is not particularly limited. From the viewpoint of ease of recovery, the average particle size may be, for example, 0.001 to 1000 μm, preferably 0.01 to 100 μm, more preferably 0.1 to 10 μm, even more preferably 0.5 to 5 μm, and particularly preferably 1 to 3 μm. The average particle size of the particles for precipitation can be measured by the BET method.

[0020] Preferably, the particles for sedimentation are magnetic particles, from the viewpoint of ease of recovery by magnetic manipulation. The magnetic particles are particles containing a magnetic material such as iron, nickel, cobalt, or an alloy thereof (e.g., ferrite). The magnetic particles preferably have an average particle size of 1 to 3 μm.

[0021] Substances having affinity for extracellular vesicle membranes are substances capable of binding to surface markers of extracellular vesicles. Examples of surface markers of extracellular vesicles include tetraspanin membrane proteins (EV membrane-specific four-spanning membrane proteins, e.g., CD9, CD63, CD81), extracellular matrix metalloproteinase inducers (e.g., CD147), heat shock protein (HSP) 70, HSP90, major histocompatibility complex (MHC) I, lysosome-associated membrane protein (LAMP) 1, intercellular adhesion molecule (ICAM)-1, integrin, ceramide, cholesterol, phosphatidylserine, Annexins, Caveolin-I, and EpCAM. The surface marker of extracellular vesicles is preferably a tetraspanin membrane protein (e.g., CD9, CD63, CD81) or an extracellular matrix metalloproteinase inducer (e.g., CD147).

[0022] Examples of substances having affinity for extracellular vesicle membranes include antibodies, aptamers, phosphatidylserine-binding proteins, and ceramide-binding proteins against the above-mentioned extracellular vesicle surface markers. In the present invention, a single substance or multiple (e.g., two, three, or four) types of substances can be used as the affinity substance for the extracellular vesicle surface markers. Preferably, the substance having affinity for the extracellular vesicle membrane may be an antibody or a binding fragment thereof against the above-mentioned extracellular vesicle surface markers. More preferably, the substance having affinity for the extracellular vesicle membrane may be an antibody against a tetraspanin membrane protein (e.g., CD9, CD63, CD81) or an extracellular matrix metalloproteinase inducer (e.g., CD147). Note that the "antibody" in the present invention includes not only a complete antibody but also an antibody fragment (e.g., Fab, Fab', F(ab')). ２ , Fv, single-chain antibodies, diabodies, etc.), and minibodies in which the variable regions of antibodies are bound.

[0023] The substance having affinity for the extracellular vesicle membrane is immobilized on the sedimentation particles by an uncharged bond, such as an amide bond or a non-covalent bond between a pair of affinity binding substances, such as biotin and streptavidin, with an amide bond being preferred.

[0024] Precipitation particles on which a substance having affinity for the extracellular vesicle membrane is immobilized via an uncharged bond can be prepared by reacting a substance having affinity for the extracellular vesicle membrane with precipitating particles capable of immobilizing a substance having affinity for the extracellular vesicle membrane via an uncharged bond. As precipitating particles capable of immobilizing a substance having affinity for the extracellular vesicle membrane via an uncharged bond, precipitating particles having a surface modification with a reactive group (e.g., amino group, carboxy group) capable of forming the above-mentioned uncharged bond upon reaction with a substance having affinity for the extracellular vesicle membrane can be used.

[0025] In a specific embodiment, the sedimentation particles may be sedimentation particles to which an antibody against a surface marker of extracellular vesicles is immobilized via an amide bond. Such sedimentation particles may be obtained by reacting particles having a surface modification with a carboxy group with an antibody against a surface marker of extracellular vesicles, and the antibody is immobilized via an amide bond (an amide bond formed by reaction between a carboxy group in the particle and a primary amine (an amino group in a side chain of a lysine residue) in the antibody). The reaction between the carboxy group in the particle and the amino group in the side chain of a lysine residue in the antibody can be preferably carried out by carbodiimide-activating the carboxy group in the particle with EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiiimide) and reacting this with the amino group in the side chain of a lysine residue in the antibody. More preferably, the reaction can be carried out by carbodiimide-activating the carboxy group in the particle with EDC, further forming an NHS ester with NHS (N-Hydroxysuccinimide) or an analog thereof (e.g., Sulfo-NHS), and then reacting this with the amino group in the side chain of a lysine residue in the antibody. The reaction of a carboxy group with a primary amine using EDC and NHS is widely known and can be carried out according to standard methods. EDC and NHS are commercially available, and the reaction of a carboxy group with a primary amine can be carried out according to the protocols attached to the commercially available products.

[0026] In another embodiment, the sedimentation particles may be sedimentation particles to which an antibody specific for a surface marker of extracellular vesicles is immobilized or to which an antibody specific for a surface marker of extracellular vesicles is immobilized via a pair of affinity binding substances. Such sedimentation particles may include, for example, particles to which streptavidin is immobilized via an uncharged bond and an antibody specific for a surface marker of extracellular vesicles to which biotin is bound via an uncharged bond. Examples of uncharged bonds include an amide bond.

[0027] More specifically, the above step (A) can be carried out by the following steps (A1) to (A4): (A1) mixing an extracellular vesicle-containing sample with sedimentation particles to which a substance having affinity for extracellular vesicle membranes is immobilized to produce a first mixture containing the extracellular vesicle-containing sample and the sedimentation particles; (A2) incubating the first mixture to produce a complex containing the extracellular vesicles and the sedimentation particles; and (A3) separating the first mixture into a supernatant and a precipitate containing the complex; and (A4) removing the supernatant to obtain a precipitate containing the complex.

[0028] The mixing in step (A1) is carried out under conditions sufficient to produce a mixture in which the sedimentation particles having the substance having affinity for the extracellular vesicle membrane immobilized thereon are thoroughly mixed. Such temperature conditions are, for example, 4 to 60°C, preferably 15 to 50°C, and more preferably 20 to 40°C. The time required to prepare the mixture is, for example, 30 seconds or less, preferably 20 seconds or less, and more preferably 15 seconds or less. The mixing time may also be 1 second or more, 3 seconds or more, or 5 seconds or more. Mixing can be carried out by inversion, stirring, or pipetting.

[0029] The incubation in step (A2) is carried out under conditions sufficient to produce sedimentation particles bound to extracellular vesicles via a substance having affinity for the extracellular vesicle membrane. The incubation time varies depending on factors such as the time required to prepare the mixture, the desired amount of extracellular vesicles to be recovered, and the incubation temperature, and is, for example, 48 hours or less, preferably 24 hours or less, more preferably 120 minutes or less, and even more preferably 60 minutes or less. From the viewpoint of rapid processing, the incubation time is even more preferably 30 minutes or less, and particularly preferably 20 minutes or less, 10 minutes or less, or 5 minutes or less. The incubation time may also be 10 seconds or more, 30 seconds or more, or 1 minute or more. The incubation temperature is the same as the temperature conditions for the above-mentioned mixing. By performing incubation under such conditions, the amount of sedimentation particles bound to extracellular vesicles can be increased.

[0030] In the step (A3), the mixture obtained in the step (A2) can be separated into a supernatant and a precipitate containing the complex. The sedimentation particles are bound to the extracellular vesicles via a substance having affinity for the extracellular vesicle membrane. Therefore, by centrifugation or magnetic manipulation (when the sedimentation particles are magnetic particles), the mixture can be separated into a supernatant (a liquid from which the extracellular vesicles bound to the sedimentation particles have been removed) and a precipitate containing the complex (a substance containing extracellular vesicles).

[0031] In the above step (A4), the supernatant can be removed to obtain a precipitate containing the complex.

[0032] The method of the present invention may further comprise washing the precipitate (sedimentation particles to which a substance having affinity for extracellular vesicle membranes is immobilized by uncharged bonds) after separating the precipitate containing the complex. The precipitate can be washed with an aqueous solution (e.g., a buffer solution). The number of washing steps is usually one to three times.

[0033] In the above step (B), nucleic acids (e.g., RNA) are eluted from extracellular vesicles. Nucleic acids can be eluted from extracellular vesicles by any method. For example, such elution can be performed using a nucleic acid extraction reagent (in other words, a solution for lysing extracellular vesicles). Various reagents are known. Alternatively, such elution can be performed by ultrasonic treatment. A protease (e.g., proteinase K) may be used to elute nucleic acids from extracellular vesicles. When recovery of RNA as the nucleic acid is desired, the solution for lysing extracellular vesicles preferably contains the same chaotropic agent as that used for adsorption of RNA to nucleic acid extraction particles in step (C) described below. By using a chaotropic agent as the lysing solution in step (B), it is not necessary to add an additional chaotropic agent in step (C). Furthermore, by using a chaotropic agent as the lysing solution, RNA eluted when extracellular vesicles are lysed can be protected from RNAse, eliminating the need to separately add an RNAse inhibitor or the like.

[0034] The chaotropic agent is preferably a chaotropic salt that generates chaotropic ions in a solution such as water. Examples of chaotropic salts include guanidine thiocyanate, guanidine hydrochloride, thiocyanate, sodium iodide, potassium iodide, sodium perchlorate, or a combination thereof. Preferred examples include guanidine thiocyanate or guanidine hydrochloride. The concentration of the chaotropic agent used as a lysis solution is not particularly limited as long as it can elute RNA from extracellular vesicles, and may be, for example, 0.5 to 15.0 M. The concentration of the chaotropic agent may be preferably 1.0 to 10.0 M, more preferably 1.0 to 8.0 M, even more preferably 1.0 to 4.0 M, and particularly preferably 1.0 to 3.0 M.

[0035] The solution for dissolving extracellular vesicles may further contain a surfactant or a chelating agent. The surfactant may be any surfactant useful for dissolving membranes. Examples include anionic surfactants and nonionic surfactants, and preferably, N-lauroyl sarcosine sodium (sarcosyl) or NP-40. The concentration of the surfactant used as the solution is not particularly limited as long as it can elute nucleic acids (e.g., RNA) from the extracellular vesicles, but a concentration of 0.1 to 5.0% is preferred. The chelating agent may be the same as the chelating agent described below in the section on mixing the extracellular vesicle-containing sample with sedimentation particles. EDTA is preferred. The concentration of the chelating agent used as the solution is not particularly limited as long as it can elute nucleic acids from the extracellular vesicles, but a concentration of 1 to 5 mM is preferred.

[0036] More specifically, the step (B) can be carried out by the following steps (B1) to (B2): (B1) (i) adding an extracellular vesicle lysis solution to the precipitate containing the complex to generate a second mixture containing the complex and the lysis solution; and (B2) incubating the second mixture to elute nucleic acids from the extracellular vesicles.

[0037] The mixing in step (B1) is carried out under conditions sufficient to produce a mixture in which the complex and the dissolution solution are thoroughly mixed. Such temperature conditions are, for example, 4 to 50°C, preferably 10 to 40°C, and more preferably 20 to 40°C. The time required to prepare the mixture is, for example, 30 seconds or less, preferably 20 seconds or less, and more preferably 15 seconds or less. The mixing time may also be 1 second or more, 3 seconds or more, or 5 seconds or more. Mixing can be performed by inversion, stirring, or pipetting.

[0038] The incubation in step (B2) is carried out under conditions sufficient to elute nucleic acids from extracellular vesicles. The incubation time varies depending on factors such as the time required to prepare the mixture, the desired amount of nucleic acid recovered, and the incubation temperature, and is, for example, 6 hours or less, preferably 2 hours or less, and more preferably 60 minutes or less. From the viewpoint of rapid processing, the incubation time is even more preferably 30 minutes or less, and particularly preferably 20 minutes or less, 10 minutes or less, or 5 minutes or less. The incubation time may also be 10 seconds or more, 30 seconds or more, or 1 minute or more. The incubation temperature is the same as the temperature conditions for the above-mentioned mixing. By mixing under such conditions, the amount of nucleic acid eluted from extracellular vesicles can be increased.

[0039] In the above step (C), the eluted nucleic acid (e.g., RNA) is recovered using particles for nucleic acid extraction. When recovery of RNA as nucleic acid is desired, the above step (C) is preferably carried out in the presence of a chaotropic agent. The concentration of the chaotropic agent is the same as that described above. The particles for nucleic acid extraction have the ability to adsorb RNA in the presence of a chaotropic agent. Therefore, when recovery of RNA as nucleic acid is desired, the eluted RNA can be bound to the particles for nucleic acid extraction by mixing the eluted RNA and the particles for nucleic acid extraction in the presence of a chaotropic agent. Therefore, the eluted RNA can be recovered from the solution obtained in the above step (B) in a form bound to the particles for nucleic acid extraction (precipitate).

[0040] Furthermore, when a chaotropic agent is used as the solution for dissolving extracellular vesicles in step (B) above, the solution containing the eluted RNA contains the chaotropic agent, and therefore, by mixing the solution containing the eluted RNA with particles for nucleic acid extraction, the eluted RNA can be adsorbed onto the particles for nucleic acid extraction.

[0041] The nucleic acid extraction particles are not particularly limited as long as they can be recovered by a predetermined procedure (e.g., magnetic manipulation or centrifugation). The nucleic acid extraction particles may also be capable of immobilizing a substance having affinity for the extracellular vesicle membrane. Examples of nucleic acid extraction particles include microparticles, nanoparticles, microbeads, nanobeads, microspheres, and nanospheres. Examples of nucleic acid extraction particles include particles with a carboxyl group surface modification and silica particles. Examples of particles with a carboxyl group surface modification include inorganic particles (e.g., metal particles, silica particles), organic particles (e.g., polymer particles such as gelatin particles), and organic-inorganic composite particles with a carboxyl group surface modification. Examples of silica particles include inorganic particles (e.g., metal particles, silica particles), organic particles (e.g., polymer particles), and organic-inorganic composite particles whose surfaces are coated with silica. The shape of the nucleic acid extraction particles may be spherical or non-spherical (e.g., ellipsoidal).

[0042] The average particle size of the particles for nucleic acid extraction is not particularly limited. From the viewpoint of ease of recovery, etc., it may be, for example, 0.001 to 1000 μm, preferably 0.01 to 100 μm, more preferably 0.1 to 10 μm, even more preferably 0.5 to 5 μm, and particularly preferably 1 to 3 μm. The average particle size of the particles for nucleic acid extraction can be measured by the BET method.

[0043] Preferably, the particles for nucleic acid extraction may be silica magnetic particles, from the viewpoint of ease of recovery by magnetic manipulation. Silica magnetic particles are particles for nucleic acid extraction that contain a magnetic material such as iron, nickel, cobalt, or an alloy thereof (e.g., ferrite). The particles for nucleic acid extraction (e.g., magnetic particles surface-modified with carboxy groups, silica magnetic particles) preferably have an average particle diameter of 1 to 3 μm.

[0044] As described above, chaotropic agents are preferably chaotropic salts that generate chaotropic ions in a solution such as water, but are not particularly limited as long as they contribute to the adsorption of RNA to nucleic acid extraction particles (e.g., silica-containing particles, particles surface-modified with carboxyl groups). Examples of chaotropic salts include guanidine thiocyanate, guanidine hydrochloride, thiocyanate, sodium iodide, potassium iodide, sodium perchlorate, or combinations thereof. Preferred are guanidine thiocyanate and guanidine hydrochloride. The concentration of the chaotropic agent in step (C) varies depending on the type of chaotropic agent, but is not particularly limited as long as it allows RNA to adsorb to the nucleic acid extraction particles, and may be, for example, 1.0 to 15.0 M. The concentration of the chaotropic agent may be preferably 1.0 to 10.0 M, more preferably 1.0 to 8.0 M, even more preferably 1.0 to 4.0 M, and particularly preferably 1.0 to 3.0 M.

[0045] Step (C) may be carried out in the presence of a lower fatty alcohol such as isopropanol, ethanol, methanol, or butanol in addition to a chaotropic agent. Isopropanol is preferred as the lower fatty alcohol. The concentration of the lower fatty alcohol in step (C) varies depending on the type of lower fatty alcohol and the type and concentration of the chaotropic agent, but is not particularly limited as long as it allows nucleic acids (e.g., RNA) to be adsorbed to particles for nucleic acid extraction (e.g., silica-containing particles, particles surface-modified with carboxy groups). For example, a final concentration of 30 to 60% isopropanol can be suitably used.

[0046] Furthermore, when a chaotropic agent is used as the solution for dissolving extracellular vesicles in step (B) above, the solution containing the eluted nucleic acid (e.g., RNA) contains the chaotropic agent, and therefore in step (C), a lower fatty alcohol and particles for nucleic acid extraction may be added to the solution containing the eluted nucleic acid and mixed.

[0047] Step (C) may be carried out in the presence of, in addition to a chaotropic agent, one or more (e.g., one, two, three, four, or five) components selected from the group consisting of surfactants, such as anionic surfactants such as N-lauroyl sarcosinate sodium (sarcosyl), and nonionic surfactants such as NP-40, and chelating agents such as EDTA. The concentrations of the surfactant and chelating agent in step (C) vary depending on their types, but are not particularly limited as long as they are concentrations that allow nucleic acids (e.g., RNA) to be adsorbed to nucleic acid extraction particles (e.g., silica-containing particles, particles surface-modified with carboxy groups). A suitable surfactant concentration is, for example, 0.1% to 5.0%. A suitable chelating agent concentration is, for example, 1 to 5 mM.

[0048] More specifically, the step (C) can be carried out by the following steps (C1) to (C3): (C1) adding particles for nucleic acid extraction to the nucleic acid eluted from the extracellular vesicles to produce a third mixture containing the nucleic acid and the particles for nucleic acid extraction; (C2) incubating the third mixture to allow the nucleic acid eluted from the extracellular vesicles to be adsorbed onto the particles for nucleic acid extraction; and (C3) recovering the particles for nucleic acid extraction to which the nucleic acid has been adsorbed from the third mixture.

[0049] The mixing in step (C1) is carried out under conditions sufficient to produce a mixture in which the eluted nucleic acid (e.g., RNA) and the particles for nucleic acid extraction are thoroughly mixed. Such temperature conditions are, for example, 4 to 60°C, preferably 15 to 50°C, and more preferably 20 to 40°C. The time required to prepare the mixture is, for example, 30 seconds or less, preferably 20 seconds or less, and more preferably 15 seconds or less. The mixing time may also be 1 second or more, 3 seconds or more, or 5 seconds or more. Mixing can be carried out by inversion, stirring, or pipetting.

[0050] The incubation in step (C2) is carried out under conditions sufficient to produce particles for nucleic acid extraction to which nucleic acids (e.g., RNA) are adsorbed. The incubation time varies depending on factors such as the time required to prepare the mixture, the desired amount of nucleic acid to be recovered, and the incubation temperature, and is, for example, 6 hours or less, preferably 2 hours or less, and more preferably 60 minutes or less. From the viewpoint of rapid processing, the incubation time is even more preferably 30 minutes or less, and particularly preferably 20 minutes or less, 10 minutes or less, or 5 minutes or less. The incubation time may also be 10 seconds or more, 30 seconds or more, or 1 minute or more. The incubation temperature is the same as the temperature conditions for the above-mentioned mixing. By mixing under such conditions, the amount of particles for nucleic acid extraction to which nucleic acids are adsorbed can be increased.

[0051] In the step (C3), nucleic acid extraction particles with adsorbed nucleic acids (e.g., RNA) can be recovered from the third mixture obtained in the step (C2). The nucleic acid extraction particles are bound to nucleic acids. Therefore, the mixture can be separated into a supernatant (a solution not containing nucleic acids) and a precipitate (a solution containing nucleic acids) containing the nucleic acid extraction particles with adsorbed nucleic acids by centrifugation or magnetic manipulation (when the nucleic acid extraction particles are silica magnetic particles).

[0052] When recovery of RNA as the nucleic acid is desired, the above steps (C1) to (C3) can be preferably carried out in the presence of a chaotropic agent, similarly to the above step (C).

[0053] Alternatively, the above steps (B) and (C) may be carried out by the following steps (BC1) to (BC3): (BC1) adding (i) a lysis solution of extracellular vesicles, and (ii) particles for nucleic acid extraction, simultaneously or separately, to the precipitate containing the complex to produce a second mixture containing the complex, the lysis solution, and particles for nucleic acid extraction; (BC2) incubating the second mixture to proceed with both elution of nucleic acids from the extracellular vesicles and adsorption of the eluted nucleic acids to the particles for nucleic acid extraction; and (BC3) recovering the particles for nucleic acid extraction to which nucleic acids have been adsorbed from the second mixture.

[0054] The mixing in step (BC1) is carried out under conditions sufficient to produce a mixture in which the precipitate, the extracellular vesicle lysate, and the particles for nucleic acid extraction are thoroughly mixed. Such temperature conditions are, for example, 4 to 60°C, preferably 15 to 50°C, and more preferably 20 to 40°C. The time required to prepare the mixture is, for example, 30 seconds or less, preferably 20 seconds or less, and more preferably 15 seconds or less. The mixing time may also be 1 second or more, 3 seconds or more, or 5 seconds or more. The precipitate may be resuspended before the addition of steps (i) and (ii) above. Mixing may be performed by inversion, stirring, or pipetting.

[0055] The incubation in step (BC2) is carried out under conditions sufficient to elute nucleic acids (e.g., RNA) from the extracellular vesicles and produce particles for nucleic acid extraction that adsorb the eluted nucleic acids. The incubation time varies depending on factors such as the time required to prepare the mixture, the desired amount of nucleic acid to be recovered, and the incubation temperature, and is, for example, 6 hours or less, preferably 2 hours or less, and more preferably 60 minutes or less. From the viewpoint of rapid processing, the incubation time is even more preferably 30 minutes or less, and particularly preferably 20 minutes or less, 10 minutes or less, or 5 minutes or less. The incubation time may also be 10 seconds or more, 30 seconds or more, or 1 minute or more. The incubation temperature is the same as the temperature conditions for the above-mentioned mixing. By mixing under such conditions, the amount of particles for nucleic acid extraction that adsorb nucleic acids can be increased.

[0056] In the step (BC3), nucleic acid extraction particles that have adsorbed nucleic acid (e.g., RNA) can be recovered from the second mixture obtained in the step (BC2). The nucleic acid extraction particles are bound to nucleic acid. Therefore, the mixture can be separated into a supernatant (a solution not containing nucleic acid) and a precipitate (a solution containing nucleic acid) that contains the nucleic acid extraction particles that have adsorbed nucleic acid by centrifugation or magnetic manipulation (when the nucleic acid extraction particles are silica magnetic particles).

[0057] When recovery of RNA as the nucleic acid is desired, the above steps (BC1) to (BC3) can be preferably carried out in the presence of a chaotropic agent, similarly to the above step (C).

[0058] The method of the present invention may further comprise washing the precipitate (particles for nucleic acid extraction having adsorbed nucleic acids such as RNA) after separating the precipitate. The precipitate can be washed using an aqueous solution (e.g., a solution containing a low concentration of a chaotropic agent, a buffer solution), ethanol, or the like. The number of washes is usually one to three times. For example, after washing with a solution containing a low concentration of a chaotropic agent, the precipitate can be washed with an alcohol such as 60 to 80% ethanol.

[0059] Nucleic acids (e.g., RNA) adsorbed to particles for nucleic acid extraction may be treated to be released from the particles for nucleic acid extraction. Such treatment may include, for example, replacing the eluent with an eluent having a low salt concentration. Examples of eluents with a low salt concentration include nuclease-free (RNAse-free) water and TE (Tris-EDTA) buffer. Elutions that release nucleic acids from particles for nucleic acid extraction are commercially available, and commercially available products may be used.

[0060] In one embodiment, the sedimentation particles may be different from the nucleic acid extraction particles. In this case, the sedimentation particles having a substance having affinity for extracellular vesicle membranes immobilized thereon and the nucleic acid extraction particles are used in combination in the method of the present invention (two-bead method). Such a method (two-bead method) can be carried out, for example, by a method comprising the following steps: (1) mixing a sample containing extracellular vesicles with sedimentation particles to which a substance having affinity for extracellular vesicle membranes has been immobilized to produce a first mixture containing the sample containing extracellular vesicles and the sedimentation particles; (2) incubating the first mixture to produce a complex containing the extracellular vesicles and the sedimentation particles; (3) separating the first mixture into a supernatant and a precipitate containing the complex; (4) removing the supernatant to obtain a precipitate containing the complex; (5) adding a lysis solution of extracellular vesicles to the precipitate containing the complex to produce a second mixture containing the complex and the lysis solution; (6) incubating the second mixture to elute nucleic acids (e.g., RNA) from the extracellular vesicles; (7) adding particles for nucleic acid extraction to the eluted nucleic acids to produce a third mixture containing the eluted nucleic acids and particles for nucleic acid extraction; (8) incubating the third mixture to adsorb the eluted nucleic acid onto the particles for nucleic acid extraction; and (9) recovering the particles for nucleic acid extraction to which the nucleic acid has been adsorbed from the third mixture. The above steps (1) to (9) can be performed in the same manner as the steps described above.

[0061] Furthermore, such a two-bead method can be carried out, for example, by a method comprising the following steps: (1) mixing a sample containing extracellular vesicles with sedimentation particles to which a substance having affinity for the extracellular vesicle membrane has been immobilized to produce a first mixture containing the sample containing extracellular vesicles and the sedimentation particles; (2) incubating the first mixture to produce a complex containing the extracellular vesicles and the sedimentation particles; (3) separating the first mixture into a supernatant and a precipitate containing the complex; (4) removing the supernatant to obtain a precipitate containing the complex; (5) simultaneously or separately adding (i) a lysis solution of extracellular vesicles and (ii) particles for nucleic acid extraction to the precipitate containing the complex to produce a second mixture containing the complex, the lysis solution, and particles for nucleic acid extraction; (6) incubating the second mixture to adsorb nucleic acids (e.g., RNA) eluted from the extracellular vesicles to the particles for nucleic acid extraction; and (7) recovering the particles for nucleic acid extraction to which nucleic acids have been adsorbed from the second mixture. The steps (1) to (7) can be carried out in the same manner as the steps described above.

[0062] Such a method (two-bead method) may also be carried out, for example, by a method comprising the following steps: (1) mixing a sample containing extracellular vesicles with (i) sedimentation particles having immobilized thereon a substance having an affinity for extracellular vesicle membranes, and (ii) particles for nucleic acid extraction to produce a first mixture containing the sample containing extracellular vesicles, the sedimentation particles, and the particles for nucleic acid extraction; (2) incubating the first mixture to produce a complex containing the extracellular vesicles and the sedimentation particles; and (3) separating the first mixture into a supernatant and a precipitate containing the complex and the particles for nucleic acid extraction; (4) removing the supernatant to obtain a precipitate containing the complex and the particles for nucleic acid extraction; (5) adding a solution of lysing extracellular vesicles to the precipitate containing the complex and the particles for nucleic acid extraction to produce a second mixture containing the complex, the solution of lysing extracellular vesicles, and particles for nucleic acid extraction; (6) incubating the second mixture to adsorb nucleic acids (e.g., RNA) eluted from the extracellular vesicles to the particles for nucleic acid extraction; and (7) Recovering the particles for nucleic acid extraction to which nucleic acids are adsorbed from the second mixture. The above steps (1) to (7) can be carried out in the same manner as the steps described above.

[0063] In another embodiment, the sedimentation particles may be the same as the nucleic acid extraction particles. In this case, the sedimentation particles to which a substance having affinity for extracellular vesicle membranes is immobilized and the nucleic acid extraction particles to which a substance having affinity for extracellular vesicle membranes is immobilized are used (single-bead method). That is, in step (A), nucleic acid extraction particles to which a substance having affinity for extracellular vesicle membranes is immobilized are used, and these nucleic acid extraction particles are also used in step (C). Such a method can be carried out, for example, by a method comprising the steps of: (1) mixing a sample containing extracellular vesicles with particles for nucleic acid extraction to which a substance having affinity for the extracellular vesicle membrane has been immobilized to produce a first mixture containing the sample containing extracellular vesicles and the particles for nucleic acid extraction; (2) incubating the first mixture to produce a complex containing the extracellular vesicles and the particles for nucleic acid extraction; and (3) separating the first mixture into a supernatant and a precipitate containing the complex; (4) removing the supernatant to obtain a precipitate containing the complex; (5) adding a solution of lysing extracellular vesicles to the precipitate containing the complex to produce a second mixture containing the complex and the solution of lysing extracellular vesicles; (6) incubating the second mixture to adsorb nucleic acids (e.g., RNA) eluted from the extracellular vesicles to the particles for nucleic acid extraction; and (7) recovering the particles for nucleic acid extraction from the second mixture. The above steps (1) to (7) can be carried out in the same manner as the steps described above.

[0064] The nucleic acid extraction particles used in the single-bead method, to which a substance having affinity for extracellular vesicle membranes is immobilized, can be prepared in the same manner as the precipitation particles to which the substance having affinity for extracellular vesicle membranes is immobilized via uncharged bonds. For example, when silica particles are used as the nucleic acid extraction particles, the nucleic acid extraction particles can be prepared by reacting silica particles surface-modified with carboxy groups with amino groups in the side chains of lysine residues in antibodies using a crosslinker such as N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiiimide (EDC). Furthermore, when particles surface-modified with carboxy groups are used as the nucleic acid extraction particles, antibodies can be immobilized to the nucleic acid extraction particles using a similar technique. In this case, in order to leave carboxyl groups for nucleic acid adsorption, it is preferable to use a smaller amount of crosslinker (e.g., EDC and NHS) and antibody than those used in the protocol attached to the commercially available product (e.g., about 1 / 4 to 3 / 4, typically about 1 / 2).

[0065] The method of the present invention can be easily performed in a single container (e.g., a tube, plate, or well). This is because the method of the present invention can separate extracellular vesicles from a sample containing extracellular vesicles as a precipitate (sedimentation particles with bound extracellular vesicles), and can recover nucleic acids (e.g., RNA) from the extracellular vesicles as a precipitate (nucleic acid extraction particles with adsorbed nucleic acids). Therefore, the extracellular vesicles and nucleic acids can be recovered by removing the supernatant, eliminating the need to transfer the container. On the other hand, in prior art techniques such as phenol / chloroform extraction or nucleic acid purification kits using the same, nucleic acids are contained in the upper liquid phase, and therefore, the upper liquid phase must be transferred to another container to recover the nucleic acids. Furthermore, in prior art techniques related to the recovery of nucleic acids from extracellular vesicles using particles for nucleic acid extraction, the separation of extracellular vesicles from a sample containing extracellular vesicles and the recovery of nucleic acids from the separated extracellular vesicles are performed in separate containers, rather than in a single container. However, the inventors have found that simply performing these processes in a single container results in a lower amount of recovered nucleic acid than when performed in separate containers. In contrast, the method of the present invention uses sedimentation particles to which a substance having affinity for the extracellular vesicle membrane is immobilized by an uncharged bond in the separation of extracellular vesicles from an extracellular vesicle-containing sample, and nucleic acid extraction particles in the recovery of nucleic acid from the extracellular vesicles. Therefore, the method of the present invention has the advantage of being easy to perform in a single container, unlike the prior art.

[0066] In the method of the present invention, a chelating agent may be used in combination with the extracellular vesicle-containing sample to improve the recovery rate of extracellular vesicles. Treating the extracellular vesicle-containing sample with a chelating agent can improve the recovery rate of extracellular vesicles (e.g., WO 2018 / 070479).

[0067] A chelating agent is a compound or salt thereof having a coordinating moiety capable of forming a coordinate bond with a metal ion. The number of coordinating moieties is preferably two or more, more preferably three or more (e.g., three or six). Examples of the coordinating atom as the coordinating moiety include an oxygen atom, a phosphorus atom, a nitrogen atom, a sulfur atom, and a chlorine atom. The coordinating atom is preferably an oxygen atom or a phosphorus atom, more preferably an oxygen atom. Examples of the coordinating group as the coordinating moiety include groups having the above-mentioned coordinating atoms. The coordinating group is preferably a carboxylic acid group or a phosphate group, more preferably a carboxylic acid group.

[0068] Examples of chelating agents include hydroxyethyliminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetra(methylenephosphonic acid) (EDTMP), glycoletherdiaminetetraacetic acid (EGTA), and salts thereof. Examples of salts include metal salts (e.g., monovalent metal salts such as sodium salts and potassium salts, and divalent metal salts such as calcium salts and magnesium salts), inorganic salts (e.g., halide salts such as fluorides, chlorides, bromides, and iodides, and ammonium salts), organic salts (e.g., ammonium salts substituted with alkyl groups), and acid addition salts (e.g., salts with inorganic acids such as sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, and phosphoric acid, and salts with organic acids such as acetic acid, oxalic acid, lactic acid, citric acid, trifluoromethanesulfonic acid, and trifluoroacetic acid).

[0069] The chelating agent may also be a chelating agent commonly used as a component contained in blood collection tubes for clinical tests. Examples of such chelating agents include EDTA, EGTA, NTA, HEDTA, EDTMP, HIDA, citric acid, and salts thereof. In the present invention, the use of such chelating agents is also desirable from the viewpoint of clinical application.

[0070] In the present invention, one type of chelating agent may be used alone, or multiple types (e.g., two, three, or four) of chelating agents may be used in combination. The concentration of the chelating agent varies depending on factors such as the type and concentration of other components used in combination with the chelating agent, and may be, for example, 10 mM to 1000 mM, preferably 20 mM to 500 mM, more preferably 30 mM to 300 mM, and even more preferably 50 mM to 200 mM.

[0071] 2. Method for Analyzing Nucleic Acids The present invention also provides a method for analyzing nucleic acids (e.g., RNA), which includes the following steps (1) and (2): (1) recovering nucleic acids by the above-described method for recovering nucleic acids; and (2) analyzing the recovered nucleic acids.

[0072] Step (1) in the analytical method of the present invention can be carried out in the same manner as in the recovery method of the present invention.

[0073] In the above step (2), the analysis of nucleic acids can be performed qualitatively or quantitatively. Such analysis can also be the analysis of one or more nucleic acids.

[0074] Examples of methods for analyzing nucleic acids include hybridization methods using probes, gene amplification methods using primers (eg, 2, 3, or 4 primers), and mass spectrometry.

[0075] It has been reported that extracellular vesicles may be involved in various diseases such as cancer (WO 2014 / 003053; WO 2014 / 152622; Taylor et al., Gynecologic Oncol, 100 (2008) pp. 13-21). Therefore, the present invention is useful, for example, for diagnosis and drug discovery based on nucleic acids contained in extracellular vesicles.

[0076] 3. Reagents and Kits The present invention also provides reagents and kits that can be used in the above-described methods of the present invention.

[0077] In one embodiment, the reagent of the present invention may be a reagent suitable for the above-mentioned two-bead method. Such a reagent may be a reagent for recovering nucleic acids (e.g., RNA), comprising: (I) (I-1) sedimentation particles to which a substance having an affinity for extracellular vesicle membranes is immobilized via an uncharged bond, or (I-2) sedimentation particles capable of immobilizing (a) a substance having an affinity for extracellular vesicle membranes and (b) a substance having an affinity for extracellular vesicle membranes via an uncharged bond; and (II) particles for nucleic acid extraction.

[0078] In the reagent of the present invention, the substance having affinity for extracellular vesicle membranes is in the form of (I-1) immobilized on particles or (I-2) released from particles. In the case of precipitated particles in the form (I-2), precipitated particles in the form (I-1) can be prepared immediately before use by reacting (a) a substance having affinity for extracellular vesicle membranes with (b) precipitated particles capable of immobilizing the substance having affinity for extracellular vesicle membranes via an uncharged bond.

[0079] In another embodiment, the reagent of the present invention may be a reagent suitable for the one-bead method described above. Such a reagent may be a nucleic acid (e.g., RNA) recovery reagent comprising particles or silica particles having a carboxyl group surface modification, to which a substance having an affinity for extracellular vesicle membranes is immobilized by an uncharged bond.

[0080] The present invention also provides a kit for analyzing nucleic acids (e.g., RNA), comprising: (A) a reagent of the present invention; and (B) a material for analyzing nucleic acids, which may include a reverse transcriptase, one or more probes, and one or more primers (e.g., a primer for reverse transcription and two or more primers for gene amplification).

[0081] In the reagent and kit of the present invention, the details of terms such as substance having affinity for extracellular vesicle membrane, uncharged bond, sedimentation particle, particle for nucleic acid extraction, etc. are as described above. The reagent and kit of the present invention may further contain the above-mentioned chaotropic agent.

[0082] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, % in the examples indicates % by weight (w / v).

[0083] Reference Example 1: Preparation of magnetic particles for immunoprecipitation Dynabeads M-280 Tosylactized (Thermo Fisher), Dynabeads epoxy (Thermo Fisher), Dynabeads Carboxylated (Thermo Fisher), carboxylated magnetic particles (Fujirebio), gelatin magnetic particles with carboxy groups (Fujirebio), and SicaStar-M COOH (Micromod) were used as magnetic particles for immunoprecipitation.

[0084] NHS (N-hydroxysuccinimide) and EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiiimide) were added to 0.01 g / mL of magnetic particles (carboxylated magnetic particles or gelatin magnetic particles having carboxy groups, SicaStar-M COOH) in 50 mM MES buffer (pH 5.5) to a final concentration of 1.0 M, respectively, and the mixture was gently stirred at 25°C for 30 minutes to form an NHS ester on the surface of the magnetic particles. The magnetic particles were collected and washed with 50 mM MES buffer (pH 5.0), and 0.2 mg / mL of mouse anti-CD9 monoclonal antibody (Fujirebio) or mouse anti-CD63 monoclonal antibody (Fujirebio), which recognize the extracellular vesicle (EV) surface markers CD9 or CD63, respectively, was added. The mixture was incubated at 25°C for 1 hour with gentle stirring, allowing the NHS ester on the magnetic particles to react with the primary amine of the antibody, and the antibody was immobilized on the magnetic particles by an amide bond. After the reaction, the magnetic particles were collected and washed with a washing solution (50 mM Tris buffer, 150 mM NaCl ２The magnetic particles were washed with a particle diluent (50 mM Tris buffer, 1 mM EDTA 2Na, 0.1% NaN ３ , 2.0% BSA, pH 7.2) to obtain a magnetic particle solution for immunoprecipitation on which each antibody was immobilized.

[0085] Dynabeads M-280 Tosylactivated, Dynabeads epoxy, and Dynabeads Carboxylated were immobilized with mouse anti-CD9 monoclonal antibody and mouse anti-CD63 monoclonal antibody, respectively, according to the attached instructions. The particles were then diluted with a particle diluent (50 mM Tris buffer, 1 mM EDTA 2Na, 0.1% NaN ３ , 2.0% BSA, pH 7.2) to obtain a magnetic particle solution for immunoprecipitation on which each antibody was immobilized.

[0086] Example 1: Evaluation of the effect of type and properties of magnetic particles on nucleic acid extraction efficiency <Objective> The following test was carried out with the objective of evaluating the effect of type and properties of magnetic particles used in immunoprecipitation on nucleic acid extraction efficiency.

[0087] <Experimental Method> 500 μL of a human serum sample was mixed with 500 μL of a buffer solution containing a chelating agent (50 mM EDTA (ethylenediaminetetraacetic acid), 50 mM EGTA (ethylene glycol tetraacetic acid), 1.0% CMC (carboxymethylcellulose) in PBS, pH 7.4 [see WO2020 / 080387]), and the mixture was applied to magnetic particles for immunoprecipitation (Dynabeads M-280 Tosylactated, Dynabeads epoxy, Dynabeads Epoxy) prepared in Reference Example 1 above, on which anti-CD9 antibody and anti-CD63 antibody were immobilized. Carboxylated magnetic particles, or gelatin magnetic particles) were added to a final concentration of 0.07 w / v% (anti-CD9 antibody-immobilized particles and anti-CD63 antibody-immobilized particles mixed at a 1:1 ratio). This was mixed by inversion at 37°C for 60 minutes to bind the immunoprecipitation particles to the EVs in the sample. The EVs-magnetic particle complexes were then recovered using a neodymium magnet. The supernatant was removed, and the sample was washed twice with 1 mL of PBS (0.05% Tergitol 15-S-30), followed by resuspension in 200 μL of PBS (0.05% Tergitol 15-S-30, pH 7.4) to obtain an EV-magnetic particle complex sample.

[0088] RNA extraction was performed on these samples using the Dynabeads SILANE viral NA kit (Thermo Fisher). Specifically, 50 μL of 18 mg / mL (concentration) Proteinase K (manufactured by Nacalai Tesque) was added to the obtained EV-magnetic particle complex sample, and then 300 μL of Lysis / Binding Buffer (containing 55-80% thiocyanic acid compound with guanidine (1:1), 3-24.9% poly(oxyethylene) p-(1,1,3,3-tetramethylbutyl)phenyl ether) (Thermo Fisher) included in the Dynabeads SILANE viral NA kit was added. The mixture was then incubated at 25°C for 5 minutes to dissolve the EV, yielding a dissolved EV sample.

[0089] Next, 150 μL of 100% isopropanol and 50 μL of 40 mg / mL Dynabeads MyOne SILANE (silica magnetic particles, pH 5.5) were added to the resulting EV lysate sample and incubated at room temperature for 10 minutes with gentle stirring to adsorb the RNA to the silica magnetic particles. The magnetic particles were attracted with a neodymium magnet, the supernatant was removed, and the sample was washed with 850 μL of Wash Solution 1 (50% isopropanol, 5-15% polyethylene glycol, 27.5-40% thiocyanic acid compound with guanidine (1:1), 1.5-22.4% poly(oxyethylene) p-(1,1,3,3-tetramethylbutyl)phenyl ether), which was repeated twice. After further washing twice with 450 μL of 70% ethanol (pH 5.0), 100 μL of Elution Buffer included in the Dynabeads SILANE viral NA kit was added, and the mixture was incubated at 70°C for 3 minutes. The supernatant was then recovered to obtain an RNA extraction sample (RNA extraction sample by the one-tube method).

[0090] As a control (two-tube method), the resulting EV lysis sample was collected, and the supernatant was transferred to a new tube. 150 μL of 100% isopropanol and 50 μL of 40 mg / mL Dynabeads MyOne SILANE (silica magnetic particles) were added, and the mixture was incubated at room temperature for 10 minutes with gentle stirring to adsorb the RNA to the silica magnetic particles. The subsequent steps (addition of silica magnetic particles, washing, and RNA extraction) were carried out in the same manner as above to obtain an RNA extraction sample using the two-tube method.

[0091] From the resulting RNA extraction samples (Tube 1 and Tube 2), a volume equivalent to 50 μL of the sample was used as a template to synthesize cDNA. Furthermore, using a volume equivalent to 2.5 μL of the sample, the expression levels of housekeeping genes (ACTB, B2M) were measured using Droplet Digital PCR (Bio-Rad) according to the attached instructions, and the measured values ​​were evaluated as the RNA extraction efficiency.

[0092] <Results> The results are shown in Figure 1. As a result, when immunoprecipitation was performed using Dynabeads Tosylactivated and Dynabeads Epoxy as magnetic particles for immunoprecipitation, it was confirmed that the RNA extraction efficiency in the one-tube method was significantly lower than that in the two-tube method. On the other hand, when other magnetic particles were used, no significant difference was observed between the two-tube method and the one-tube method, indicating that nucleic acid extraction can be performed even in the presence of immunoprecipitation particles. These results show that in nucleic acid extraction using silica magnetic particles, there are magnetic particles that can coexist in the reaction system, and that by using a specific combination, it is possible to consistently perform EV recovery and nucleic acid extraction from samples.

[0093] When antibodies are immobilized on Dynabeads Tosylactated and Dynabeads Epoxy, the binding mode is an amine bond (secondary amine), and when antibodies are immobilized on other magnetic particles, the binding mode is an amide bond. Based on these findings, it is believed that the binding mode on immunoprecipitation particles is an amide bond, which is effective when extracting nucleic acids in one tube.

[0094] Example 2 <Purpose> It was revealed in Example 1 that magnetic particles to which antibodies were immobilized via amide bonds could be used in a nucleic acid extraction process using silica magnetic particles. Based on this, it was hypothesized that nucleic acid extraction from EV immunoprecipitation could be performed using a single type of magnetic particle by immobilizing antibodies via amide bonds to silica magnetic particles, and the following test was carried out to verify this hypothesis.

[0095] <Experimental Method> In this experiment, an RNA extraction sample was obtained in the same manner as in Example 1 (single bead method), except that SicaStar-M COOH, which has a carboxyl group on the silica surface, was used as the magnetic particles and no silica magnetic particles were added.

[0096] Specifically, 500 μL of a human serum sample was mixed with 500 μL of a buffer solution containing a chelating agent, and immunoprecipitation magnetic particles (SicaStar-M COOH) with immobilized anti-CD9 and anti-CD63 antibodies prepared in Reference Example 1 above were added at a final concentration of 0.07 w / v%. This was mixed by inversion at 37°C for 60 minutes to bind the immunoprecipitation particles to the EV in the sample. Thereafter, an EV lysate sample was obtained in the same manner as in Example 1. 150 μL of 100% isopropanol was added to the obtained EV lysate sample and incubated at room temperature for 10 minutes with gentle stirring to adsorb the RNA to the silica magnetic particles. Thereafter, the sample was washed twice with isopropanol and ethanol, and an RNA extraction sample (RNA extraction sample by the single-bead method) was obtained using Elution Buffer in the same manner as in Example 1.

[0097] For comparison, an RNA extraction sample (RNA extraction sample by the one-tube method) was obtained in the same manner as in Example 1, except that SicaStar-M was used as the magnetic particles for immunoprecipitation and Dynabeads SILANE viral NA kit (Thermo Fisher) was used as the silica magnetic particles. The extracted RNA was analyzed by droplet digital PCR using the same procedure as in Example 1, and the expression levels of the obtained ACTB and B2M genes were measured, and the measured values ​​were evaluated as RNA extraction efficiency.

[0098] <Results> The results are shown in Figure 2. A comparison of the one-bead method and the one-tube method confirmed that they showed equivalent RNA extraction efficiency for the B2M gene. These results demonstrate that by using silica magnetic particles to which antibodies are immobilized via amide bonds, it is possible to perform EV immunoprecipitation and nucleic acid extraction using only one type of magnetic particle.

[0099] Example 3 <Purpose> It is believed that loss of trace amounts of EV-derived nucleic acids can be reduced by consistently performing EV recovery and nucleic acid extraction from specimens. Therefore, the usefulness of the present invention was demonstrated by comparing it with the conventional, commonly used phenol-chloroform extraction method.

[0100] <Experimental Method> EV-magnetic particle complex samples were obtained using the same procedure as in Example 1. In this experiment, EVs were collected using gelatin particles. Nucleic acid extraction was performed from the obtained EV-magnetic particle complex samples using Dynabeads SILANE viral NA kit or TRIzol RNA isolation reagent (Thermo Fisher) according to the attached manual. For Dynabeads SILANE, two conditions, the two-tube method and the one-tube method, were used as in Example 1. For TRIzol RNA isolation reagent, the EV-magnetic particle complexes were collected, the supernatant was removed, and then 1 mL of TRIzol Reagent was added and incubated at room temperature for 5 minutes to separate the proteins. Then, 0.2 mL of chloroform was added, and the mixture was vortexed for 15 seconds. The mixture was then centrifuged (4°C, 15 minutes, 12,000 g) to separate the two phases, and the aqueous phase containing RNA was collected. Subsequent procedures were carried out according to the attached manual. The extracted RNA was analyzed by droplet digital PCR using the same procedure as in Example 1, and the expression levels of the ACTB and B2M genes obtained were measured. The measured values ​​were evaluated as the RNA extraction efficiency.

[0101] <Results> The results are shown in Figure 3. As a result, the highest RNA extraction efficiency was observed when nucleic acid extraction was performed using the one-tube method with Dynabead SILANE. Generally, RNA extraction methods using silica magnetic particles are considered to have lower RNA recovery efficiency than phenol-chloroform extraction methods. However, this result showed that the one-tube method, which can minimize RNA loss during handling, is effective for extracting RNA from samples containing trace amounts of RNA, such as EV.

[0102] Example 4 <Purpose> The following experiment was carried out with the objective of evaluating whether the properties of silica magnetic particles used in nucleic acid extraction, that is, the size and surface structure, affect the efficiency of nucleic acid extraction.

[0103] <Experimental Method> An EV-magnetic particle complex sample was obtained using the same procedure as in Example 1. In this experiment, EVs were collected using gelatin particles. RNA was extracted from the obtained EV-magnetic particle complex sample using the one-tube method in accordance with the manual provided with the Dynabeads SILANE viral NA kit. In addition to the Dynabeads SILANE provided with the kit, SicaStar-M, which has a different surface structure, was also used as the silica magnetic particles. The extracted RNA was analyzed by droplet digital PCR using the same procedure as in Example 1, and the expression levels of the ACTB and B2M genes obtained were measured, and the measured values ​​were evaluated as the RNA extraction efficiency.

[0104] <Results> The results are shown in Figure 4. When two types of silica magnetic particles with different surface structures were compared, it was found that RNA extraction was possible with either type of magnetic particle. This result suggests that a variety of silica magnetic particles can be used in the method of the present invention.

[0105] Example 5 <Purpose> In the one-tube method, magnetic particles for immunoprecipitation are present in the same reaction site for nucleic acid extraction using silica magnetic particles. Therefore, the following experiment was conducted to evaluate whether the amount of particles for immunoprecipitation affects the efficiency of nucleic acid extraction.

[0106] <Experimental Method> EV-magnetic particle complex samples were obtained using the same procedure as in Example 1. In this experiment, gelatin particles were added to 500 μL of a human serum sample at a final concentration of 0.07 w / v% (1x) and 5 times the concentration, 0.35 w / v% (5x), and EVs were recovered. RNA was extracted from the obtained EV-magnetic particle complex samples using a Dynabead SILANE viral NA kit by the one-tube method, and the expression levels of the ACTB and B2M genes were evaluated using the procedure in Example 1.

[0107] <Results> The results are shown in Figure 5. Even when five times the amount of immunoprecipitation magnetic particles was present, RNA extraction equivalent to that achieved with one amount was possible, demonstrating that the effect of immunoprecipitation magnetic particles on nucleic acid extraction is minimal. This suggests that, for example, increasing the amount of immunoprecipitation particles added can be used to speed up EV recovery.

[0108] Example 6 <Purpose> The following test was carried out with the aim of evaluating whether nucleic acid extraction from gelatin particles is possible by the one-tube method using magnetic particles with carboxy groups bound thereto (SPRI magnetic beads, manufactured by Beckman Coulter) as magnetic particles for nucleic acid extraction.

[0109] <Experimental Method> The experiment was performed using the same method as described in Example 1. Specifically, EV-magnetic particle complex samples were obtained using gelatin magnetic particles as magnetic particles for immunoprecipitation to which anti-CD9 antibodies and anti-CD63 antibodies were immobilized. Nucleic acids were then extracted using carboxylated magnetic particles for nucleic acid extraction (SPRI magnetic beads, manufactured by Beckman Coulter) and silica particles (Dynabeads MyOne SILANE, manufactured by Thermo Fisher). For the carboxylated magnetic particles for nucleic acid extraction (SPRI magnetic beads), the reagents included in the RNAdvance Blood Kit (Beckman Coulter, Inc.) were used, and nucleic acid extraction was performed according to the attached protocol. Nucleic acid extraction was performed using silica particles (Dynabeads MyOne SILANE) according to the protocol included with the Dynabeads SILANE Viral NA Kit (Thermo Fisher). In the two-tube method, the lysis buffer and proteinase K included with each kit were added to a tube containing an EV-magnetic particle complex sample, followed by magnetic collection. The supernatant was transferred to a new tube and subjected to nucleic acid extraction to obtain an RNA extraction sample. In the one-tube method, lysis buffer and proteinase K were added to a tube containing an EV-magnetic particle complex sample, and then carboxylated magnetic particles or silica particles for nucleic acid extraction were added to this tube to perform nucleic acid extraction to obtain an RNA extraction sample.

[0110] From the obtained RNA extraction samples (1 tube, 2 tube), a volume equivalent to 500 μL of the sample was used as a template to synthesize cDNA. Furthermore, using a volume equivalent to 40 μL of the sample, the expression levels of housekeeping genes (ACTB, B2M) were measured using Droplet Digital PCR (Bio-Rad) according to the attached instructions, and the measured values ​​were evaluated as the RNA extraction efficiency.

[0111] <Results> The results are shown in Figure 6. As a result, in the nucleic acid extraction method using carboxylated magnetic particles for nucleic acid extraction, no significant difference was observed between the two-tube method and the one-tube method, demonstrating that nucleic acid extraction can be performed even in the presence of immunoprecipitation particles.

Claims

1. A method for recovering nucleic acids, comprising: (A) separating extracellular vesicles from a sample containing extracellular vesicles using sedimentation particles to which a substance having affinity for the extracellular vesicle membrane is fixed by an uncharged bond; (B) eluting nucleic acids from the extracellular vesicles; and (C) recovering the eluted nucleic acids using nucleic acid extraction particles.

2. The method of claim 1, wherein the nucleic acid is RNA and (C) is carried out in the presence of a chaotropic agent.

3. The method according to claim 1, wherein the substance having affinity for extracellular vesicle membranes is an antibody against a surface marker of extracellular vesicles.

4. The method of claim 1, wherein the surface marker of the extracellular vesicles is CD9 and / or CD63.

5. The method of claim 1, wherein the uncharged bond is an amide bond.

6. The method according to claim 1, wherein the sedimentation particles are particles having a surface modification with a carboxy group, obtained by reacting an antibody against a surface marker of extracellular vesicles, to which the antibody is immobilized via an amide bond, and the amide bond is a bond formed by reaction between the carboxy group and an amino group in the antibody.

7. The method according to claim 1, wherein the particles for nucleic acid extraction are particles having a surface modification with a carboxy group or silica particles.

8. The method of claim 1, wherein said method is carried out in a single vessel.

9. The method according to claim 1, wherein the sedimentation particles are different from the nucleic acid extraction particles.

10. The method of claim 9, comprising: (1) mixing a sample containing extracellular vesicles with sedimentation particles having immobilized thereon a substance having affinity for the extracellular vesicle membrane to produce a first mixture containing the sample containing extracellular vesicles and the sedimentation particles; (2) incubating the first mixture to produce a complex containing the extracellular vesicles and the sedimentation particles; and (3) separating the first mixture into a supernatant and a precipitate containing the complex; (4) removing the supernatant to obtain a precipitate containing the complex; (5) simultaneously or separately adding (i) a lysis solution of extracellular vesicles and (ii) particles for nucleic acid extraction to the precipitate containing the complex to produce a second mixture containing the complex, the lysis solution, and particles for nucleic acid extraction; (6) incubating the second mixture to adsorb nucleic acids eluted from the extracellular vesicles to the particles for nucleic acid extraction; and (7) recovering the particles for nucleic acid extraction to which nucleic acids are adsorbed from the second mixture.

11. The method according to claim 9, comprising: (1) mixing a sample containing extracellular vesicles with sedimentation particles having immobilized thereon a substance having affinity for extracellular vesicle membranes to produce a first mixture containing the sample containing extracellular vesicles and the sedimentation particles; (2) incubating the first mixture to produce a complex containing the extracellular vesicles and the sedimentation particles; (3) separating the first mixture into a supernatant and a precipitate containing the complex; (4) removing the supernatant to obtain a precipitate containing the complex; (5) adding a lysis solution of extracellular vesicles to the precipitate containing the complex to produce a second mixture containing the complex and the lysis solution; (6) incubating the second mixture to elute nucleic acids from the extracellular vesicles; (7) adding particles for nucleic acid extraction to the eluted nucleic acids to produce a third mixture containing the eluted nucleic acids and particles for nucleic acid extraction; (8) incubating the third mixture to adsorb the eluted nucleic acids to the particles for nucleic acid extraction; and (9) Recovering the nucleic acid-absorbed particles for nucleic acid extraction from the third mixture.

12. The method according to claim 1, wherein the sedimentation particles are the same as the particles used for nucleic acid extraction.

13. The method of claim 11, comprising: (1) mixing a sample containing extracellular vesicles with particles for nucleic acid extraction to which a substance having affinity for the extracellular vesicle membrane is immobilized to produce a first mixture containing the sample containing extracellular vesicles and the particles for nucleic acid extraction; (2) incubating the first mixture to produce a complex containing the extracellular vesicles and the particles for nucleic acid extraction; and (3) separating the first mixture into a supernatant and a precipitate containing the complex; (4) removing the supernatant to obtain a precipitate containing the complex; (5) adding a solution of lysing extracellular vesicles to the precipitate containing the complex to produce a second mixture containing the complex and the solution of lysing extracellular vesicles; (6) incubating the second mixture to adsorb nucleic acids eluted from the extracellular vesicles to the particles for nucleic acid extraction; and (7) recovering the particles for nucleic acid extraction from the second mixture.

14. The method according to claim 1, wherein either the sedimentation particles or the nucleic acid extraction particles, or both the sedimentation particles and the nucleic acid extraction particles, are magnetic particles.

15. A method for analyzing nucleic acids, comprising: (1) recovering nucleic acids by the method of any one of claims 1 to 14; and (2) analyzing the recovered nucleic acids.

16. A nucleic acid recovery reagent comprising: (I) (I-1) sedimentation particles to which a substance having an affinity for extracellular vesicle membranes is immobilized by an uncharged bond, or (I-2) sedimentation particles capable of immobilizing (a) a substance having an affinity for extracellular vesicle membranes and (b) a substance having an affinity for extracellular vesicle membranes via an uncharged bond; and (II) particles for nucleic acid extraction.

17. The reagent according to claim 16, wherein the sedimentation particles to which a substance having affinity for extracellular vesicle membranes is immobilized via an uncharged bond are sedimentation particles to which a substance having affinity for extracellular vesicle membranes is immobilized via an amide bond, or the sedimentation particles capable of immobilizing a substance having affinity for extracellular vesicle membranes via an uncharged bond are sedimentation particles having a surface modification with a carboxy group.

18. A nucleic acid recovery reagent comprising particles or silica particles having a surface modification with carboxy groups, to which a substance having an affinity for extracellular vesicle membranes is immobilized by an uncharged bond.

19. The reagent according to any one of claims 16 to 18, wherein the nucleic acid is RNA and further comprises a chaotropic agent.

20. A kit for analyzing nucleic acids, comprising: (A) a reagent according to any one of claims 16 to 18; and (B) a substance for analyzing nucleic acids; and (C) optionally, a chaotropic agent.

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