A RNA purification partner and its application

Abstract

The present invention relates to a RNA purification partner and its application. The RNA purification partner is an ionic liquid composed of cations and anions. The purification method is chromatography. The purification partner is used to simultaneously improve the elution efficiency of RNA on the chromatography medium and improve the stability of RNA. The chromatography is, for example, ion exchange chromatography, affinity chromatography, etc. Improving the stability of RNA refers to stabilizing the secondary structure of RNA and inhibiting the enzymatic hydrolysis of RNA by RNAse. The RNA purification partner of the present invention can also be used alone as a stabilizer for RNA.

Description

Technical Field

[0001] The invention belongs to the technical field of separation and purification of ribonucleic acid, and relates to a ribonucleic acid purification partner and application thereof. Background Art

[0002] Ribonucleic acid (RNA) is a long-chain polymer formed by condensation of ribonucleotides through phosphodiester bonds. It has a wide range of applications in biology, medicine, and nanotechnology. In organisms, RNA is the key to the central dogma of driving protein synthesis. RNA-related research such as structure, biochemistry, and vaccinology requires high-purity RNA. Therefore, sample purification is a key step in RNA research.

[0003] At present, a variety of liquid chromatography techniques have been used to purify RNA in vivo or RNA obtained by in vitro transcription, including affinity chromatography, size exclusion chromatography, ion exchange chromatography, reversed-phase ion pair chromatography, etc. Among them, anion exchange chromatography adsorbs biomolecules through the electrostatic attraction between positively charged ligands and negatively charged biomolecules on the chromatographic medium, and then desorbs and elutes the biomolecules adsorbed on the medium by increasing the salt concentration in the eluent and shielding the charge. The presence of a large number of negatively charged phosphate groups on the RNA chain makes it negatively charged in a wide pH range, making it suitable for purification using anion exchange chromatography. Although anion exchange chromatography can be used for RNA purification, due to the strong negative charge of RNA, the high-salt elution conditions of conventional ion exchange may not completely dissociate RNA from the positively charged ligand, resulting in a low elution recovery rate. In response to similar problems, some optimization methods have been reported in related literature to promote the elution of DNA from solid-phase adsorption surface materials.

[0004] However, these optimized methods for eluting DNA are not applicable to RNA, because compared with protein or DNA, RNA is more unstable and its structure is more susceptible to degradation by temperature, pH and ribonucleases, making it difficult to store for a long time, and the structural integrity of RNA is essential for its biological activity. Therefore, how to maintain the stability of purified RNA is an important issue that needs to be paid attention to in RNA research. At present, in addition to preventing RNA degradation by low-temperature storage (-80℃~-20℃), improving the stability of the RNA structure itself and inhibiting the degradation of RNA by ribonucleases are the two main strategies for stabilizing RNA. For example, the structural stability of the mRNA itself can be improved by capping the 5' end of the mRNA, modifying the coding region with pseudouracil, or adding a poly A structure to its 3' end. Or use cations such as Mg 2+ , K + 、Na +The structural stability of RNA is improved by shielding the negatively charged phosphate groups in the RNA backbone and chelating the 2'hydroxyl group. The activity of ribonucleases in liquid chromatography buffer or RNA storage solution is inhibited by using nuclease inhibitors such as diethyl pyrocarbonate (DEPC), vanadyl ribonucleoside complex (RVC), guanidine isothiocyanate or RNasin. However, the existing RNA stabilizers only have a single function and cannot stabilize the RNA structure and inhibit the activity of ribonucleases at the same time.

[0005] Therefore, how to improve the elution efficiency during RNA purification while maintaining the stability of the purified RNA has become an urgent problem to be solved by technicians in this field. Summary of the invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a RNA purification partner and its application. The RNA purification partner of the present invention can also be used alone as a stabilizer for RNA.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a RNA purification partner, wherein the RNA purification partner is an ionic liquid composed of cations and anions, the purification method is chromatography, and the purification partner is used to simultaneously improve the elution efficiency of RNA on the chromatography medium and improve the stability of RNA.

[0009] The chromatography includes, for example, ion exchange chromatography, affinity chromatography and the like.

[0010] The elution efficiency refers to the recovery rate after RNA elution, and the elution efficiency = the amount of RNA after elution / the amount of RNA before elution × 100%.

[0011] Improving the stability of RNA refers to stabilizing the secondary structure of RNA and inhibiting the enzymatic degradation of RNA by RNAse.

[0012] The secondary structure refers to a single-stranded region structure, a stem-loop structure or a double-stranded structure formed by complementary base pairing in a single strand of RNA.

[0013] The ribonuclease refers to an enzyme that hydrolyzes the phosphodiester bond of RNA, including ribonuclease A, ribonuclease T1 or ribonuclease H.

[0014] Preferably, the ribonucleic acid is a long chain molecule formed by condensation of ribonucleotides through phosphodiester bonds, including any one of mRNA, tRNA, rRNA, miRNA, snRNA, snoRNA, siRNA, saRNA, tmRNA, crRNA, tracrRNA, gRNA, ribozyme, viroid or terminal polymerase RNA, or a combination of at least two thereof.

[0015] Preferably, the ribonucleic acid is extracted from animals, plants or microorganisms, or obtained by in vitro transcription from DNA.

[0016] Preferably, the microorganism comprises yeast.

[0017] Preferably, the ionic liquid comprises an imidazolium-type ionic liquid and / or a choline-type ionic liquid.

[0018] Preferably, the cation in the imidazolium-type ionic liquid is [C n mim] + (1-alkyl-3-methylimidazole), n=2-8 (2, 3, 4, 5, 6, 7 or 8), for example [C2mim] + (1-ethyl-3-methylimidazole), [C4mim] + (1-butyl-3-methylimidazole), [C6mim] + (1-hexyl-3-methylimidazole), [C8mim] + (1-octyl-3-methylimidazole), etc.

[0019] Preferably, the cation in the choline ionic liquid is [Cho] + (choline).

[0020] Preferably, the anion in the ionic liquid comprises [Tf2N] - (bis(trifluoromethanesulfonyl imide) ion), [SCN] - (thiocyanate ion), [N(CN)2] - (dicyanimide ion), [TfO] - (trifluoromethanesulfonate ion), [MeOSO3] - (methylsulfate ion), [Br] - (bromide ion), [Cl] - (chloride ion), [BF4] - (tetrafluoroborate ion), [EtOSO3] - (ethyl sulfate ion) or [dca] - (dichloroacetate ion) or a combination of at least two thereof.

[0021] The combination of at least two such as [Tf2N]- and [EtOSO3] - Combination of [Cl] - and [SCN] - Combination of [dca] - and [TfO] - Any other combination is also possible.

[0022] Preferably, the chromatography medium comprises an anion exchange chromatography medium.

[0023] Preferably, the ligand in the anion exchange chromatography medium comprises TMAM (—CH2N + (CH3)3)、TEAE(—(CH2)2N + (C2H5)3), HEDMAM (—CH2N + (CH3)2C2H4OH), QAE(—CH2N + (C2H5)2—CH2CH(OH)CH3), DEAE(—(CH2)2N(C2H5)2), MP(—C5H5NCH3), GE(—C2H4C(NH2)=N + H2), DEAHP(—CH2CH(OH)CH2NH(C2H5)2), AE(—C2H4N + H3) or PEI(—(C2H4NH) n C2H4N + Any one of H3).

[0024] Preferably, when the ribonucleic acid is extracted from yeast, the ionic liquid comprises any one or a combination of at least two of [Cho][EtOSO3], [C8mim][MeOSO3], [C6mim][Tf2N], [C4mim][dca], [Cho][Cl], [Cho][BF4] or [C4mim][Cl].

[0025] The combination of at least two of the above mentioned can be, for example, a combination of [Cho][EtOSO3] and [C8mim][MeOSO3], a combination of [C6mim][Tf2N] and [C4mim][dca], a combination of [Cho][BF4] and [C4mim][Cl], or any other combination.

[0026] Preferably, when the ribonucleic acid is mRNA (preferably obtained by in vitro transcription), the ionic liquid includes any one or a combination of at least two of [Cho][Cl], [Cho][Br], [C4mim][Cl], [C2mim][Br], [C6mim][Tf2N] or [C4mim][dca].

[0027] Preferably, the ionic liquid comprises [Cho][BF4] and / or [C4mim][Cl].

[0028] Preferably, the concentration ratio of [Cho][BF4] to [C4mim][Cl] is (3-6):(4-7).

[0029] Specific numerical values ​​in the above (3-6) include 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.

[0030] Specific numerical values ​​in the above (4-7) include 4, 4.5, 5, 5.5, 6, 6.5, 7, etc.

[0031] In a second aspect, the present invention provides use of the RNA purification partner as described in the first aspect in preparing a chromatography eluent for purifying RNA.

[0032] In a third aspect, the present invention provides a method for purifying RNA, which comprises injecting the RNA to be purified into a chromatography column containing a chromatography medium, eluting with an eluent containing the RNA purification partner as described in the first aspect, collecting the eluted components, and completing the purification.

[0033] Preferably, the eluent is obtained by mixing water or a buffer solution with a RNA purification partner, and the buffer solution includes any one of acetate buffer, phosphate buffer, Tris-HCl buffer, HEPES buffer or carbonate buffer, or a combination of at least two thereof.

[0034] Preferably, the concentration of the buffer solution is 5-200 mM, for example, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, 200 mM, etc.

[0035] Preferably, the pH value of the buffer solution is 6.0-8.5, for example, 6.0, 6.2, 6.4, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.5, etc.

[0036] Preferably, the concentration of the RNA purification partner in the buffer solution is 0.05M-2M, for example, 0.05M, 0.08M, 0.1M, 0.2M, 0.4M, 0.6M, 1M, 1.2M, 1.4M, 1.6M, 1.8M, 2.0M, etc.

[0037] Preferably, the eluent also contains an inorganic salt, which includes any one of sodium chloride, potassium chloride or ammonium sulfate, or a combination of at least two of them. The combination of at least two is, for example, a combination of sodium chloride and ammonium sulfate, a combination of potassium chloride and ammonium sulfate, sodium chloride and potassium chloride, etc. Any other combination is also acceptable.

[0038] Preferably, the concentration of the inorganic salt in the eluent is 0.5-2M, for example, 0.5M, 0.6M, 0.8M, 1M, 1.2M, 1.4M, 1.6M, 1.8M, 2.0M, etc.

[0039] Preferably, the elution method includes isocratic elution or gradient elution, and the gradient elution includes linear gradient elution or step gradient elution.

[0040] Preferably, the elution further comprises flushing the chromatography medium with an equilibrium buffer, wherein the equilibrium buffer comprises any one of acetate buffer, phosphate buffer, Tris-HCl buffer, HEPES buffer or carbonate buffer, or a combination of at least two thereof.

[0041] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention creatively proposes to use ionic liquid as a RNA purification partner, which has the following advantages:

[0044] (1) It can improve the elution efficiency of RNA on anion exchange chromatography and also improve the stability of RNA in the eluted fractions obtained after elution;

[0045] (2) Stabilize RNA structure by stabilizing RNA molecular structure and inhibiting ribonuclease activity, which is not achievable by existing RNA stabilizers;

[0046] (3) It can be used as a stabilizing liquid for RNA from various sources. It has good solubility and is easy to use, and has broad application prospects.

[0047] In addition, the effects of different ionic liquids are different. In terms of improving the structural stability of yeast RNA, [Cho][EtOSO3] has a better effect. In terms of reducing the stability of ribonuclease A, [C8mim][MeOSO3], [C6mim][Tf2N] and [C4mim][dca] have better effects. In terms of improving the elution efficiency of yeast RNA, each ionic liquid can increase the elution efficiency to more than 99% (the conventional elution liquid in the prior art can only achieve an elution efficiency of 71-77%). In terms of improving the stability of yeast RNA after elution, [Cho][Cl] has a better effect. In terms of improving the elution efficiency of mRNA, [C4mim][Cl] has a better effect. In terms of improving the stability of mRNA after elution, [Cho][BF4] and [C4mim][Cl] have better effects.

[0048] The present invention also unexpectedly discovered that, when the concentration of both is 1 M, the combination of [C4mim][Cl] and [Cho][BF4] has a better effect than using any single component ionic liquid. This result shows that [C4mim][Cl] and [Cho][BF4] cooperate with each other and have an unexpected synergistic effect in improving the mRNA elution efficiency and elution stability. DETAILED DESCRIPTION

[0049] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0050] In the following examples, unless otherwise specified, all reagents and consumables were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.

[0051] The yeast RNA (CAS No.: 63231-63-0) involved in the following examples and comparative examples was purchased from Sigma, with the catalog number: R6625.

[0052] Example 1

[0053] This embodiment provides a yeast RNA solution containing a choline-type ionic liquid, and the preparation method thereof is as follows:

[0054] 1) dissolving a certain amount of choline ionic liquid [Cho][EtOSO3] in 20 mM Tris-HCl buffer at pH 7.5, and adjusting the pH to pH 7.5 with 4 mol / L NaOH or 2 mol / L HCl to prepare a [Cho][EtOSO3] solution with a concentration of 0.4 M;

[0055] 2) 1 mL of the above [Cho][EtOSO3] solution was added to 1 mL of a 1 mg / mL yeast RNA solution (pH 7.5) to obtain a yeast RNA solution to which 0.2 M [Cho][EtOSO3] was added.

[0056] Example 2

[0057] This embodiment provides a yeast RNA solution containing a choline-type ionic liquid. The preparation method thereof is different from that of Example 1 only in that [Cho][EtOSO3] is replaced with [Cho][dca] of equal concentration, and other conditions refer to those of Example 1.

[0058] Example 3

[0059] This embodiment provides a yeast RNA solution containing imidazole-type ionic liquid, and the preparation method thereof is as follows:

[0060] 1) Dissolve a certain amount of choline-type ionic liquid [C4mim][Tf2N] in 20 mM, pH 7.5 Tris-HCl buffer, and adjust the pH to pH 7.5 with 4 mol / L NaOH or 2 mol / L HCl to prepare a 0.2 M [C4mim][Tf2N] solution;

[0061] 2) 1 mL of the above [C4mim][Tf2N] solution was added to 1 mL of a 1 mg / mL yeast RNA solution (pH 7.5) to obtain a yeast RNA solution to which 0.1 M [C4mim][Tf2N] was added.

[0062] Example 4

[0063] This embodiment provides a yeast RNA solution containing an imidazolium-type ionic liquid. The preparation method thereof is different from that of Example 3 only in that [C4mim][Tf2N] is replaced with [C6mim][SCN] of equal concentration, and other conditions refer to Example 3.

[0064] Example 5

[0065] This embodiment provides a yeast RNA solution containing an imidazolium-type ionic liquid. The preparation method thereof is different from that of Example 3 only in that [C4mim][Tf2N] is replaced with an equal concentration of [C8mim][MeOSO3], and other conditions refer to Example 3.

[0066] Example 6

[0067] This embodiment provides an eluent containing a choline-type ionic liquid, and the preparation method thereof is as follows:

[0068] A certain amount of choline ionic liquid [Cho][Cl] was dissolved in 50 mM, pH 6.5, phosphate buffer containing 2 mM EDTA, and the pH was adjusted to pH 6.5 with 4 mol / L NaOH or 2 mol / L HCl to prepare an eluent containing 1 M [Cho][Cl].

[0069] Example 7

[0070] This embodiment provides an eluent containing a choline-type ionic liquid. The preparation method thereof is different from that of embodiment 6 only in that the concentration of [Cho][Cl] in the eluent is changed to 2M, and other conditions refer to those of embodiment 6.

[0071] Example 8

[0072] This embodiment provides an eluent containing a choline-type ionic liquid. The preparation method thereof is different from that of Embodiment 6 only in that the concentration of [Cho][Cl] in the eluent is changed to 1.5 M. The other conditions refer to those of Embodiment 6.

[0073] Example 9

[0074] This embodiment provides an eluent containing a choline-type ionic liquid. The preparation method thereof is different from that of Embodiment 6 only in that [Cho][Cl] is replaced with [Cho][Br] of equal concentration, and other conditions refer to those of Embodiment 6.

[0075] Example 10

[0076] This embodiment provides an eluent containing a choline-type ionic liquid. The preparation method thereof is different from that of Example 6 only in that [Cho][Cl] is replaced with [Cho][BF4] of equal concentration, and other conditions refer to those of Example 6.

[0077] Embodiment 11

[0078] This embodiment provides an eluent containing an imidazole-type ionic liquid, and the preparation method thereof is as follows:

[0079] A certain amount of choline ionic liquid [C4mim][Cl] was dissolved in 50 mM, pH 6.5, phosphate buffer containing 2 mM EDTA, and the pH was adjusted to pH 6.5 with 4 mol / L NaOH or 2 mol / L HCl to prepare an eluent containing 1 M [C4mim][Cl].

[0080] Example 12

[0081] This embodiment provides an eluent containing an imidazolium-type ionic liquid. The preparation method thereof is different from that of Embodiment 11 only in that the concentration of [C4mim][Cl] in the eluent is changed to 2M, and the other conditions refer to those of Embodiment 11.

[0082] Example 13

[0083] This embodiment provides an eluent containing an imidazolium-type ionic liquid. The preparation method thereof is different from that of Embodiment 11 only in that the concentration of [C4mim][Cl] in the eluent is changed to 1.5 M. The other conditions refer to those of Embodiment 11.

[0084] Embodiment 14

[0085] This embodiment provides an eluent containing an imidazolium-type ionic liquid. The preparation method thereof is different from that of Embodiment 11 only in that [C4mim][Cl] is replaced with an equal concentration of [C2mim][Br], and other conditions refer to Embodiment 11.

[0086] Embodiment 15

[0087] This embodiment provides an eluent containing an imidazolium-type ionic liquid. The preparation method thereof is different from that of Embodiment 11 only in that [C4mim][Cl] is replaced with an equal concentration of [C6mim][Tf2N], and other conditions refer to Embodiment 11.

[0088] Example 16

[0089] This embodiment provides an eluent containing an imidazolium-type ionic liquid. The preparation method thereof is different from that of Embodiment 11 only in that [C4mim][Cl] is replaced with an equal concentration of [C4mim][dca], and other conditions refer to Embodiment 11.

[0090] Embodiment 17

[0091] This embodiment provides an eluent containing 0.7M [C4mim] [Cl] and 0.3M [Cho] [BF4], and the preparation method thereof is as follows:

[0092] A certain amount of imidazole ionic liquid [C4mim][Cl] and a certain amount of choline ionic liquid [Cho][BF4] were dissolved in 50mM, pH 6.5, phosphate buffer containing 2mM EDTA, and the pH was adjusted to pH 6.5 with 4mol / L NaOH or 2mol / L HCl to prepare an eluent containing both 0.6M [C4mim][Cl] and 0.4M [Cho][BF4].

[0093] Embodiment 18

[0094] This example provides an eluent containing both 0.5M [C4mim][Cl] and 0.5M [Cho][BF4], and its preparation method refers to Example 17.

[0095] Embodiment 19

[0096] This example provides an eluent containing both 0.4M [C4mim][Cl] and 0.6M [Cho][BF4], and its preparation method refers to Example 17.

[0097] Embodiment 20

[0098] This embodiment provides an eluent containing a choline-type ionic liquid and 1M NaCl, and the preparation method thereof is as follows:

[0099] A certain amount of choline ionic liquid [Cho][Br] was dissolved in 50 mM, pH 6.5, phosphate buffer containing 1 M NaCl and 2 mM EDTA, and the pH was adjusted to pH 6.5 with 4 mol / L NaOH or 2 mol / L HCl to prepare an eluent containing 0.2 M [Cho][Cl] and 1 M NaCl.

[0100] Embodiment 21

[0101] This embodiment provides an eluent containing an imidazole-type ionic liquid and 1M ammonium sulfate, and its preparation method is as follows:

[0102] A certain amount of choline ionic liquid [C2mim][dca] was dissolved in 50 mM, pH 6.5, phosphate buffer containing 1 M ammonium sulfate and 2 mM EDTA, and the pH was adjusted to pH 6.5 with 4 mol / L NaOH or 2 mol / L HCl to prepare an eluent containing 0.2 M [C2mim][Cl] and 1 M ammonium sulfate.

[0103] Embodiment 22

[0104] This embodiment provides an eluent containing only imidazole-type ionic liquid, and the preparation method thereof is as follows:

[0105] A certain amount of choline type ionic liquid [C2mim][Br] was dissolved in water to prepare an eluent containing 0.05 M [C2mim][Br].

[0106] Comparative Example 1

[0107] This comparative example provides a yeast RNA solution, which differs from Example 1 only in that the solution does not contain an ionic liquid. The specific preparation method is as follows:

[0108] To 1 mL of a 1 mg / mL yeast RNA solution (pH 7.5), 1 mL of a 20 mM Tris-HCl buffer, pH 7.5, was added to obtain a yeast RNA solution.

[0109] Comparative Example 2

[0110] This comparative example provides a conventional eluent, which differs from Example 6 only in that the eluent does not contain an ionic liquid but contains 1M NaCl. The preparation method thereof is as follows:

[0111] A certain amount of NaCl was dissolved in 50 mM, pH 6.5, phosphate buffer containing 2 mM EDTA, and the pH was adjusted to pH 6.5 with 4 mol / L NaOH or 2 mol / L HCl to prepare an eluent containing 1 M NaCl.

[0112] Comparative Example 3

[0113] This comparative example provides a conventional eluent, which differs from Example 6 only in that the eluent does not contain an ionic liquid but contains 1.5 M NaCl. The preparation method thereof is as follows:

[0114] A certain amount of NaCl was dissolved in 50 mM, pH 6.5, phosphate buffer containing 2 mM EDTA, and the pH was adjusted to pH 6.5 with 4 mol / L NaOH or 2 mol / L HCl to prepare an eluent containing 1.5 M NaCl.

[0115] Comparative Example 4

[0116] This comparative example provides a conventional eluent, which differs from Example 6 only in that the eluent does not contain an ionic liquid but contains 2M NaCl. The preparation method thereof is as follows:

[0117] A certain amount of NaCl was dissolved in 50 mM, pH 6.5, phosphate buffer containing 2 mM EDTA, and the pH was adjusted to pH 6.5 with 4 mol / L NaOH or 2 mol / L HCl to prepare an eluent containing 2 M NaCl.

[0118] Test Example 1-Yeast RNA Stability Test

[0119] The T of RNA in the yeast RNA solution of Examples 1-5 and Comparative Example 1 m The value was measured to reflect the stability of RNA structure in each group of solutions, T m The value is defined as the temperature corresponding to the loss of half of the RNA secondary structure. The higher the temperature, the more stable the RNA secondary structure.

[0120] The specific method is as follows: first, the ionic liquid that is not bound to RNA in each group of solutions is removed by G25 desalting column, and then circular dichroism is used to measure the changes in the RNA secondary structure spectrum at 210nm-320nm under the condition of 20℃-90℃ gradient heating (2℃ / min). The heating curve is analyzed using Global 3 software to calculate the T of RNA. mThe results are shown in Table 1.

[0121] Table 1

[0122] Group <![CDATA[T m Value(℃)]]> Group <![CDATA[T m Value(℃)]]> Example 1 55.6 Example 4 51.7 Example 2 54.0 Example 5 50.2 Example 3 52.3 Comparative Example 1 42.5

[0123] The results show that: from the comparison results of Examples 1-5 and Comparative Example 1, it can be seen that both choline-type and imidazole-type ionic liquids can increase the T m value, which is beneficial to maintaining the stability of the RNA secondary structure. Under the same concentration, the effect of choline-type ionic liquids on improving the stability of RNA structure is better than that of imidazole-type ionic liquids, among which the choline-type ionic liquid [Cho][EtOSO3] has the best effect (Example 1).

[0124] Test Example 2-Ribonuclease Stability Test

[0125] Differential scanning calorimetry (DSC) was used to measure the T m After adding 0.5 mg / mL ribonuclease A to the corresponding solutions of Examples 1-5, 6, 9-10, 11, 14-16, Comparative Example 1, and 3, the samples were placed in MicrolCal TM In the VP-DSC instrument, the temperature range of the instrument was 20℃-90℃ (1℃ / min), and the T of ribonuclease in each group of solutions was measured under this condition. m Value (thermal denaturation temperature), the higher the value, the higher the stability of the ribonuclease. The results are shown in Table 2.

[0126] Table 2

[0127] Group <![CDATA[T m Value(℃)]]> Group <![CDATA[T m Value(℃)]]> Example 1 60.4 Example 6 61.3 Example 2 58.2 Example 9 58.7 Example 3 54.3 Example 10 58.9

[0128] Example 4 55.1 Embodiment 11 57.2 Example 5 52.9 Embodiment 14 58.6 Comparative Example 1 63.5 Embodiment 15 52.1 Comparative Example 2 64.0 Example 16 51.0

[0129] The results show that: from the comparison of the yeast RNA solutions of each group (Examples 1-5 and Comparative Example 1), it can be seen that both choline and imidazole ionic liquids can reduce the T of ribonuclease A. m value, making ribonuclease A more unstable and inactivated in the ionic liquid environment. Combined with the results of Test Example 1, it can be seen that choline and imidazole ionic liquids can simultaneously achieve the effect of improving the stability of RNA and reducing the stability of ribonucleases. Among them, under the same concentration, the effect of imidazole ionic liquids in reducing the stability of ribonucleases is better than that of choline ionic liquids, among which [C8mim][MeOSO3] has the best effect (Example 5).

[0130] The same conclusion was drawn from the comparison of the eluents of each group (Examples 6, 9-11, 14-16 and Comparative Example 3): both choline and imidazole ionic liquids can reduce the T of ribonuclease A. m value, making ribonuclease A more unstable and inactivated in the ionic liquid environment. Among them, when the ionic liquid in the eluent is [C6mim][Tf2N] or [C4mim][dca], the effect of reducing the stability of ribonuclease is best (Examples 15 and 16).

[0131] Test Example 3-Yeast RNA Elution Efficiency Test

[0132] At 25°C, the chromatography column (chromatographic medium is POROS DEAE) was equilibrated with 50 mM PB + 0.2 mM EDTA, pH 6.5 buffer, and then 0.9 mg / mL yeast RNA was injected into the chromatography column. After the injection was completed, the chromatography column was eluted with 50 mM PB + 0.2 mM EDTA, pH 6.5 buffer, and then eluted with the eluents in Examples 6-16 and Comparative Examples 2-4, respectively. The eluted components were collected and the elution efficiency was calculated, elution efficiency = total amount of RNA in the eluted components / total amount of RNA injected into the chromatography column × 100%. The results are shown in Table 3.

[0133] Table 3

[0134] Group Elution efficiency (%) Group Elution efficiency (%) Example 6 >99 Example 13 >99 Example 7 >99 Embodiment 14 >99 Example 8 >99 Embodiment 15 >99 Example 9 >99 Example 16 >99 Example 10 >99 Comparative Example 2 77 Embodiment 11 >99 Comparative Example 3 75 Example 12 >99 Comparative Example 4 71

[0135] The results showed that when using traditional NaCl as the elution liquid, the elution efficiency of yeast RNA on the anion exchange medium was low, only 71%-77%, while when using ionic liquid as the purification partner for elution, the elution efficiency of RNA could be significantly increased to more than 99%.

[0136] Test Example 4 - Determination of the stability of yeast RNA after elution

[0137] According to the method of Test Example 3, yeast RNA was eluted using each group of elution solutions, and the elution fractions were collected after elution. The T value of RNA in each elution fraction was determined according to the method of Test Example 1. m The results are shown in Table 4.

[0138] Table 4

[0139]

[0140]

[0141] The results showed that after elution with an ionic liquid as a purification partner, the RNA T mThe value is significantly higher than the result of elution using a conventional elution solution without ionic liquid, indicating that ionic liquids can stabilize the secondary structure of RNA during the elution process and significantly improve the stability of RNA after elution.

[0142] From the comparison results of Examples 6, 9, 10, 11, 14, 15, and 16, it can be seen that, under the same concentration, the effect of [Cho][Cl] on improving RNA elution stability is better than that of other ionic liquids.

[0143] From the comparison results of Examples 6-8 and Examples 11-13, it can be seen that the concentration of the ionic liquid in the eluent has a certain influence on the RNA elution stability, and the best effect is achieved when the concentration is 2M.

[0144] Test Example 5-mRNA elution efficiency test

[0145] Under 25°C, the chromatography column (chromatographic medium is DEAE Sepharose FF) was equilibrated with 50mM PB + 0.2mM EDTA, pH 6.5 buffer, and 142μg / mL of mRNA expressing green fluorescent protein (transcribed from green fluorescent protein DNA, and the transcription method is a conventional method in the art) was injected into the chromatography column. After the injection was completed, the chromatography column was eluted with 50mM PB + 0.2mM EDTA, pH 6.5 buffer, and then eluted with the eluents of Examples 6-7, Examples 9-12, Examples 17-19 and Comparative Examples 2 and 4, respectively. The eluted components were collected, and the elution efficiency was calculated. The results are shown in Table 5.

[0146] Table 5

[0147]

[0148]

[0149] The results showed that the elution efficiency of mRNA can be significantly improved when ionic liquid is used as a purification partner. The elution efficiency is the highest when the ionic liquid is [C4mim][Cl].

[0150] In addition, from the comparison results of Examples 11-12, it can be seen that the concentration of the ionic liquid in the eluent has a great influence on the mRNA elution efficiency, and the best effect is achieved when the concentration is 2M.

[0151] From the comparison results of Examples 10, 11, and 17, it can be seen that when the concentration is 1 M, the combination of [C4mim][Cl] and [Cho][BF4] has a better effect than using any single component ionic liquid. This result shows that [C4mim][Cl] and [Cho][BF4] cooperate with each other and have an unexpected synergistic effect in improving the mRNA elution efficiency.

[0152] Test Example 6 - Determination of the stability of mRNA after elution

[0153] After eluting the mRNA with different eluents according to the method described in Test Example 5, the eluted fractions were collected and placed at 4°C for 72 hours. The mRNA content in the eluted fractions was determined by the grayscale change of agarose electrophoresis, and the remaining percentage of mRNA (%) was calculated to reflect the degradation of mRNA. The T of mRNA in the eluted fractions was determined according to the method described in Test Example 1. m The values ​​are used to reflect the structural stability of mRNA. The results are shown in Table 6.

[0154] Table 6

[0155]

[0156]

[0157] The results showed that after elution using an elution solution containing an ionic liquid as a purification partner, the stability of mRNA in the elution fraction obtained was significantly higher than that obtained using a conventional elution solution not containing an ionic liquid.

[0158] From the comparison results of Examples 6, 9, 10, and 11, it can be seen that, under the same concentration, the effects of [Cho][BF4] and [C4mim][Cl] on improving the mRNA elution stability are better than those of other ionic liquids.

[0159] The concentration of the ionic liquid in the eluent has a certain influence on the stability of mRNA elution. From the comparison between Examples 6-7 and 11-12, it can be seen that the effect is better when the concentration is 2M.

[0160] From the comparison results of Examples 10, 11, and 17, it can be seen that when the concentration is 1 M, the combination of [C4mim][Cl] and [Cho][BF4] has a better effect than using any single component ionic liquid. This result shows that [C4mim][Cl] and [Cho][BF4] cooperate with each other and have an unexpected synergistic effect in improving the elution stability of mRNA.

[0161] Test Example 7-mRNA elution efficiency and post-elution stability test

[0162] At 25°C, the chromatography column (chromatographic medium is QSepharose FF) was equilibrated with 50 mM PB + 0.2 mM EDTA, pH 6.5 buffer, and 142 μg / mL of mRNA expressing green fluorescent protein was injected into the chromatography column. After the injection was completed, the chromatography column was eluted with 50 mM PB + 0.2 mM EDTA, pH 6.5 buffer, and then eluted with the eluents of Examples 20, 21 and Comparative Example 2, respectively, and the eluted fractions were collected. The elution efficiency was calculated, and the T of the mRNA in the eluted fractions was determined according to the method described in Test Example 1. m The results are shown in Table 7.

[0163] Table 7

[0164] Group Elution efficiency (%) <![CDATA[T m Value(℃)]]> Embodiment 20 44 43.1 Embodiment 21 58 46.2 Comparative Example 2 41 41.5

[0165] The results showed that the use of ionic liquids as purification partners can not only stabilize the secondary structure of RNA, but also inhibit the activity of ribonucleases and improve the elution efficiency of RNA on anion exchange chromatography, which has great practical value.

[0166] Test Example 8 - mRNA stability test after elution (other chromatography)

[0167] Under 25°C, the chromatography column (chromatographic medium is oligo(dT)) was equilibrated with 20 mM Tris-HCl + 1.0 M NaCl, pH 7.5 buffer, and 180 μg / mL of mRNA expressing green fluorescent protein was injected into the chromatography column. After the injection was completed, the chromatography column was eluted with 50 mM PB + 0.2 mM EDTA, pH 6.5 buffer, and then eluted with the eluent of Example 22 and deionized water, respectively. The eluted fractions were collected, and the T of the mRNA in the eluted fractions was determined according to the method described in Test Example 1. m value.

[0168] The results showed that the mRNA T after elution with the eluent of Example 22 was m The value is 42.1℃, and the T value of mRNA after elution with water is m The value was 39.5℃, indicating that using ionic liquids as purification partners for other chromatography can also improve the stability of RNA after elution.

[0169] The applicant declares that the present invention illustrates a RNA purification partner and its application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0170] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0171] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for purifying ribonucleic acid, characterized in that: The RNA purification method comprises injecting the RNA to be purified into a chromatography column containing a chromatography medium, eluting with an eluent containing a RNA purification partner, collecting the eluted components, and completing purification; The RNA purification partner is an ionic liquid composed of cations and anions, the purification method is chromatography, the chromatography medium includes an anion exchange chromatography medium; the ligand in the anion exchange chromatography medium is DEAE; The purification partner is used to simultaneously improve the elution efficiency of RNA on the chromatography medium and improve the stability of RNA; The ribonucleic acid is mRNA; The eluent is obtained by mixing a buffer solution and a RNA purification partner, wherein the buffer solution is a phosphate buffer; the concentration of the RNA purification partner in the buffer solution is 0.05 M-2 M; The ionic liquid is a combination of [Cho][BF4] and [C4mim][Cl]; the concentration ratio of [Cho][BF4] to [C4mim][Cl] is (3-6):(4-7).

2. The method for purifying RNA according to claim 1, wherein: The concentration of the buffer solution is 5-200 mM.

3. The method for purifying RNA according to claim 1, wherein: The pH value of the buffer solution is 6.0-8.

5.

4. The method for purifying RNA according to claim 1, wherein: The elution method includes isocratic elution or gradient elution, and the gradient elution includes linear gradient elution or step gradient elution.

5. The method for purifying RNA according to claim 1, wherein: The method further comprises washing the chromatography medium with an equilibrium buffer before elution, wherein the equilibrium buffer is a phosphate buffer.

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