Method for enriching short-chain nucleic acids

Abstract

The present invention relates to a method for enriching nucleic acids having a length of less than 300 nucleotides, comprising the following steps: i) providing a fluid phase, preferably an aqueous phase P1, containing (α1) at least one nucleic acid having a length of less than 300 nucleotides, and (α2) at least one component different from the nucleic acid (α1), ii) contacting the phase P1 with an anion exchange matrix to bind the nucleic acid (α1) to the anion exchange matrix, iii) optionally washing the anion exchange matrix with a wash buffer, wherein the nucleic acid (α1) remains bound to the anion exchange matrix, and iv) removing, preferably eluting, the nucleic acid (α1) bound to the anion exchange matrix from the anion exchange matrix to obtain a fluid phase, preferably an aqueous phase P2, containing the nucleic acid (α1). The present invention also relates to a kit for enriching nucleic acids having a length of less than 300 nucleotides, to the use of the kit, to the use of an anion exchange matrix, and to a method for treating a disease.

Description

[0001] This invention patent application is a divisional application of the invention patent application with international application number PCT / EP2006 / 069484, international application date December 8, 2006, application number 200680046308.3 entering the Chinese national phase, and titled "Method for Enriching Short-Chain Nucleic Acids".

[0002] The present invention relates to a method for enriching nucleic acids with a length of less than 300 nucleotides, a kit for enriching nucleic acids with a length of less than 300 nucleotides, the use of the kit, the use of an anion exchange matrix and a method for treating diseases.

[0003] Several years ago, due to the discovery that small ribonucleic acids (RNAs) performed the substantial regulatory function of gene expression, scientific research has increasingly focused on small RNAs shorter than 300 nucleotides, especially shorter than 100 nucleotides. More specifically, many researchers have been interested in microRNAs (miRNAs). MiRNAs are small non-coding RNAs of about 22 nucleotides in length that are evolutionarily conservative, and their complex role in regulating gene expression is becoming increasingly apparent. MiRNAs have been found in all eukaryotic organisms and nearly all tissues studied, including fungi, plants, insects, and mammals. In addition to miRNAs, other small RNAs have also been found to play an important role in cellular function. These small RNAs include, for example, small interfering RNAs (siRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs).

[0004] These short RNAs shorter than 300 nucleotides must be purified as pure as possible and in high yield from the biological system to be studied so that their cellular effects can be studied. Therefore, there is an urgent need to provide methods for purifying and isolating such short RNAs from complex biological systems, especially from cell lysates.

[0005] Generally, for separating the nucleic acid in biological samples, they must be separated from remaining cellular components such as proteins, carbohydrates, lipids and other components. Prior art has disclosed multiple methods for separating nucleic acids from very different raw materials, such as from cell culture, from tissues of plant and animal origin and from body fluids. For example, one method is included in extracting a generally aqueous original solution (Chomczynski and Sacchi, 1987) with the help of organic solvents such as phenol and chloroform, then precipitates nucleic acid from aqueous phase with the help of alcohols such as ethanol or isopropanol (Sambrook, J., Fritsch, EFin T.Maniatis, CSH, " Molecular Cloning ", 1989). Another method comprises that nucleic acid is fixed to solid phase, for example, by silicon adsorption technology. Disadvantageously, all these methods can not or are not enough to separate or at least purify relatively small nucleic acids.

[0006] To solve this problem, the prior art has disclosed methods for specifically enriching small RNA populations, which are based on silica membrane technology. In these methods, a relatively small amount of alcohol is added to the cell lysate after the cells are lysed, and at least some of the relatively long nucleic acids can bind to the silica membrane under chaotropic binding conditions. However, the amount of alcohol used in the purification methods described in the prior art is too low, so that the small nucleic acids cannot also be effectively bound to the silica membrane, so these small RNAs appear in the effluent. The alcohol concentration of the effluent is then increased, and then a second silica membrane is bound. After the washing step, the small RNA is eluted together with all other nucleic acids that were not bound to the first column (see, for example, Ambion, Austin, USA). Kit or from QIAGEN, Hilden, Germany Lipid Tissue Mini Kit, for instructions, see "User Manual").

[0007] The disadvantages of the Qiagen and Ambion methods are that they require two solid phases and cannot isolate only the desired small RNAs in a single binding step. Furthermore, for example, miRNAs, which are approximately 22 nucleotides in size, cannot be isolated without simultaneously isolating tRNAs and other larger nucleic acids. While this method can enrich small RNAs, particularly miRNAs, to a certain degree under certain conditions, these small RNAs can still be contaminated with other nucleic acids, particularly transfer RNAs (tRNAs).

[0008] The object of the present invention is to overcome the disadvantages of the prior art.

[0009] More specifically, the object of the present invention is to provide a method for enriching small nucleic acids, in particular miRNAs, which method is capable of enriching small nucleic acids from complex biological compositions such as cell lysates using as few method steps as possible.

[0010] Another object of the present invention is to provide a method for purifying small nucleic acids. For example, this method can not only remove specific nucleic acids with a length of 25 nucleotides or less from nucleic acids or other components with a length greater than 300 nucleotides in complex biological compositions such as cell lysates, but can also specifically remove these small nucleic acids from other nucleic acids with a length less than 300 and greater than 25 nucleotides, such as tRNA.

[0011] The present invention also provides a method that can be used to produce RNA of a desired size as individually as possible.

[0012] A further object of the present invention is to provide a method which does not require changing the reaction vessel - through a "one-pot reaction" - thereby minimizing the risk of mixing of the samples to be analyzed.

[0013] A further object of the present invention is to provide a kit with the aid of which small nucleic acids, in particular small RNAs, can be advantageously purified from complex biological compositions as described above.

[0014] A method for enriching nucleic acids having a length of less than 300 nucleotides, preferably less than 200 nucleotides, more preferably less than 100 nucleotides, even more preferably less than 50 nucleotides, and most preferably less than 25 nucleotides contributes to solving the problem mentioned at the outset, the method comprising the following steps:

[0015] i) providing a fluid phase, preferably an aqueous phase P1, containing

[0016] (α1) at least one nucleic acid having a length of less than 300 nucleotides, preferably less than 200 nucleotides, more preferably less than 100 nucleotides, even more preferably less than 50 nucleotides, and most preferably less than 25 nucleotides, and

[0017] (α2) at least one component different from the nucleic acid (α1),

[0018] ii) contacting the phase P1 with an anion exchange matrix so that the nucleic acid (α1) is bound to the anion exchange matrix,

[0019] iii) optionally washing the anion exchange matrix with a wash buffer, wherein the nucleic acid (α1) remains bound to the anion exchange matrix, and

[0020] iv) removing, preferably eluting, the nucleic acid (α1) bound to the anion exchange matrix from the anion exchange matrix to obtain a fluid phase, preferably an aqueous phase P2, containing the nucleic acid (α1).

[0021] Surprisingly, it has been found that small nucleic acids less than 300 nucleotides in length can be enriched from complex biological compositions that may contain numerous other components in addition to the small nucleic acids by binding to an anion exchange matrix followed by washing and elution, without the need to first dilute the longer nucleic acids as required by the above-mentioned Qiagen and Ambine methods.

[0022] According to a preferred embodiment of the method of the present invention, the nucleic acid (α1) to be purified is single-stranded or double-stranded RNA, preferably double-stranded RNA. More specifically, the preferred RNA having a length of less than 300 nucleotides is selected from the group consisting of miRNA, pre-miRNA, siRNA, snRNA, snoRNA, tRNA, 5S-rRNA, 5.8S-rRNA, or a mixture of at least two thereof, in particular a mixture of miRNA and tRNA. More preferably, the nucleic acid (α1) is a miRNA having a length of 15-30 nucleotides, more preferably 17-24 nucleotides, and most preferably 20-23 nucleotides.

[0023] The terms "5S-rRNA" and "5.8S-rRNA" refer to non-coding RNAs found within eukaryotic ribosomes. The term "tRNA" refers to a RNA composed of approximately 80 nucleotides and containing conjugated base pairs (adenine and uracil; cytosine and guanine). These base pairs form the tRNA's cloverleaf-like structure. The term "siRNA" refers to a RNA of approximately 22 nucleotides in length that is produced by the cleavage of double-stranded RNA (dsRNA) by an enzymatic "cutting machine" and incorporation into the "RISC" (RNA-induced silencing complex) enzyme complex. The term "snRNA" refers to a catalytically active RNA of approximately 100-300 base pairs found within the nucleus of eukaryotic cells. These snRNAs are typically bound to proteins to form "snRNPs" (small nuclear ribonucleoproteins) and are responsible for cleaving introns from pre-mRNA to produce mRNA. The term "snoRNA" refers to a class of RNA molecules involved in chemical modifications of ribosomal RNA (rRNA) and other RNA genes, such as methylated RNA. They form part of "snoRNPs" (small nucleolar nuclear ribonucleoproteins). The term "miRNA" refers to small nucleic acids that regulate developmental processes in plants and animals. They specifically bind to mRNA and inhibit its translational activity, for example, preventing the overproduction of growth factors. MiRNAs are single-stranded RNA molecules generated from double-stranded precursors.

[0024] The component (α2) other than the nucleic acid (α1) having a length of not more than 300 nucleotides is a specific nucleic acid (α2') having a length of at least 300 nucleotides and a component (α2") other than the nucleic acid.

[0025] The nucleic acid (α2′) having a length of at least 300 nucleotides includes in particular a single-stranded or double-stranded DNA molecule or a single-stranded or double-stranded RNA molecule, such as mRNA, 18S-rRNA or 28S-rRNA.

[0026] Components other than nucleic acids (α2") are in particular those components which are released during cell lysis. These components therefore include in particular proteins, lipids, polypeptides or polysaccharides.

[0027] The fluid phase provided in method step i), preferably the aqueous phase P1, can be a cell-free sample material, plasma, serum, a body fluid (such as blood, urine, semen, saliva, cerebrospinal fluid, sputum), a surface biopsy sample, wastewater, sludge or a cell lysate (such as a cell lysate from animal or plant tissue, from microorganisms such as bacteria, fungi or yeast, from tissue culture or cell culture, or from a body fluid such as blood).

[0028] According to one embodiment of the method of the present invention, the fluid phase provided in step i), preferably the aqueous phase P1, is a cell lysate obtained by a method comprising the following steps:

[0029] 1) providing cells,

[0030] II) lysing the cells to obtain a cell lysate, and

[0031] III) optionally at least partially isolating at least one component (α2) other than nucleic acid (α1) from the cell lysate.

[0032] The cells provided in method step I) may be optionally fixed tissue sections or optionally fixed tissue fragments, adherent cultured cells, suspension cultured cells or cells in body fluids.

[0033] If the cells are adherent cells or cells within a tissue assembly, step I) of the method may optionally include washing the adherent cells or tissue, detaching the adherent cells or removing the cells from the tissue using a suitable enzymatic solution containing a coordinating compound such as EDTA or a mixture thereof, and optionally isolating a specific cell population from the cell suspension thus obtained by, for example, a cell sorter, pelleting the detached or separated cells, washing the cell pellet thus obtained and optionally resuspending it in a suitable suspension buffer. However, the adherent cells may also be lysed without first detaching them, which is preferably followed by a washing step.

[0034] If the cells are suspension culture cells or cells in body fluids, method step I) preferably comprises pelleting the suspension cells, which is then advantageously followed by removal of specific cell populations, e.g. by cell sorting, washing the pellet thus obtained and optionally resuspending it in a suitable suspension buffer.

[0035] The pelleted cells may be optionally resuspended in a suspension buffer preferably containing one or more buffer substances and, optionally, one or more coordinating compounds. The pH of the suspension buffer can vary over a wide range and is suitable for carrying out the method of the present invention, preferably within a pH range of 3-11, more preferably within a pH range of 5-10, and most preferably within a pH range of 7-9. Buffer systems known to those skilled in the art for adjusting pH can be used. According to the present invention, buffer systems based on tris(hydroxymethyl)aminomethane (TRIS), morpholinopropanesulfonic acid (MOPS), or 2-[4-(2-hydroxyethyl)-1-piperazino]ethanesulfonic acid (HEPES) are preferably used, containing the buffer component at a concentration in the range of 0.5-100 mmol / l, more preferably within a range of 1-50 mmol / l, and most preferably within a range of 2.5-25 mmol / l. Buffer systems based on alkali metal acetate / acetic acid or mixtures of alkali metal acetate / acetic acid buffer systems and tris(hydroxymethyl)aminomethane buffer systems may also be used. Similarly, the coordinating compound that can be used may be any compound that is capable of specifically coordinating with calcium ions. The preferred coordination compound is ethylenediaminetetraacetic acid (EDTA), the content of which in the suspension buffer is preferably 0.01-20 mmol / l, more preferably 0.1-15 mmol / l, and most preferably 0.5-5 mmol / l.

[0036] The amount of suspension buffer used depends on the number of cells provided. Usually, the amount of suspension buffer used is 6 10-2000 μl per cell, more preferably 50-1000 μl, most preferably 100-500 μl.

[0037] Particularly suitable suspension buffers for use in the present invention are those containing 0.5-100 mmol / l, more preferably 1-50 mmol / l, most preferably about 2.5-25 mmol / l tris(hydroxymethyl)aminomethane and 0.01-20 mmol / l, more preferably 0.1-15 mmol / l, most preferably 0.5-5 mmol / l EDTA, with a pH range of 7-9, more preferably about 8.

[0038] In method step II), the provided cells are lysed. Any lysis method known to the skilled person that is suitable for releasing specific RNA material from the cells can be used for lysis of the cells. Possible lysis methods include, in particular, lysis by heat, lysis by mechanical force, lysis by enzymes such as protein kinase K, lysis by contacting the cells with a lysis buffer containing a detergent or a chaotropic compound, or lysis by a hypotonic solution. If appropriate, the above methods can also be combined, for example, mechanical disruption of the cells in a lysis buffer containing a detergent or a chaotropic compound, or using a lysis buffer containing protein kinase K and a chaotropic compound.

[0039] According to the present invention, it is particularly preferred to lyse the cells with a lysis buffer comprising a detergent, an enzyme, a chaotropic compound or a mixture of at least two of these components.

[0040] A large number of suitable detergents have been disclosed in the prior art. Particularly preferred detergents according to the present invention are selected from the group consisting of sodium dodecyl sulfate (SDS), polyethylene glycol-phenol ethers such as Triton X-100, Tween, NP-40 or mixtures thereof, with SDS and Triton X-100 being particularly preferred detergents. If the detergent used is SDS, according to the present invention, it is preferred to use 1-30 mol, preferably 2-20 mol, most preferably 3-6 mol of NaOH or KOH, more preferably NaOH, per mol of SDS to lyse the cells. If the lysis buffer contains a detergent, according to the present invention, it is also preferred to use 10 mol of NaOH or KOH per 10 mol of SDS in step II). 6 The cells are lysed in the presence of 0.01-100 μmol, more preferably 0.1-50 μmol, and most preferably 0.25-5 μmol of detergent. When a detergent is used to lyse the cells, if the detergent is a liquid compound at room temperature and pressure, the cells are typically lysed in the presence of 0.005-5% (v / v), more preferably 0.01-1% (v / v), and most preferably 0.025-0.5% (v / v) detergent. If the detergent is a solid compound at room temperature and pressure, the cells are typically lysed in the presence of 0.01-1% by weight, more preferably 0.25-5% by weight, and most preferably 0.05-0.4% by weight detergent.

[0041] Preferred chaotropic compounds are especially chaotropic salts. For the purposes of the present invention, chaotropic salts preferably represent salts that have a high affinity (competitiveness, attraction) for water to form a larger, tight hydration envelope (shell-like water molecules). Preferred chaotropic salts are especially guanidine isothiocyanate or guanidine hydrochloride, with guanidine isothiocyanate being particularly preferred. If chaotropic salts are used to lyse cells, according to the method of the present invention, cells are lysed in method step II) when the chaotropic salt concentration is 0.5-10 mol / l, more preferably 1-5 mol / l, and most preferably 2-3.5 mol / l. If chaotropic salts are contained in the lysis buffer, it is also advantageous that the lysis buffer optionally contains a water-miscible organic solvent, such as a water-miscible alcohol such as ethanol or isopropanol, in a content of 10-60% by volume, more preferably 20-50% by volume.

[0042] Preferred enzymes are especially proteases, among which trypsin, proteinase K, chymotrypsin, papain, pepsin, pronase and endoproteinase Lys-C are more preferred, and proteinase K is most preferred. The enzyme concentration in the lysis buffer is preferably in the range of 0.01 to 10% by weight, more preferably 0.1 to 5% by weight, most preferably 0.2 to 1% by weight, based in each case on the total weight of the lysis buffer.

[0043] The concentration of the detergent, chaotropic salt, or enzyme in the lysis buffer also depends on the amount of cells to be lysed and the manner in which the cells are provided in step I). If the cells to be lysed are first suspended in a suspension buffer, the lysis buffer contains a concentration of detergent, chaotropic salt, or enzyme that is higher than the concentration of the component required during cell lysis. This enriched lysis buffer is then added to the cell suspension in an amount sufficient to provide the cell suspension with a concentration of chaotropic salt, detergent, or enzyme that is sufficient to achieve the most complete lysis of the cells possible, as described above. However, if, for example, the lysis buffer is applied directly to adherent cells or to contact cell clusters, the lysis buffer preferably contains a concentration of detergent, chaotropic salt, or enzyme that is present during cell lysis.

[0044] According to a specific embodiment of the method of the present invention, the cells are lysed in the presence of 0.1-1 mol / l, more preferably 0.2-0.8 mol / l, and most preferably 0.3-0.7 mol / l of an alkali metal salt, preferably sodium chloride, potassium chloride, and lithium chloride, with sodium chloride being particularly preferred. If the cells to be lysed are first suspended in a suspension buffer, an appropriate amount of alkali metal salt may already be added to the suspension buffer, or a lysis buffer containing a relatively high concentration of alkali metal salt may be added to the suspension buffer. However, for example, if the lysis buffer is applied directly to adherent cells or contact cell clusters, the lysis buffer preferably contains an alkali metal salt within the above-mentioned concentration range.

[0045] A lysis buffer particularly suitable for use in the present invention and which can be added to the cell suspension is a buffer containing 1-200 mmol / l, more preferably 5-150 mmol / l, and most preferably about 10-100 mmol / l NaOH and 0.01-1% (v / v), more preferably 0.025-0.5% (v / v), and most preferably 0.05-0.4% (v / v) SDS, and having a pH range of 5-7, more preferably about 5.5. The lysis buffer is preferably added to the cell suspension in a volume ratio of 3:1-1:3, more preferably 2:1-1:2, and most preferably about 1:1.

[0046] Lysis buffers that are particularly suitable for use in the present invention and that can be added to cell pellets, to adherent cells or to tissue sections or tissue fragments are:

[0047] a buffer containing 0.1-1 mol / l, more preferably 0.25-0.75 mol / l, most preferably about 0.4-0.6 mol / l NaCl and 0.1-10% (v / v), more preferably 0.5-5% (v / v), most preferably 0.75-1.5% (v / v) Triton X-100 and a pH range of 6-8, more preferably about 7, or

[0048] a buffer solution containing 0.5-10 mol / l, more preferably 1-5 mol / l, most preferably about 1.5-3 mol / l guanidine isothiocyanate, 1-50 mmol / l, more preferably 5-40 mmol / l, most preferably 10-20 mmol / l sodium citrate and 10-60% (v / v), more preferably 20-50% (v / v), most preferably 30-40% (v / v) ethanol, and having a pH in the range of 6-8, more preferably about 7,

[0049] The amount of the lysis buffer to be added to the cells to be lysed is preferably 6 Cells 50-2000 μl, more preferably 100-1000 μl, most preferably 150-300 μl.

[0050] If the cells are present in tissue sections or tissue fragments, providing the cells in method step 1) preferably comprises exposing the tissue sections or tissue fragments to liquid nitrogen immediately after removal from the plant or animal. The tissue sections or tissue fragments are then preferably immediately exposed to a lysis buffer and, if appropriate, homogenized by a suitable homogenizer.

[0051] After the cells are contacted with the lysis buffer, they can be lysed at a temperature ranging from 15 to 40° C., and then particularly preferably at room temperature, for 1 to 60 minutes, more preferably 2 to 15 minutes.

[0052] Before the cell lysate is brought into contact with the anion exchange matrix in the form of the aqueous phase P1 in method step ii), according to one embodiment of the method according to the invention, it is also advantageous to previously separate one or more components (α2) other than nucleic acids (α1) from the cell lysate. In principle, the separation can be carried out by any separation method known to the skilled person, such as precipitation, separation by dialysis or chromatography, or extraction, particularly preferably extraction, in particular extraction with acidic phenol or a mixture of phenol and chloroform, most preferably extraction with acidic phenol. This involves bringing the acidic phenol into contact with the cell lysate and mixing them thoroughly, for example using a vortex mixer, preferably in a volume ratio of 3:1 to 1:3, more preferably 2:1 to 1:2, and most preferably about 1:1. The composition is then centrifuged and the aqueous phase is separated from the organic phase. In this way, one or more components other than nucleic acids (α1) are diluted in the separated aqueous phase, which is then subjected to method step ii) as phase P1.

[0053] In method step ii) of the method according to the invention, the aqueous phase P1 is then brought into contact with an anion exchange matrix in order to bind the nucleic acid (α1) to the anion exchange matrix.

[0054] In principle, any material which has at least some of its functional groups in cationic form under the conditions, in particular the pH conditions, under which the aqueous phase P1 contacts the anion exchange matrix can be used as an anion exchange matrix.

[0055] Anion exchange matrices preferably comprise solids of electrically neutral matrix materials. Such matrices are defined by their size, form, porosity, mechanical properties, and positively charged functional groups preferably covalently bonded to the solid backbone. The three most commonly used types of matrix materials are orthosilicic acid, polysaccharides, and synthetic polyolefins, with the polyolefins employed primarily being polystyrene or poly(meth)acrylic resins. Poly(meth)acrylic resins include polymers of various substituted (meth)acrylamides (=poly(meth)acrylamides) and polymers of (meth)acrylates (=poly(meth)acrylates), the (meth)acrylate monomers optionally bearing alkyl substituents on the C-2 or C-3 atoms. Particularly preferred functional groups bonded to the matrix are those selected from the group consisting of primary, secondary, or tertiary amino groups, phosphino groups, hydrazino groups, and imino groups. The most preferred anion exchange matrix is ​​a matrix having covalently bonded diethylaminoethyl (DEAE, [CH3CH2)2N-CH2-CH2-] a) backbone materials, in particular DEAE cellulose, and linear or branched polyethyleneimines containing [-CH2-CH2-NH-] groups and / or [-CH2-CH2-N(CH2CH2-NH2)-] groups. Anion exchange matrices can also be used as fillers, for example in separate separation columns. However, they can also be used as coatings for other materials that do not consist of materials with anion exchange properties—in particular particles, filters, membranes, monoliths or other organic or inorganic surfaces such as microtiter plates—or other reaction vessels that have anion exchange matrices.

[0056] According to the present invention, a particularly preferred anion exchange matrix is ​​in the form of a coating on magnetic or non-magnetic particles, particularly preferably magnetic, most preferably superparamagnetic, ferrimagnetic or ferromagnetic particles. Magnetic particles have the advantage over non-magnetic particles of forming magnetic aggregates, which allows them to be removed gently, quickly and efficiently from the aqueous phase P1.

[0057] Preferred magnetic particles that can be coated with an anion exchange matrix are available, for example, from Dynal, Advanced Magnetics Inc., Biotechnologies Ltd., Amersham, Promega, Scigen, Advanced Genetic Technologies and Seradyn. Suitable magnetic particles are in particular the particles described in WO-A-83 / 03920 and the particles sold as DYNA-BEADS by Dynal AS, Oslo, Norway. If the anion exchange matrix employed is polyethyleneimine, epoxide-functionalized magnetic particles are particularly preferred, such as those available under the trade name "M-PVAE0x" from Chemagen AG, Baesweiler, Germany. Carboxylate-functionalized particles can also be used, which are also available from ChemoGen under the trade names "M-PVAC11" or "M-PVA C12". The average diameter of the magnetic particles is preferably 0.1-100 μm, more preferably 0.5-50 μm, most preferably 1-10 μm, and their specific surface area is preferably in the range of 0.5-250 m 2 / g, more preferably 1-50m 2 / g.

[0058] According to one embodiment of the method according to the invention, the nucleic acid (α1) is attached to the anion exchange matrix in method step ii) at a pH of preferably 2-10, more preferably 3-7, most preferably 4-6.

[0059] If the pH of the aqueous phase P1 used in process step ii) deviates from these pH values, as is particularly the case when using an alkaline lysis buffer containing SDS, it may be necessary to adjust the pH of the aqueous phase P1 to the desired value, for example by adding a neutralizing buffer before or during contacting the aqueous phase P1 with the anion exchange matrix. The neutralizing buffer preferably contains an alkali metal salt of acetic acid, more preferably potassium acetate, in a concentration range of 10 to 10,000 mmol / l, more preferably 50 to 5000 mmol / l, and most preferably 100 to 1000 mmol / l. The pH of the neutralized solution is preferably 2 to 8, more preferably 4 to 6. The pH of the alkali metal salt of acetic acid solution is preferably adjusted to the above-mentioned range by adding acetic acid.

[0060] According to one embodiment of the method according to the invention, in step ii), the nucleic acid (α1) is preferably attached to the anion exchange matrix in the presence of an alkali metal salt, preferably potassium chloride, sodium chloride or lithium chloride, more preferably sodium chloride, the concentration of the alkali metal salt during attachment preferably being in the range of 0.01-10 mol / l, more preferably 0.05-5 mol / l, and most preferably 0.25-0.75 mol / l. The concentration of these salts during attachment can be adjusted by adding a suitably concentrated salt solution to the initially charged aqueous phase P1 (e.g., cell lysate), or by suspending the cells in the presence of a suspension buffer or lysing the cells in the presence of a lysis buffer, both buffers having a suitable salt concentration.

[0061] According to another embodiment of the method according to the invention, in step ii), the nucleic acid (α1) is preferably attached to the anion exchange matrix in the presence of a chaotropic substance, in particular a chaotropic salt such as guanidine isothiocyanate, the concentration of the chaotropic salt during attachment preferably being 0.1-10 mol / l, more preferably 0.5-5 mol / l, and most preferably 1-3 mol / l. In this embodiment of the method according to the invention, it is also advantageous to carry out the attachment in the presence of a water-miscible organic solvent, in particular an alcohol such as ethanol or isopropanol, in a concentration of 10-70% by volume, more preferably 20-60% by volume.

[0062] In method step ii), when an anion exchange matrix is ​​used as the column packing, the aqueous phase P1, such as a cell lysate, having the aforementioned favorable pH conditions and containing the aforementioned salts at a defined concentration, is preferably passed through the column material to attach the nucleic acid (α1) to the anion exchange matrix. The temperature during the passage is preferably 1-30° C., more preferably 2-25° C., for example, room temperature. The passage of the aqueous phase P1 through the column material can be achieved, if appropriate, by overpressure, vacuum, centrifugation, or by capillary force.

[0063] When particles coated with an anion exchange matrix are used, the attachment is preferably carried out while continuously stirring the aqueous phase P1 in contact with the particles, for example by stirring with a shaker. In this case, the attachment is also preferably carried out at a temperature of about 1-30°C, more preferably 2-25°C, for example at room temperature.

[0064] After the nucleic acid (α1) has been attached to the anion exchange matrix, it can optionally be washed with a wash buffer in method step iii). If the anion exchange matrix is ​​used as a column filler, the wash buffer is preferably passed through the column for washing, although overpressure, vacuum, centrifugation, or capillary force can also be used. For example, if non-magnetic particles coated with an anion exchange matrix are used, the particles are first removed from the aqueous phase P1 by, for example, filtration or centrifugation, and then washed with a wash buffer. When magnetic particles coated with an anion exchange matrix are used, the reaction vessel containing the magnetic particles in contact with the aqueous phase P1 is preferably exposed to a magnet so that the magnetic particles adhere to the inner wall of the reaction vessel under the influence of the magnetic field, thereby washing. Under these conditions, the aqueous phase P1 can be conveniently removed and replaced with a wash buffer. Apparatus suitable for this process can be purchased, for example, from Daniel AG in Oslo, Norway.

[0065] The washing buffer can be, for example, RNase-free water, a mixture of water and a water-soluble organic solvent, such as water and 1-80% by volume of a water-soluble alcohol, for example, a mixture of 1-80% by volume of ethanol or isopropanol, or an aqueous salt solution, especially an acetate aqueous solution, such as an aqueous sodium acetate solution, the concentration of which is 1-50 mmol / l, more preferably 5-25 mmol / l, and the pH of the washing buffer is particularly preferably 4-9.

[0066] The washing step can be repeated one, two, three times, or even more times as needed, using fresh wash buffer each time.

[0067] After the nucleic acids (α1) have been attached to the anion exchange matrix in method step ii) and optionally washed in method step iii), the nucleic acids (α1) bound to the anion exchange matrix can be removed from the anion exchange matrix in method step iv), resulting in a fluid phase containing the nucleic acids (α1), preferably an aqueous phase P2.

[0068] The removal is preferably performed by eluting the anion exchange matrix with an elution buffer capable of releasing the binding between the functional groups of the anion exchange matrix and the nucleic acid (α1), and the resulting eluate is the fluid phase P2 containing the nucleic acid (α1).

[0069] If an anion exchange matrix is ​​used as the column filler, elution is preferably performed by flowing an elution buffer through the column. Alternatively, overpressure, vacuum, centrifugation, or capillary forces may be utilized. For example, if non-magnetic particles coated with an anion exchange matrix are used, these particles are first removed from the aqueous phase P2 or from the wash buffer, for example by filtration or centrifugation, and then contacted with the elution buffer. When magnetic particles coated with an anion exchange matrix are used, the reaction vessel containing the magnetic particles in contact with the aqueous phase P1 or the wash buffer is preferably exposed to a magnet so that the magnetic particles adhere to the inner wall of the reaction vessel under the action of the magnetic field, thereby performing washing. In these cases, the aqueous phase P1 or the wash buffer can be conveniently removed and replaced with the wash buffer.

[0070] The elution buffer is preferably an aqueous salt solution, in particular an aqueous solution containing an alkali metal halide such as NaCl, KCl or LiCl, an alkaline earth metal halide such as CaCl2 or MgCl2, an ammonium salt such as ammonium chloride or ammonium sulfate, or a mixture of at least two of these salts. The elution buffer may also optionally contain a buffer system such as alkali metal acetate / acetic acid or a buffer system based on tris(hydroxymethyl)aminomethane.

[0071] According to a specific embodiment of the method of the present invention, the elution buffer contains a water-soluble calcium salt such as CaCl2, a water-soluble magnesium salt such as MgCl2, a water-soluble ammonium salt such as ammonium sulfate or ammonium chloride, or a mixture of at least two of these salts. If the elution buffer contains CaCl2, the concentration of the salt is preferably 1-1000mmol / l, more preferably 5-500mmol / l, and most preferably 10-100mmol / l. If the elution buffer contains MgCl2, the concentration of the salt is preferably 1-1000mmol / l, more preferably 5-500mmol / l, and most preferably 10-100mmol / l. If the elution buffer contains ammonium sulfate and / or ammonium chloride, the total concentration of these salts is preferably 1-1000mmol / l, more preferably 5-500mmol / l, and most preferably 20-300mmol / l. The pH of the elution buffer is preferably 5-12, preferably 6-10, and more preferably 7-10.

[0072] The elution buffer preferably contains only calcium salts, in particular CaCl2, and / or ammonium salts, preferably ammonium sulfate and / or ammonium chloride, because these salts are particularly suitable for selectively enriching miRNA over tRNA. Thus, by using an elution buffer consisting of water and CaCl2 and an elution buffer consisting of water and ammonium sulfate or ammonium chloride, a good enrichment of miRNA and simultaneous dilution of tRNA is achieved, preferably with a CaCl2 concentration of up to 60 mmol / l and an ammonium sulfate or ammonium chloride concentration of up to 170-200 mmol / l, and therefore, these elution buffers are particularly suitable for selectively enriching miRNA from a composition containing miRNA and tRNA.

[0073] Elution buffers particularly suitable for use in the present invention are:

[0074] - an elution buffer EP1 containing 1-10000 mmol / l, more preferably 10-5000 mmol / l, most preferably 50-1000 mmol / l TRIS, 1-1000 mmol / l, preferably 5-800 mmol / l, most preferably 10-500 mmol / l alkali metal salt, preferably NaCl or KCl, 1-400 mmol / l, more preferably 10-300 mmol / l, most preferably 50-200 mmol / l ammonium salt, preferably ammonium sulfate or ammonium chloride, and 0.1-200 mmol / l, more preferably 0.5-100 mmol / l, most preferably 1-50 mmol / l magnesium salt, preferably magnesium chloride, all dissolved in water and preferably at a pH of 7-11, more preferably 8-10;

[0075] - elution buffer EP2, which contains 1-1000 mmol / l, more preferably 5-500 mmol / l, most preferably 10-100 mmol / l of a magnesium salt, preferably magnesium chloride, dissolved in water and preferably has a pH of 6-10, more preferably 7-9;

[0076] - elution buffer EP3, which contains 1-1000 mmol / l, more preferably 5-500 mmol / l, most preferably 10-100 mmol / l of a calcium salt, preferably calcium chloride, dissolved in water and preferably has a pH of 6-10, more preferably 7-9;

[0077] - elution buffer EP4, which contains 1-1000 mmol / l, more preferably 5-500 mmol / l, most preferably 20-300 mmol / l of an ammonium salt, preferably ammonium chloride or ammonium sulfate, dissolved in water and preferably has a pH of 6-10, more preferably 7-9;

[0078] - Elution buffer EP5 containing 1-2000 mmol / l, more preferably 10-1000 mmol / l, most preferably 100-500 mmol / l of an alkali metal salt, preferably potassium chloride, sodium chloride or lithium chloride, all dissolved in water, and preferably having a pH of 6-10, more preferably 7-9.

[0079] More specifically, elution buffers EP1 to EP4 are suitable for purifying miRNA from a composition containing miRNA and tRNA, while elution buffer EP5 is particularly suitable for substantially purifying nucleic acids from a composition containing nucleic acids having a length of less than 300 nucleotides, in particular nucleic acids having a length of less than 100 nucleotides, and also containing long-chain nucleic acids. 2+ , Ca 2+ and NH4 + The enrichment of miRNA to tRNA enriched from a composition containing miRNA and tRNA is selectively adjusted by adjusting the concentration of the miRNA to tRNA.

[0080] According to a specific embodiment of the method of the present invention, the relative amount of RNA with a length of less than 300 nucleotides, preferably less than 100 nucleotides, most preferably less than 25 nucleotides in said phase P2 based on the total amount of RNA in said phase P2 is preferably at least 2 times, more preferably at least 4 times, even more preferably at least 6 times, even more preferably at least 10 times, and most preferably at least 20 times greater than the relative amount of RNA with a length of less than 300 nucleotides, preferably less than 100 nucleotides, most preferably less than 25 nucleotides in said phase P1 based on the total amount of RNA in said phase P1.

[0081] In other specific embodiments of the method of the present invention, in particular in embodiments using any one of the elution buffers EP1-EP4, the relative amount of miRNA in the aqueous phase P2 based on the total amount of miRNA and tRNA in the aqueous phase P2 is preferably at least 2 times greater than the relative amount of miRNA in the aqueous phase P1 based on the total amount of miRNA and tRNA in the aqueous phase P1, more preferably at least 4 times, even more preferably at least 6 times, even more preferably at least 10 times, and most preferably at least 20 times greater.

[0082] A contribution to solving the problem mentioned at the outset is also made by a kit for enriching nucleic acids having a length of less than 300 nucleotides, preferably less than 100 nucleotides, most preferably less than 25 nucleotides, comprising:

[0083] (β1) lysis buffer or lysis buffer concentrate,

[0084] (β2) anion exchange matrix,

[0085] (β3) elution buffer,

[0086] (β4) optional suspension buffer,

[0087] (β5) optional neutralization buffer,

[0088] (β6) optional wash buffer, and

[0089] (β7) Optional extractant, for example phenol, an alcohol such as ethanol, or a mixture thereof.

[0090] Such a kit can be used to perform the above-described method.

[0091] Preferred suspension buffers (β4), lysis buffers (β1), neutralization buffers (β5), wash buffers (β6), and elution buffers (β3) are those described above as preferred buffers that can be used in conjunction with the methods of the present invention. Lysis buffer concentrates are buffers containing a compound that effectively lyses, in particular a detergent or a chaotropic salt, at a concentration higher than that used during cell lysis. This type of lysis buffer concentrate is particularly useful if a cell suspension is to be used as a starting material for isolating short-chain nucleic acids, and the lysis conditions required for lysis can be adjusted by adding a specified amount of the lysis buffer concentrate.

[0092] Suitable anion exchange matrices (β2) are similar materials as those described above as preferred anion exchange matrices in the method according to the invention for enriching nucleic acids, for example, in particular magnetic or nonmagnetic particles coated with anion exchange matrices.

[0093] According to one embodiment of the kit of the present invention, the kit contains magnetic particles coated with anion exchange matrix as the anion exchange matrix (β2) and any buffer selected from EP1, EP2, EP3 or EP4 as the elution buffer (β3).

[0094] In addition, the use of the above-mentioned kit for the method of the present invention to purify nucleic acids with a length of less than 300 nucleotides, preferably less than 200 nucleotides, more preferably less than 100 nucleotides, even more preferably less than 50 nucleotides, and most preferably less than 25 nucleotides also contributes to solving the problem mentioned at the beginning.

[0095] The use of anion exchange matrices for the purification of nucleic acids having a length of less than 300 nucleotides, preferably less than 200 nucleotides, more preferably less than 100 nucleotides, even more preferably less than 50 nucleotides and most preferably less than 25 nucleotides also contributes to solving the problem mentioned at the outset, wherein the anion exchange matrix and the nucleotides are preferably the compounds mentioned at the outset as preferred components of the method according to the invention for purifying nucleic acids.

[0096] Finally, a method for treating a disease also contributes to solving the problem mentioned at the outset, said method comprising the following steps:

[0097] (γ1) diagnosing a disease by a diagnostic method comprising enriching nucleic acids having a length of less than 300 nucleotides, preferably less than 200 nucleotides, more preferably less than 100 nucleotides, even more preferably less than 50 nucleotides, and most preferably less than 25 nucleotides according to the purification method described at the beginning, and

[0098] (γ2) Therapeutic treatment of diagnosed diseases.

[0099] The disease to be treated may be any disease whose cause or progression is in any way related to the species and amount of nucleic acids present in specific body cells or body fluids, wherein the length of the nucleic acids is less than 300 nucleotides, preferably less than 200 nucleotides, more preferably less than 100 nucleotides, even more preferably less than 50 nucleotides, and most preferably less than 25 nucleotides, and in particular diseases associated with the species and amount of miRNAs, either the change in the species and amount of these nucleic acids compared to healthy people is the cause of the disease or the change in the species and amount of these nucleic acids compared to healthy people is the result of the disease.

[0100] The invention will be explained in more detail based on the following non-limiting figures and examples.

[0101] Figure 1 Depicted are 15% silver nitrate-stained polyacrylamide gels used to separate the eluate from Example 1 (duplicate gels; a = wash buffer after the first wash, b = wash buffer after the second wash, c = eluate).

[0102] Figure 2 Depicted is a 15% silver nitrate-stained polyacrylamide gel used to separate the eluate from Example 2 (duplicate gels).

[0103] Figure 3 Depicted is a 15% silver nitrate-stained polyacrylamide gel used to separate the eluate from Example 3 (duplicate gels).

[0104] Figure 4 The silver nitrate-stained 15% polyacrylamide gel used to separate the eluate from Example 4 is described (duplicate gels were used).

[0105] Figure 5 Depicted is a 15% silver nitrate-stained polyacrylamide gel used to separate the eluate from Example 5 (duplicate gels). Example

[0106] In the following examples, miRNAs spiked in the cellular background.

[0107] Example 1

[0108] Put 10 6 Jurkat cells were mixed with 1 μg of miR177 antisense miRNA and lysed with 550 μl of lysis buffer containing 0.5 M NaCl and 1% (v / v) Triton X-100. After incubation on ice for 10 minutes, 550 μl of acidic phenol was added. The mixture was vortexed and centrifuged at 20,800 × g for 5 minutes. The aqueous phase was removed and mixed with 652 μg of polyethyleneimine-coated magnetic particles.

[0109] 4 g of epoxide-functionalized magnetic particles (M-PVA E0x particles from Chemergen, Besweiler, Germany) were suspended in 50 ml of a 10% aqueous solution of high molecular weight polyethyleneimine (Sigma-Aldrich, Aldrich No. 40,872-7), pH 10, transferred to a round-bottom flask, and heated at 60° C. with stirring for 10 hours to obtain particles. The particles were then demagnetized and the mixture was washed six times with deionized water.

[0110] After shaking on a plate shaker for 5 minutes, the supernatant was discarded and the cells were washed twice with 500 μl of water. The pH was adjusted to 4.7, 5.5, 7.0 or 8.5 ( Figure 1 Elution was performed with 20 μl of a buffer containing 1 mol / l Tris / Cl, pH 9.5, 400 mmol / l KCl, 100 mmol / l ammonium sulfate, and 30 mmol / l MgCl2. An aliquot of the eluate was loaded onto a 15% polyacrylamide gel and stained with silver nitrate ( Figure 1 c channel).

[0111] Using 0.5 mol / l NaCl as the lysis buffer can effectively purify miRNA so that only miRNA and tRNA remain in the eluate after elution, while all other nucleic acid types have been diluted through the purification process.

[0112] Example 2

[0113] As described in Example 1, 10 6Jurkat cells were mixed with 1 μg of let7a antisense RNA, lysed, and bound to magnetic particles. After washing twice with water, the cells were eluted with 20 μl of a buffer containing 100 mmol / l NaCl, 250 mmol / l NaCl, 400 mmol / l NaCl, 100 mmol / l KCl, 250 mmol / l KCl, and 400 mmol / l KCl. An aliquot of the eluate was loaded onto a 15% polyacrylamide gel and stained with silver nitrate ( Figure 2 ).

[0114] This experiment revealed that different molar concentrations of salt can be used during elution. When NaCl, KCl, and LiCl (data not shown) were used as elution buffers, tRNA and miRNA could be purified simultaneously with high yields.

[0115] Example 3

[0116] The process was as described in Example 2, using a buffer containing 10-100 mmol / l MgCl2 as the elution buffer. An aliquot of the eluate was loaded onto a 15% polyacrylamide gel and stained with silver nitrate ( Figure 3 ).

[0117] This experiment demonstrates that miRNA can be purified using MgCl2 as an elution buffer. If a low molar concentration of MgCl2 is used as an elution buffer, tRNA and longer nucleic acids can be considerably diluted while miRNA can be recovered in very good yields.

[0118] Example 4

[0119] The process was as described in Example 2, using a buffer containing 10-85 mmol / l CaCl2 as the elution buffer. An aliquot of the eluate was loaded onto a 15% polyacrylamide gel and stained with silver nitrate ( Figure 4 ).

[0120] When the molar concentration of CaCl2 reaches up to about 50 mmol / l, miRNA can be eluted with very good recovery, while the eluate contains only trace amounts of tRNA. If the molar concentration is further increased, tRNA can also be eluted with good recovery.

[0121] Example 5

[0122] The process is as described in Example 2, using a buffer containing 25-400 mmol / l ammonium sulfate or 25-400 mmol / l ammonium chloride as the elution buffer. An aliquot of the eluate is loaded onto a 15% polyacrylamide gel and stained with silver nitrate ( Figure 5 ).

[0123] Ammonium salts, in particular, exhibited optimal elution properties, along with calcium chloride, enabling high miRNA yields while also achieving very low tRNA yields. While tRNA yields remained relatively low at ammonium salt concentrations up to approximately 170-200 mmol / l in the eluate, miRNA yields were very good at these molar concentrations. Even at concentrations up to approximately 400 mmol / l, significant amounts of tRNA were found in the eluate.

Claims

1. A method for enriching nucleic acids of less than 300 nucleotides in length from a plasma or serum sample, the method comprising the following steps: i) Lysing cells contained in the sample with a lysis buffer containing 0.5 mol / l sodium chloride to provide a fluid phase P1 containing (a1) at least one nucleic acid a1 having a length of less than 300 nucleotides, and (a2) at least one component different from the nucleic acid a1, ii) contacting the phase P1 with an anion exchange matrix to bind the nucleic acid a1 to the anion exchange matrix, wherein the anion exchange matrix comprises a functional group selected from the group consisting of an amino group, a hydrazine group, and an imine group, wherein the anion exchange matrix is ​​present in the form of a coating on magnetic particles, The binding of nucleic acid a1 to the anion exchange matrix is ​​carried out at a pH of 4-6 in the presence of 0.05-0.75 mol / l alkali metal salt. wherein the binding of the nucleic acid is performed by continuously stirring the fluid phase P1 in contact with the particles, iii) separating the magnetic particles from the phase P1 as magnetic aggregates and washing the anion exchange matrix with a washing buffer, wherein the nucleic acid a1 remains bound to the anion exchange matrix, and iv) eluting the nucleic acid a1 bound to the anion exchange matrix from the anion exchange matrix using an elution buffer to obtain an aqueous phase P2 containing the nucleic acid a1, The elution buffer contains: 1 mol / l Tris / Cl, pH 9.5, 400 mmol / l KCl, 100 mmol / l ammonium sulfate and 30 mmol / l MgCl2, wherein the nucleic acid a1 is a mixture of miRNA and tRNA; the elution buffer contains 100 mmol / l NaCl, 250 mmol / l NaCl, 400 mmol / l NaCl, 100 mmol / l KCl, 250 mmol / l KCl or 400 mmol / l KCl, wherein the nucleic acid a1 is a mixture of miRNA and tRNA; The elution buffer contains 10-50 mmol / l CaCl2, wherein the nucleic acid a1 is miRNA; or The elution buffer contains 25-200 mmol / l of ammonium chloride or ammonium sulfate, wherein the nucleic acid a1 is miRNA.

2. The method according to claim 1, wherein The anion exchange matrix has amino groups.

3. The method according to claim 1, wherein The particles are superparamagnetic, ferrimagnetic or ferromagnetic particles.

4. The method according to claim 1, wherein The alkali metal salt is potassium chloride, sodium chloride or lithium chloride. The method of claim 1 , wherein the fluid phase P1 is an aqueous phase.

6. Use of a kit for enriching nucleic acids having a length of less than 300 nucleotides in the method according to any one of claims 1 to 5, the kit comprising: (b1) lysis buffer or lysis buffer concentrate, (b2) an anion exchange matrix, wherein the anion exchange matrix has a functional group selected from the group consisting of an amino group, a hydrazine group, and an imine group, and wherein the anion exchange matrix is ​​in the form of a coating on magnetic particles, (b3) elution buffer, (b4) optional suspension buffer, (b5) optional neutralization buffer, (b6) optional wash buffer, and (b7) Optional extractant.

7. The use according to claim 6, characterized in that The anion exchange matrix (b2) has an amino group.

8. The use according to claim 6, characterized in that The anion exchange matrix (b2) is present in the form of a coating on superparamagnetic, ferrimagnetic or ferromagnetic particles.

9. The use according to any one of claims 6 to 8, wherein The elution buffer (b3) contains calcium chloride at a concentration of 1-1000 mmol / l.

10. The use according to any one of claims 6 to 8, wherein The elution buffer (b3) contains magnesium chloride at a concentration of 1-1000 mmol / l.

11. The use according to any one of claims 6 to 8, wherein The elution buffer (b3) contains ammonium sulfate or ammonium chloride at a concentration of 1-1000 mmol / l.

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