Method for chromatographic purification of RNA from cellulose

Abstract

The invention provides methods and compositions for enriching single-stranded RNA.

Description

Technical Field

[0001] The present invention relates to a method for purifying RNA, and in particular to a method for rapidly purifying single-stranded RNA. Background Art

[0002] mRNA therapy, which uses mRNA as a therapeutic agent to treat diseases, is an emerging gene therapy approach. It can be used to treat genetic diseases or repair tissues through the expression of functional proteins, and can also be used for immunotherapy through the expression of antigens, antibodies, or receptors, offering significant potential for therapeutic applications. mRNA is a single-stranded RNA (ssRNA). Its application in medical settings requires reproducible and efficient ssRNA production and purification to remove reactants and byproducts during the production process. As is well known, ssRNA production relies on DNA-dependent RNA polymerase transcription, and the resulting RNA is often an impure mixture of RNA. Transcription products contain not only NTPs, T7 RNA polymerase, inorganic pyrophosphatase, and RNase inhibitors, but also truncated RNA byproducts such as aborted transcripts and transcription termination products, as well as redundant transcription byproducts. Furthermore, nonspecific RNA polymerase activity dependent on the RNA template may also produce dsRNA (double-stranded RNA) byproducts. The production of mRNA also includes the 5'UTR capping enzyme modification of ssRNA, the 5'UTR methyltransferase modification, and the polyA modification reaction steps of 3'UTR. These reaction product steps also include various enzyme proteins, NTP substrates, etc. that need to be purified and removed.

[0003] Traditional methods such as agarose gel electrophoresis, polyacrylamide gel electrophoresis, phenol-chloroform extraction, LiCl precipitation, ethanol or isopropanol precipitation, and commercial silica-based RNA purification kits can remove free nucleotides, proteins, salts, and short RNA oligonucleotides from reaction products with varying degrees of efficiency. However, these methods are incapable of achieving reproducible, scalable, and efficient production. Existing research indicates that only by increasing mRNA purity through chromatographic purification and reducing immune activation and other side effects caused by byproducts in the synthesis of transcribed mRNA can the translation efficiency of mRNA be significantly improved.

[0004] Studies have shown that high-performance liquid chromatography can achieve efficient and scalable purification and production of pharmaceutical RNA at the milligram to gram level. Currently reported chromatographic separation technologies related to sample preparation of mRNA or other long-chain ssRNA include reversed-phase ion-pair chromatography (RP-IP), ion exchange chromatography (IE), affinity chromatography (AC), molecular sieve separation (SEC), etc. McKenna SA et al. reported a chromatographic purification method for purifying 120-400nt long-chain RNA from transcript compounds using gel filtration molecular sieve. This method can only separate short-fragment ssRNA, and its separation effect decreases significantly as the length of ssRNA increases. Weissman D and Nwokeoji AO respectively reported cases of separating ssRNA, dsRNA, and incompletely paired dsRNA using ion-pair reversed-phase HPLC. However, the relative loading capacity of the reversed-phase chromatographic filler in this method is limited, which is not conducive to the scale-up of the RNA purification process. The toxicity of certain organic substances in the chromatographic separation buffer also affects its production application. Keel AY, Di Tomasso G, et al. reported on the use of MS2 and ARiBO RNA ligand affinity chromatography to purify RNA. These methods require the addition of RNA ligand sequences to the therapeutic RNA sequence, which may affect the therapeutic efficiency of the target RNA. A disadvantage of ion exchange (IEX) HPLC purification methods is that most published literature focuses on the purification of long RNAs of several hundred bases. Purification of RNAs longer than 1000 nt requires denaturing and / or alkaline mobile phases, which can lead to RNA instability, precipitation, degradation, and low product recovery.

[0005] In addition to the above-mentioned HPLC purification method, another method is to purify RNA by specific adsorption through cellulose chromatography. As early as the 1990s, Maran A and Mellits KH reported a method for separating ssRNA and dsRNA using cellulose. Since then, other documents have reported methods for purifying and separating RNA using cellulose chromatography. These documents all use ethanol as the mobile phase to purify and separate ssRNA. For example, CN109072232A discloses a method for providing single-stranded RNA. In the RNA positive purification process, the ethanol concentration of the binding buffer ranges from greater than 35%, preferably 38%-42%; the salt concentration is 15-70mM, and optimally 20-60mM. The disadvantage of the above-mentioned cellulose-based purification method is that the RNA needs to be pretreated and separated by centrifugal precipitation before purification through cellulose chromatography. This pretreatment step is obviously not conducive to the scale-up of the process.

[0006] There is still a need in the art for an RNA extraction process with simplified steps and amenable to scale-up. Summary of the Invention

[0007] The inventors studied sample pretreatment methods under different organic solvents and salt concentrations, as well as the efficiency of RNA chromatographic purification under different pretreatment methods, and developed a solvent and method that can directly perform cellulose chromatographic purification without the need for a sample pretreatment step of centrifugation and precipitation.

[0008] In a first aspect, the present invention provides a composition for enriching single-stranded RNA using a cellulose material, comprising 40-70% (v / v) of an organic alcohol and a salt, wherein the organic alcohol is selected from n-propanol, isopropanol or ethanol.

[0009] In one or more embodiments, the single-stranded RNA is a single-stranded RNA having a cellular function. The cellular function is a function of translating a protein.

[0010] In one or more embodiments, the single-stranded RNA further comprises a double-stranded single-stranded RNA. The double-stranded RNA is a non-specific double-stranded secondary structure formed by one or more single-stranded RNAs.

[0011] In one or more embodiments, the RNA is free or substantially free of double-stranded RNA.

[0012] In one or more embodiments, the organic alcohol (n-propanol, isopropanol or ethanol) content is 40%, 45%, 50%, 55%, 60%, 65%, 70% or a range therebetween, for example, 40-60%, 50-60%, 60%-70%.

[0013] In one or more embodiments, the salt is selected from sodium chloride, potassium chloride, lithium chloride.

[0014] In one or more embodiments, the salt concentration is 100-300 mM, preferably 120-250 mM, more preferably 125-250 mM.

[0015] In one or more embodiments, the composition further comprises EDTA. The concentration of EDTA may be 0.01-1 mM, preferably 0.1-0.5 mM, more preferably 0.2 mM.

[0016] In one or more embodiments, the composition further comprises HEPES or Tris. The concentration of HEPES or Tris may be 1-40 mM, preferably 10-30 mM, more preferably 20 mM.

[0017] In one or more embodiments, the pH of the composition is 6-8, preferably 6.5-7.5.

[0018] In one or more embodiments, the composition comprises 40%-70% n-propanol, isopropanol, or ethanol, 100-300 mM sodium chloride, potassium chloride, or lithium chloride, 0.01-1 mM EDTA, and 1-40 mM HEPES or Tris, or equivalent concentrates or dilutions thereof.

[0019] In one or more embodiments, the composition comprises 50%-60% n-propanol, isopropanol, or ethanol, 120-250 mM sodium chloride, potassium chloride, or lithium chloride, 0.1-0.5 mM EDTA, and 10-30 mM HEPES or Tris, or equivalent concentrates or dilutions thereof.

[0020] In one or more embodiments, the composition is a solution.

[0021] In one or more embodiments, when the composition is used to enrich single-stranded RNA, it can be mixed with a sample containing single-stranded RNA at a volume ratio of 1:1. The sample containing single-stranded RNA is, for example, a product system obtained by in vitro biosynthesis of RNA or modification of RNA. In one or more embodiments, the in vitro biosynthesis includes in vitro transcription synthesis.

[0022] In a second aspect, the present invention provides a composition for enriching single-stranded RNA using a cellulosic material, the composition comprising single-stranded RNA, an organic alcohol, and a salt, wherein the organic alcohol is selected from n-propanol, isopropanol, or ethanol accounting for 20-35% (v / v) of the composition. The composition is a mixture.

[0023] In one or more embodiments, the single-stranded RNA is a single-stranded RNA having a cellular function. The cellular function is a function of translating a protein.

[0024] In one or more embodiments, the single-stranded RNA further comprises a double-stranded single-stranded RNA. The double-stranded RNA is a non-specific double-stranded secondary structure formed by one or more single-stranded RNAs.

[0025] In one or more embodiments, the RNA is free or substantially free of double-stranded RNA.

[0026] In one or more embodiments, the single-stranded RNA is single-stranded RNA produced in vivo and extracted.

[0027] In one or more embodiments, the single-stranded RNA is a single-stranded RNA produced by in vitro biosynthesis or modification. In one or more embodiments, the in vitro biosynthesis includes in vitro transcription synthesis.

[0028] In one or more embodiments, the composition further comprises non-RNA components synthesized by in vitro transcription of RNA.

[0029] In one or more embodiments, the single-stranded RNA synthesized by in vitro transcription includes RNA synthesized by RNA polymerase using DNA as a template. In one or more embodiments, the RNA polymerase is selected from T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and Syn5 RNA polymerase.

[0030] In one or more embodiments, the modified single-stranded RNA comprises a capped, methylated, and / or tailed single-stranded RNA. In one or more embodiments, the modification is performed enzymatically, and the enzyme is preferably selected from vaccinia virus capping enzyme, vaccinia virus mRNA dioxymethyltransferase, Escherichia coli poly(A) RNA polymerase, and a ribonuclease inhibitor.

[0031] In one or more embodiments, the organic alcohol content is (v / v) 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, 35%, or any range therebetween, such as 20-30%, 25-30%, 30-35%. The above content is volume percentage.

[0032] In one or more embodiments, the salt is selected from sodium chloride, potassium chloride, lithium chloride.

[0033] In one or more embodiments, the salt concentration is 50-250 mM, preferably 60-125 mM, more preferably 62.5-125 mM, and even more preferably 100-125 mM.

[0034] In one or more embodiments, the composition further comprises EDTA. The concentration of EDTA may be 0.01-0.5 mM, preferably 0.05-0.3 mM, more preferably 0.1 mM.

[0035] In one or more embodiments, the composition further comprises HEPES or Tris. The concentration of HEPES or Tris may be 1-20 mM, preferably 6-15 mM, more preferably 10 mM.

[0036] In one or more embodiments, the pH of the composition is 6-8, preferably 6.5-7.5.

[0037] In one or more embodiments, the mixture comprises 20%-35% n-propanol, isopropanol, or ethanol, 50-150 mM sodium chloride, potassium chloride, or lithium chloride, 0.01-0.5 mM EDTA, and 1-20 mM HEPES or Tris, or equivalent concentrates or dilutions thereof.

[0038] In one or more embodiments, the mixture comprises 20%-35% n-propanol, isopropanol, or ethanol, 60-125 mM sodium chloride, potassium chloride, or lithium chloride, 0.05-0.3 mM EDTA, and 6-15 mM HEPES or Tris, or equivalent concentrates or dilutions thereof.

[0039] In one or more embodiments, the mixture comprises 20%-30% n-propanol, isopropanol, or ethanol, 62.5-125 mM sodium chloride, potassium chloride, or lithium chloride, 0.05-0.3 mM EDTA, and 6-15 mM HEPES or Tris, or equivalent concentrates or dilutions thereof.

[0040] In one or more embodiments, the composition comprises 25%-30% n-propanol, isopropanol, or ethanol, 60-125 mM sodium chloride, potassium chloride, or lithium chloride, 0.05-0.3 mM EDTA, and 6-15 mM HEPES or Tris, or equivalent concentrates or dilutions thereof.

[0041] In one or more embodiments, the composition is free of precipitate.

[0042] In one or more embodiments, the composition further comprises water, and the volume percentage of water in the composition may be 65-80%.

[0043] A third aspect of the present invention provides a method for enriching single-stranded RNA, comprising:

[0044] (1) contacting a mixture containing single-stranded RNA, an organic alcohol, and a salt with a cellulose material, wherein the organic alcohol is selected from n-propanol, isopropanol, or ethanol accounting for 20-35% (v / v) of the mixture;

[0045] (2) Using an eluent, a liquid containing single-stranded RNA is obtained from the cellulose material.

[0046] In one or more embodiments, the mixture is as described in the second aspect herein.

[0047] In one or more embodiments, the single-stranded RNA is a single-stranded RNA having a cellular function. The cellular function is a function of translating a protein.

[0048] In one or more embodiments, the single-stranded RNA further comprises a double-stranded single-stranded RNA. The double-stranded RNA is a non-specific double-stranded secondary structure formed by one or more single-stranded RNAs.

[0049] In one or more embodiments, the length of the single-stranded RNA is at least 200 bp, such as at least 300 bp, at least 500 bp, at least 1000 bp, at least 2000 bp, at least 3000 bp, at least 4000 bp, at least 5000 bp.

[0050] In one or more embodiments, the single-stranded RNA is single-stranded RNA produced in vivo and extracted.

[0051] In one or more embodiments, the single-stranded RNA is a single-stranded RNA produced by in vitro biosynthesis or modification. In one or more embodiments, the in vitro biosynthesis includes in vitro transcription synthesis.

[0052] In one or more embodiments, the single-stranded RNA synthesized by in vitro transcription includes RNA synthesized by RNA polymerase using DNA as a template. In one or more embodiments, the RNA polymerase is selected from T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and Syn5 RNA polymerase.

[0053] In one or more embodiments, the modified single-stranded RNA comprises a capped, methylated, and / or tailed single-stranded RNA. In one or more embodiments, the modification is performed enzymatically, and the enzyme is preferably selected from vaccinia virus capping enzyme, vaccinia virus mRNA dioxymethyltransferase, Escherichia coli poly(A) RNA polymerase, and a ribonuclease inhibitor.

[0054] In one or more embodiments, the contacting of step (1) comprises mixing the mixture and a cellulosic material or loading the mixture onto a column packed with a cellulosic material.

[0055] In one or more embodiments, the contact time of step (1) is 30-150 minutes, and the temperature is room temperature.

[0056] In one or more embodiments, the organic alcohol (n-propyl alcohol, isopropyl alcohol or ethanol) content in the mixture is (v / v) 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, 35% or a range between any of the above values, such as 20-30%, 25-30%, 30-35%. In a preferred embodiment, the n-propyl alcohol or isopropyl alcohol content in the mixture is (v / v) 20-35%, preferably 20-30%, more preferably 25-30%.

[0057] In one or more embodiments, the salt is selected from sodium chloride, potassium chloride, lithium chloride.

[0058] In one or more embodiments, the salt concentration is 50-150 mM, preferably 60-125 mM, more preferably 62.5-125 mM, and most preferably 100-125 mM.

[0059] In one or more embodiments, the mixture further comprises EDTA. The concentration of EDTA may be 0.01-0.5 mM, preferably 0.05-0.3 mM, more preferably 0.1 mM.

[0060] In one or more embodiments, the mixture further comprises HEPES or Tris. The concentration of HEPES or Tris may be 1-20 mM, preferably 6-15 mM, more preferably 10 mM.

[0061] In one or more embodiments, the pH of the mixture is 6-8, preferably 6.5-7.5.

[0062] In one or more embodiments, the mixture comprises 20%-35% n-propanol, isopropanol, or ethanol, 50-150 mM sodium chloride, potassium chloride, or lithium chloride, 0.01-0.5 mM EDTA, and 1-20 mM HEPES or Tris, or equivalent concentrates or dilutions thereof.

[0063] In one or more embodiments, the mixture comprises 20%-35% n-propanol, isopropanol, or ethanol, 60-125 mM sodium chloride, potassium chloride, or lithium chloride, 0.05-0.3 mM EDTA, and 6-15 mM HEPES or Tris, or equivalent concentrates or dilutions thereof.

[0064] In one or more embodiments, the mixture comprises 20%-30% n-propanol, isopropanol, or ethanol, 62.5-125 mM sodium chloride, potassium chloride, or lithium chloride, 0.05-0.3 mM EDTA, and 6-15 mM HEPES or Tris, or equivalent concentrates or dilutions thereof.

[0065] In one or more embodiments, step (2) comprises:

[0066] (2a) eluting the liquid containing the single-stranded RNA from the cellulose material using an elution liquid, wherein the elution speed is preferably 30-100 cm / h, or

[0067] (2b) Single-stranded RNA is obtained from the cellulose material using an eluent, and a liquid containing the single-stranded RNA is obtained by solid-liquid separation.

[0068] In one or more embodiments, the eluent contains 4-18%, preferably 6-16% or 8-10%, more preferably 8% or 10% of an organic alcohol selected from n-propanol, isopropanol or ethanol.

[0069] In one or more embodiments, the organic alcohol in the eluent is the same as the organic alcohol in the mixture.

[0070] In one or more embodiments, the eluent further contains a salt, preferably selected from sodium chloride, potassium chloride and lithium chloride, with a salt concentration of 50-150 mM, preferably 60-125 mM, more preferably 62.5-125 mM, and most preferably 100-125 mM.

[0071] In one or more embodiments, the eluent further comprises EDTA. The EDTA concentration may be 0.01-0.5 mM, preferably 0.05-0.3 mM, more preferably 0.1 mM.

[0072] In one or more embodiments, the eluent further comprises HEPES or Tris. The concentration of HEPES or Tris may be 1-20 mM, preferably 6-15 mM, more preferably 10 mM.

[0073] In one or more embodiments, the pH of the eluent is 6-8, preferably 6.5-7.5.

[0074] In one or more embodiments, the eluent comprises 6%-16% n-propanol, isopropanol or ethanol, 60-125 mM sodium chloride, 0.05-0.3 mM EDTA and 6-15 mM HEPES or Tris.

[0075] In one or more embodiments, the cellulosic material has a particle size of 20 microns to 250 microns, preferably 50 to 100 microns.

[0076] In one or more embodiments, the RNA loading of the cellulosic material is 2.5-6.25 mg / g, preferably 3.75-6.25 mg / g.

[0077] In one or more embodiments, the cellulosic material is pretreated before contacting the mixture, and the pretreatment solution comprises 20-35% organic alcohol, 50-150 mM salt (sodium chloride, potassium chloride or lithium chloride). Optionally, the pretreatment solution further comprises 0.01-0.5 mM EDTA and / or 1-20 mM HEPES or Tris.

[0078] In one or more embodiments, the single-stranded RNA or the mixture containing the single-stranded RNA is not subjected to a pre-purification treatment before step (1).

[0079] In one or more embodiments, the pre-purification treatment comprises precipitation of the nucleic acid, such as by precipitation with LiCl.

[0080] A fourth aspect of the present invention further provides a method for enriching single-stranded RNA, comprising:

[0081] (1) mixing the composition described herein with a sample containing single-stranded RNA at a volume ratio of 1:1, and contacting the resulting mixture with a cellulose material,

[0082] (2) Using an eluent, a liquid containing single-stranded RNA is obtained from the cellulose material.

[0083] In one or more embodiments, the single-stranded RNA is a single-stranded RNA having a cellular function. The cellular function is a function of translating a protein.

[0084] In one or more embodiments, the single-stranded RNA further comprises a double-stranded single-stranded RNA. The double-stranded RNA is a non-specific double-stranded secondary structure formed by one or more single-stranded RNAs.

[0085] In one or more embodiments, the length of the single-stranded RNA is at least 200 bp, such as at least 300 bp, at least 500 bp, at least 1000 bp, at least 2000 bp, at least 3000 bp, at least 4000 bp, at least 5000 bp.

[0086] In one or more embodiments, the sample containing single-stranded RNA is a sample containing single-stranded RNA produced in vivo and subjected to RNA extraction.

[0087] In one or more embodiments, the sample containing single-stranded RNA is a sample containing single-stranded RNA produced by in vitro biosynthesis of RNA or modified RNA. In one or more embodiments, the in vitro biosynthesis includes in vitro transcription synthesis.

[0088] In one or more embodiments, the single-stranded RNA synthesized by in vitro transcription includes RNA synthesized by RNA polymerase using DNA as a template. In one or more embodiments, the RNA polymerase is selected from T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and Syn5 RNA polymerase.

[0089] In one or more embodiments, the modified single-stranded RNA comprises a capped, methylated, and / or tailed single-stranded RNA. In one or more embodiments, the modification is performed enzymatically, and the enzyme is preferably selected from vaccinia virus capping enzyme, vaccinia virus mRNA dioxymethyltransferase, Escherichia coli poly(A) RNA polymerase, and a ribonuclease inhibitor.

[0090] In one or more embodiments, the contacting of step (1) comprises mixing the mixture with a cellulosic material or loading the mixture onto a column packed with a cellulosic material.

[0091] In one or more embodiments, the contact time of step (1) is 30-150 minutes, and the temperature is room temperature.

[0092] In one or more embodiments, the organic alcohol (n-propyl alcohol, isopropyl alcohol or ethanol) content in the mixture is (v / v) 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, 35% or a range between any of the above values, such as 20-30%, 25-30%, 30-35%. In a preferred embodiment, the n-propyl alcohol or isopropyl alcohol content in the mixture is (v / v) 20-35%, preferably 20-30%, more preferably 25-30%.

[0093] In one or more embodiments, the salt is selected from sodium chloride, potassium chloride, lithium chloride.

[0094] In one or more embodiments, the salt concentration is 50-150 mM, preferably 60-125 mM, more preferably 62.5-125 mM, and most preferably 100-125 mM.

[0095] In one or more embodiments, the mixture further comprises EDTA. The concentration of EDTA may be 0.01-0.5 mM, preferably 0.05-0.3 mM, more preferably 0.1 mM.

[0096] In one or more embodiments, the mixture further comprises HEPES or Tris. The concentration of HEPES or Tris may be 1-20 mM, preferably 6-15 mM, more preferably 10 mM.

[0097] In one or more embodiments, the pH of the mixture is 6-8, preferably 6.5-7.5.

[0098] In one or more embodiments, the mixture comprises 20%-35% n-propanol, isopropanol, or ethanol, 50-150 mM sodium chloride, potassium chloride, or lithium chloride, 0.01-0.5 mM EDTA, and 1-20 mM HEPES or Tris, or equivalent concentrates or dilutions thereof.

[0099] In one or more embodiments, the mixture comprises 20%-35% n-propanol, isopropanol, or ethanol, 60-125 mM sodium chloride, potassium chloride, or lithium chloride, 0.05-0.3 mM EDTA, and 6-15 mM HEPES or Tris, or equivalent concentrates or dilutions thereof.

[0100] In one or more embodiments, the mixture comprises 20%-30% n-propanol, isopropanol, or ethanol, 62.5-125 mM sodium chloride, potassium chloride, or lithium chloride, 0.05-0.3 mM EDTA, and 6-15 mM HEPES or Tris, or equivalent concentrates or dilutions thereof.

[0101] In one or more embodiments, step (2) comprises:

[0102] (2a) eluting the liquid containing the single-stranded RNA from the cellulose material using an elution liquid, wherein the elution speed is preferably 30-100 cm / h, or

[0103] (2b) Single-stranded RNA is obtained from the cellulose material using an eluent, and a liquid containing the single-stranded RNA is obtained by solid-liquid separation.

[0104] In one or more embodiments, the eluent contains 4-18%, preferably 6-16% or 8-10%, more preferably 8% or 10% of an organic alcohol selected from n-propanol, isopropanol or ethanol.

[0105] In one or more embodiments, the organic alcohol in the eluent is the same type of organic alcohol as that in the mixture.

[0106] In one or more embodiments, the eluent further contains a salt, preferably selected from sodium chloride, potassium chloride and lithium chloride, with a salt concentration of 50-150 mM, preferably 60-125 mM, more preferably 62.5-125 mM, and most preferably 100-125 mM.

[0107] In one or more embodiments, the eluent further comprises EDTA. The EDTA concentration may be 0.01-0.5 mM, preferably 0.05-0.3 mM, more preferably 0.1 mM.

[0108] In one or more embodiments, the eluent further comprises HEPES or Tris. The concentration of HEPES or Tris may be 1-20 mM, preferably 6-15 mM, more preferably 10 mM.

[0109] In one or more embodiments, the pH of the eluent is 6-8, preferably 6.5-7.5.

[0110] In one or more embodiments, the eluent comprises 6%-16% n-propanol, isopropanol or ethanol, 60-125 mM sodium chloride, 0.05-0.3 mM EDTA and 6-15 mM HEPES or Tris.

[0111] In one or more embodiments, the cellulosic material has a particle size of 20 microns to 250 microns, preferably 50 to 100 microns.

[0112] In one or more embodiments, the RNA loading of the cellulosic material is 2.5-6.25 mg / g, preferably 3.75-6.25 mg / g.

[0113] In one or more embodiments, the cellulosic material is pretreated before contacting the mixture, and the pretreatment solution comprises 20-35% organic alcohol, 50-150 mM salt (sodium chloride, potassium chloride or lithium chloride). Optionally, the pretreatment solution further comprises 0.01-0.5 mM EDTA and / or 1-20 mM HEPES or Tris.

[0114] In one or more embodiments, the sample containing single-stranded RNA is not pre-purified before step (1).

[0115] In one or more embodiments, the pre-purification treatment comprises precipitation of the nucleic acid, such as by precipitation with LiCl.

[0116] The fifth aspect of the present invention provides single-stranded RNA or a liquid containing single-stranded RNA obtained by the method described in the third or fourth aspect.

[0117] In one or more embodiments, the single-stranded RNA or the liquid containing single-stranded RNA does not contain or substantially does not contain double-stranded RNA.

[0118] In one or more embodiments, the single-stranded RNA or the system containing the single-stranded RNA comprises the single-stranded RNA in double-stranded form. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 It is the Akta spectrum of ethanol, isopropanol, and n-propanol purification.

[0120] Figure 2 is a dot blot analysis of dsRNA residues.

[0121] Figure 3 Capillary electrophoresis analysis of the integrity of cellulose-purified RNA is shown.

[0122] Figure 4 This is the AKTA profile of capped mRNA purification using ethanol, isopropanol, and n-propanol mobile phases.

[0123] Figure 5 This is a dot blot detection image of dsRNA residue.

[0124] Figure 6 This is a graph showing the integrity of cellulose-purified RNA after capping.

[0125] Figure 7Capillary electrophoresis analysis of dsRNA elution samples from pseudouridine-modified mRNA purification. The arrow indicates the dsRNA detection peak.

[0126] Figure 8 The EGFP positive rate and fluorescence intensity of EGFP-mRNA treated with different purification methods on PBMC are shown. Note: Cap sample is the control, which is the eGFP mRNA sample purified by NEB RNA purification kit.

[0127] Figure 9 This is a map of RNA purification >2000nt.

[0128] Figure 10 This is a dot blot detection image of dsRNA residue.

[0129] Figure 11 Shown are the effects of salt concentration and isopropanol concentration on post-transcription RNA samples.

[0130] Figure 12 It is shown that the method of the present invention is used for RNA purification in a mixed system after transcription, capping and tailing.

[0131] Figure 13 It is the RNA purification map.

[0132] Figure 14 RNA integrity assay is shown after collection following isopropanol elution.

[0133] Figure 15 It is a dsRNA residual detection.

[0134] Figure 16 The green fluorescence positive rate of PBMC cells after PB mRNA-mediated eGFP expression cassette transposon knock-in was shown.

[0135] Figure 17 A diagram showing the use of cellulose matrix for chromatographic purification of transcript RNA.

[0136] Figure 18 The process of cellulose chromatography to purify RNA is shown. A, prior art, B, exemplary embodiment of the present application. DETAILED DESCRIPTION

[0137] After research, the inventors screened out two organic solvents that have not been used in cellulose purification, isopropanol and n-propanol, which are also suitable for RNA purification from cellulose. Based on isopropanol, n-propanol and ethanol, the inventors studied the optimal conditions for RNA sample pretreatment. Under these conditions, the pretreated samples will not produce RNA or protein precipitation and can be directly used for cellulose purification, so there is no need for additional precipitation and separation steps.

[0138] The present invention achieves the above effects through the following method, including:

[0139] (1) contacting a mixture containing single-stranded RNA, an organic alcohol, and a salt with a cellulose material, wherein the organic alcohol is selected from n-propanol, isopropanol, or ethanol accounting for 20-35% (v / v) of the mixture;

[0140] (2) Using an eluent, a liquid containing single-stranded RNA is obtained from the cellulose material.

[0141] As used herein, "single-stranded RNA" or "ssRNA" includes any form of single-stranded RNA, such as mRNA, microRNA, and shRNA. Single-stranded RNA can be single-stranded RNA produced in vivo or in cells and extracted. Methods and reagents for extracting single-stranded RNA from viruses, cells or tissues are well known in the art, such as the TRIZOL method. Single-stranded RNA also includes "double-stranded single-stranded RNA", which refers to a non-specific secondary structure formed by one or more single-stranded RNA molecules, rather than a double-stranded RNA with base complementarity generated by an RNA template. After thermal denaturation, the intramolecular or intermolecular secondary structure of the double-stranded single-stranded RNA is unstable and can be restored to a single molecule of the target single-stranded RNA. Herein, "double-stranded RNA" or "dsRNA" refers to a double-stranded RNA with sequence complementarity generated by an RNA template.

[0142] Single-stranded RNA can also be produced by in vitro synthesis or modification. "In vitro synthesis" as used herein includes both biosynthesis and chemical synthesis. Biosynthesis primarily refers to single-stranded mRNA obtained by transcription from DNA. This transcription is typically performed by RNA polymerase. RNA polymerases that can be used to obtain the biosynthetic RNA described herein include T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and Syn5 RNA polymerase. Reagents and methods for obtaining RNA by in vitro DNA transcription are known in the art, such as rNTPs, DNA template, nuclease-free water, inhibitors, RNA polymerase, MgCl2, etc.; exemplary methods are described in Example 1 herein.

[0143] As used herein, "in vitro modification" includes, but is not limited to, RNA capping, methylation, base substitution, base transversion, or tailing. Such modifications can be performed enzymatically or non-enzymatically. Common modification enzymes include, but are not limited to, vaccinia virus capping enzyme, vaccinia virus mRNA dioxymethyltransferase, Escherichia coli poly(A) RNA polymerase, and ribonuclease inhibitors. Reagents and methods for in vitro RNA modification are known in the art, such as GTP, SAM, vaccinia capping enzyme, 2-O-methyltransferase, and the like; exemplary methods are described in Example 1 herein.

[0144] Without wishing to be bound by any theory, the single-stranded RNA that can be purified by the methods herein can be of any length, typically at least 200 bp, e.g., at least 300 bp, at least 500 bp, at least 1000 bp, at least 2000 bp, at least 3000 bp, at least 4000 bp, at least 5000 bp.

[0145] Herein, the cellulose material can be any cellulose filler product known in the art that can be used for RNA purification. The form of the cellulose material is varied, such as granules, spheres, columns, cones, etc. The diameter of the cellulose material is not limited, for example 20 microns to 250 microns, preferably 50-100 microns. The cellulose material can be pretreated before contacting the RNA, for example, after incubation with a pretreatment solution and then optionally solid-liquid separation (for example, applying a driving force such as centrifugation). Usually, the composition of the pretreatment solution is the same as the loading buffer (except for RNA, protein and other transcription products or substrates). Therefore, the pretreatment solution herein contains 20-35% organic alcohol and 50-150mM salt. Optionally, the pretreatment solution also contains 0.01-0.5mM EDTA and / or 1-20mM HEPES or Tris. According to the method of the present invention, single-stranded RNA is enriched, and the RNA loading capacity of the cellulose material can reach 2.5-6.25mg / g, preferably 3.75-6.25mg / g.

[0146] The compositions or mixtures described herein are primarily aqueous solutions, wherein the organic alcohol is primarily n-propanol, isopropanol, or ethanol. The inventors have discovered that, in the presence of 20-35% organic alcohol and salt, single-stranded RNA containing impurities such as proteins and NTPs can be directly contacted with cellulose for purification without requiring a pre-purification process of precipitation and redissolution. Figure 18 , B). In the prior art ( Figure 18 A), single-stranded RNA must first undergo a precipitation step to remove impurities such as proteins and NTPs before it can be used for cellulose purification. Unless otherwise specified, the percentages of organic alcohols mentioned in this article are by volume (v / v).

[0147] Herein, salt is any form of salt known in the art to be useful for RNA purification, primarily inorganic salts such as sodium chloride, potassium chloride, and lithium chloride. In one or more embodiments, the salt concentration is 50-150 mM, preferably 60-125 mM, more preferably 62.5-125 mM, and most preferably 100-125 mM.

[0148] The composition or mixture containing an organic alcohol and a salt may further contain a chelating agent such as EDTA or a buffer such as HEPES or Tris. The EDTA concentration may be 0.01-0.5 mM, preferably 0.05-0.3 mM, and more preferably 0.1 mM. The HEPES or Tris concentration may be 1-20 mM, preferably 6-15 mM, and more preferably 10 mM. The pH of the composition or mixture may be 6-8, preferably 6.5-7.5.

[0149] In one or more embodiments, the composition or mixture comprises 20%-35% n-propanol, isopropanol or ethanol, 60-125 mM sodium chloride, 0.05-0.3 mM EDTA and 6-15 mM HEPES or Tris. Preferably, the composition or mixture comprises 25-30% n-propanol, isopropanol or ethanol, 125 mM sodium chloride, 0.1 mM EDTA and 10 mM HEPES or Tris.

[0150] A mixture containing the above components and single-stranded RNA can be obtained by mixing the prepared pre-composition with a solution containing single-stranded RNA (e.g., a reaction solution for extracting or synthesizing RNA). The mixing ratio is not limited, as long as the final concentration of each component in the resulting mixture is as described herein.

[0151] In the above method, the contacting in step (1) includes mixing the composition or mixture with the cellulosic material or loading the composition or mixture onto a column filled with the cellulosic material. For example, the RNA-containing mixture and cellulose particles having a certain pore size are placed in a container and mixed and stirred (30-60 minutes at room temperature). Alternatively, the RNA-containing mixture can be applied to a portion or all of the cellulose column for a continuous period (30-150 minutes at room temperature).

[0152] Then, in step (2), an eluent is used to separate the single-stranded RNA from the cellulose material, thereby obtaining a liquid containing the single-stranded RNA. For example, the eluent can be used to elute the liquid containing the single-stranded RNA from the cellulose material (e.g., a column filled with the cellulose material) (elution rate 30-100 cm / h); alternatively, the eluent can be used to separate the single-stranded RNA from the cellulose material (e.g., cellulose particles) into a solution and the liquid containing the single-stranded RNA can be obtained by solid-liquid separation (e.g., applying a driving force such as centrifugation). Eluents capable of separating single-stranded RNA from cellulose materials are known in the art, for example, eluents containing 4-18%, preferably 6-12% or 8-10%, more preferably 8% or 10% of an organic alcohol selected from n-propanol, isopropanol, or ethanol. Typically, the organic alcohol in the eluent is the same as the organic alcohol in the mixture containing the single-stranded RNA. When the single-stranded RNA is less than 2000 nt in length, the content of the organic alcohol (e.g., isopropanol) in the eluent is preferably 10-20%, for example, 16%. When the length of the single-stranded RNA is greater than 2000 nt, the content of the organic alcohol (eg, isopropanol) in the eluent is preferably 5-10%, such as 8% or 10%.

[0153] In some most preferred embodiments, the single-stranded RNA is capped single-stranded RNA, the mixture comprises 30% isopropanol, 125mM sodium chloride, 0.1mM EDTA and 10mM HEPES or Tris, and the eluent comprises 10% isopropanol, 125mM sodium chloride, 0.1mM EDTA and 10mM HEPES or Tris; or the single-stranded RNA is tailed single-stranded RNA, the mixture comprises 25% isopropanol, 125mM sodium chloride, 0.1mM EDTA and 10mM HEPES or Tris, and the eluent comprises 10% isopropanol, 125mM sodium chloride, 0.1mM EDTA and 10mM HEPES or Tris.

[0154] The single-stranded RNA obtained by any method of the present invention can be further processed, such as precipitated and / or modified. For example, the single-stranded RNA obtained by the method of the present invention can be precipitated using conventional methods (e.g., using "sodium acetate / isopropanol" precipitation or "LiCl" precipitation) to produce a single-stranded RNA preparation in a dried form. The dried single-stranded RNA can be stored (e.g., at -70°C) or can be dissolved in a suitable solvent (e.g., water or TE buffer (10mM TRIS, 1mM EDTA)) and then stored (e.g., at -70°C) or used. In addition, the single-stranded RNA can be further modified, for example, by removing uncapped 5'-triphosphates and / or adding a cap structure, which is then stored (e.g., at -70°C) or used.

[0155] Advantages of the present invention

[0156] 1. In the prior art, RNA samples need to be pre-treated and centrifuged before being redissolved for cellulose chromatography purification. When industrialized production is carried out, this will increase the purification steps, increase the difficulty of production, and reduce process efficiency. The sample pretreatment of the method of the present invention is simpler and has fewer steps. After adding a buffer containing an organic solvent, the sample can be directly loaded for cellulose chromatography purification. This ensures that no precipitated substances appear in the synthetic product, and that under these conditions, only the RNA target product can be adsorbed onto the cellulose chromatography filler, while other reaction by-products flow through without being adsorbed, thereby achieving one-step purification of the transcribed and synthesized RNA. The method of the present invention is also applicable to capped RNA products and tailed RNA products. Since the steps of centrifugal precipitation and sample redissolution are reduced, production efficiency is improved, the use of centrifugal equipment is avoided, and production costs are reduced.

[0157] 2. The inventors discovered that the byproduct dsRNA is not entirely a dsRNA byproduct generated by RNA template dependence. The capillary electrophoresis identification and cell test results of the present invention indicate that these "dsRNA" (referred to herein as double-stranded single-stranded RNA) are merely nonspecific secondary structures formed between target single-stranded RNA molecules. After thermal denaturation, the intermolecular secondary structure is unstable and can be restored to a single molecule of the target single-stranded RNA. If the purification parameters are over-adjusted during purification to remove these RNAs as byproducts, the purification recovery rate will be reduced and the process cost will be increased. Therefore, the method of the present invention can better balance the dsRNA byproducts in the single-stranded RNA purification product, simplify the purification steps without affecting the transfection effect of the single-stranded RNA purification product, and is more conducive to production applications.

[0158] 3. The RNA loading capacity per gram of cellulose in the present invention can reach 6.25 mg, which is much higher than the 1-2.5 mg range described in existing patents.

[0159] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein may be used to implement or test the present invention, preferred methods and materials are now described. All publications and patents specifically mentioned herein are incorporated herein by reference in their entirety for all purposes, including describing and disclosing the chemical substances, equipment, statistical analyses, and methods reported in the publications that may be used in connection with the present invention. All references cited in this specification should be considered as an indication of the state of the art in the art. Nothing herein should be construed as an admission that the present invention cannot be antedated by virtue of prior invention.

[0160] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental procedures in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning Laboratory Manual, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0161] Example

[0162] Example 1, Preparation of RNA Transcription and / or Modification Synthetic Products

[0163] RNA Transcription: Prepare a suitable T7 RNA polymerase reaction system at room temperature and add the reaction components in the order shown in the following example. The reaction system can be scaled up or down proportionally. Incubate at 37°C for 6-16 hours. In this example, the transcribed RNAs are green fluorescent eGFP mRNA (Cap-eGFP mRNA, transcription template as shown in SEQ ID NO: 1) and Piggybac transposase mRNA (PB mRNA, transcription template as shown in SEQ ID NO: 2), with sequence lengths of 1156 nt and 2175 nt, respectively.

[0164] Table 1-1, RNA transcription synthesis system

[0165]

[0166]

[0167] Capping RNA: This step is suitable for capping 10 μg RNA (≥100 nt) and can be amplified as needed. Transfer 10 μg RNA to a 1.5 mL centrifuge tube and dilute to 14 μl with nuclease-free water. Heat at 65°C for 10 minutes, then remove the tube and place on ice for 5 minutes. Add the following components in sequence and incubate at 37°C for 30-90 minutes.

[0168] Table 1-2, RNA capping synthesis system

[0169]

[0170] The transcribed and / or capped and / or tailed products can be used directly for chromatographic purification or LiCl precipitation and re-dissolved in RNase-free aqueous solution. The LiCl precipitation procedure is as follows: Add two volumes of LiCl (7.5M Lithium Chloride, 50mM EDTA) to the transcription system to a final concentration of 2.5M lithium chloride. Chill the mixture at -20°C for half an hour (30 minutes or overnight). Centrifuge at 16,000 x g at 4°C for 20-30 minutes, and remove the supernatant with a pipette. Gently wash the pellet with 70% ethanol and centrifuge at 16,000 x g at 4°C for 10 minutes. Remove the supernatant and resuspend the pellet in nuclease-free water.

[0171] Example 2: Screening of new organic solvents and conditions for preparing cellulose mobile phase for RNA purification

[0172] To date, all reported mobile phases for RNA purification from cellulose have used ethanol as the organic solvent, and no studies have investigated the use of other organic solvents in this field. To expand the application scope of cellulose-based RNA purification, we screened two organic solvents, isopropanol and n-propanol, for use in the preparation of mobile phases for RNA purification from cellulose. Ethanol was used as a positive organic solvent control. The effects of varying concentrations of ethanol, isopropanol, and n-propanol on target RNA recovery and dsRNA removal were tested. An AKTA Avant 150 purifier coupled with a cellulose column was used for this experiment. An XK16 / 20 25 mL cellulose column was loaded. LiCl precipitation of a transcribed RNA approximately 1000 nt in length, eGFP mRNA, was investigated. The RNA solution was diluted 1:1 with a 70% (v / v) organic solvent dilution buffer (20 mM HEPES, 250 mM NaCl, 0.2 mM EDTA, 70% (v / v) ethanol, isopropanol, or n-propanol, with the balance being water, pH 7.2). A cellulose column was equilibrated with an equilibration buffer (10 mM HEPES, 125 mM NaCl, 0.1 mM EDTA, 35% (v / v) ethanol or isopropanol or n-propanol, pH 7.2) containing 35% (v / v) organic solvent concentration, 3 mg of RNA was loaded onto the cellulose column, and the eluted RNA was collected by reducing the concentration of the organic solvent (ethanol or isopropanol or n-propanol) in the eluate buffer to 16% (v / v), 10% (v / v) and 0% (v / v), respectively.

[0173] Sample RNA was recovered using NEB's Monarch RNA Purification Kit Spin Column. The recovered RNA was then displaced into an aqueous solution for concentration determination. Sample concentrations were directly measured using a Nanodrop instrument, and RNA yields were calculated for each fraction. J2 anti-dsRNA monoclonal antibody (Scicons) was purchased and used for dot blot analysis of RNA in the eluate and dsRNA content in the initial input RNA sample. RNA integrity was analyzed by capillary electrophoresis using a PerkinElmer Labchip detector. Statistical data for recovery calculations using various methods are shown in Table 2 below.

[0174] Table 2 The yield of transcribed RNA washed out of different concentrations of organic phase for cellulose purification using ethanol, isopropanol and n-propanol

[0175]

[0176] The elution curve of the chromatogram (UV absorbance at 260 nm) shows that when the concentration of the three organic solvent eluates, ethanol, isopropanol, and n-propanol, is reduced to 16% (v / v), most of the target RNA is eluted and appears as a single UV peak ( Figure 1 The RNA yields collected in this part were 73.2% in the ethanol eluate, 71.8% in the isopropanol eluate, and 78.3% in the n-propanol eluate. The n-propanol eluate had the highest RNA yield (Table 2). The dsRNA residual test results showed that when the organic solvent concentration of the eluate was 16% (v / v), most of the dsRNA was removed from this part compared to the initial input RNA ( Figure 2 ). There is a small amount of dsRNA remaining in the RNA eluted with n-propanol, and the dsRNA residue in the RNA eluted with ethanol and isopropanol is the least. When the concentration of the organic solvent in the eluate is reduced to 10% (v / v), only a small part of the RNA is washed off the cellulose column, and this part is shown to contain a large amount of dsRNA. The column is also shown to contain a large amount of dsRNA when it is regenerated. In addition to the reported use of ethanol for the separation of dsRNA and ssRNA, this confirms that isopropanol and n-propanol can also separate dsRNA and ssRNA, and the separation effect of isopropanol is better than that of n-propanol. Comparative analysis of the integrity of the RNA after cellulose purification and the initial input RNA shows that the RNA integrity is not affected ( Figure 3 ).

[0177] Example 3. Screening of solvents and conditions for purification of modified RNA

[0178] Based on the above experimental data, we also tested the RNA after cellulose purification and capping to verify whether the concentrations of the three organic solvent eluates, ethanol, isopropanol, and n-propanol, are also suitable for the capped RNA. 3 mg of RNA capped with LiCl precipitation was diluted with an organic solvent dilution buffer (20 mM HEPES, 250 mM NaCl, 0.2 mM EDTA, 70% (v / v) ethanol or isopropanol or n-propanol, pH 7.2). The diluted RNA was loaded onto a cellulose column in a buffer containing 35% organic solvent (10 mM HEPES, 125 mM NaCl, 0.1 mM EDTA, 35% (v / v) ethanol or isopropanol or n-propanol, pH 7.2). The organic solvent concentration of the eluate (10 mM HEPES, 125 mM NaCl, 0.1 mM EDTA, 16% (v / v), 10% (v / v), and 0% (v / v) ethanol or isopropanol or n-propanol, pH 7.2) was reduced to 16% (v / v), 10% (v / v), and 0% (v / v), and the eluted RNA was collected, respectively.

[0179] The elution curve (UV absorbance at 260 nm) of the capped RNA chromatogram is consistent with the elution curve of the transcribed RNA. When the concentration of the three organic solvent eluates, ethanol, isopropanol, and n-propanol, is reduced to 16% (v / v), most of the target RNA is eluted and appears as a single UV peak ( Figure 4 The yield of capped RNA collected in this portion was 73.4% in the ethanol eluate, 71.4% in the isopropanol eluate, and 80.6% in the n-propanol eluate, with the n-propanol eluate having the highest RNA yield (Table 3).

[0180] The results of dsRNA residual detection using J2 anti-dsRNA monoclonal antibody showed that when the organic solvent concentration of the eluate was 16% (v / v), more than 85% of the dsRNA content was removed in this part compared with the initial input RNA ( Figure 5 ). When the eluate contained isopropanol as an organic solvent, the dsRNA residue was the least, followed by ethanol and n-propanol. Under the same concentration of organic solvent, isopropanol showed a higher resolution when used as an organic solvent to separate dsRNA and ssRNA. When the concentration of the organic solvent in the eluate was 10% (v / v), only a small portion of RNA was washed off the cellulose column, and this portion showed a large amount of dsRNA. This once again confirmed that organic solvents such as isopropanol and n-propanol can also be successfully used as mobile phases and cellulose as stationary phases for capped RNA purification to remove dsRNA impurities, and isopropanol is the best for separating dsRNA and ssRNA. Comparison of the integrity of the RNA after cellulose purification with that of the initial input RNA showed that the RNA was not affected and had good integrity ( Figure 6).

[0181] We further analyzed the eluted dsRNA samples using capillary electrophoresis. The results showed that the results of RNA cellulose purification using ethanol, isopropanol, and n-propanol mobile phases were very similar, with no significant differences. The dsRNA samples eluted under 0% organic phase and aqueous solution conditions did contain a certain proportion of RNA detection peaks containing secondary structures, as detailed in [1]. Figure 7 .

[0182] Table 3 Yield of capped RNA from cellulose purification using ethanol, isopropanol, and n-propanol at different organic phase concentrations

[0183]

[0184] Example 4. Cellular Function of Purified RNA

[0185] The different Cap eGFP mRNA products purified under the above different organic buffer conditions were transfected into cells to verify their translation efficiency. The primary PBMC cell transfection procedure is as follows: Prepare electroporation solution: Take LONZA electroporation solution, remove the EGFP mRNA to be transfected, calculate the amount required for electroporation, and the EGFP mRNA-electroporation dose is 20 μg. Mix the mixture and electroporation solution evenly and prepare it before electroporation. Prepare 12-well plates: Take CD3 / CD28 antibodies from the -20°C refrigerator and thaw them for use. Add antibodies to DPBS at a concentration of 5 μg / mL, mix well, add to the wells, and place in a 37°C incubator. Centrifuge fresh cells at 1200 rpm for 5 minutes, remove and discard the supernatant, add DPBS, centrifuge at 1200 rpm for 3 minutes, discard the DPBS, add an appropriate amount of DPBS, gently pipette to mix, and count the cells. Take a 1.5 mL centrifuge tube and add 6.5×10 6 Centrifuge at 1200 rpm for 3 minutes, discard the supernatant, and prepare an electroporation kit (Lonza). Add 100 μL of each electroporation reagent in the appropriate proportions to the sample, mix thoroughly, and transfer the mixture to a cuvette. Place the cuvette in an electroporator for electroporation. Use the micropipette provided in the kit to transfer the electroporated cell suspension to a 12-well plate pre-filled with culture medium. After 4 hours, transfer the plate to a coated plate and incubate at 37°C in a 5% CO2 incubator. Detect EGFP positivity two days after electroporation.

[0186] from Figure 8 As can be seen in the figure, in PBMC cells, the eGFP samples purified using the three methods showed comparable eGFP fluorescence positivity after electroporation. However, when purified using 16% isopropanol, the resulting EGFP-mRNA showed significantly higher fluorescence intensity and positivity after electroporation. These results demonstrate that using isopropanol as the organic solvent in the buffer solution is superior to ethanol in purification.

[0187] Example 5: Effect of RNA Chain Length on Isopropanol Mobile Phase Cellulose Purification

[0188] Since isopropanol organic solvent shows high resolution in the separation of ssRNA and dsRNA, isopropanol was selected as an organic solvent for testing to verify whether the increase in RNA length would affect the separation of ssRNA and dsRNA by cellulose-purified RNA. The tailed RNA mixture, the aqueous RNA sample precipitated with LiCl, and RNA with a length >2000nt were loaded onto the cellulose column. Since the RNA sample contained 50% organic solvent, all RNA could bind to the cellulose column at this time. The isopropanol concentration of the eluate on the cellulose column was reduced to 16% (v / v), 8% (v / v), 6% (v / v), and 0% (v / v) to release ssRNA and dsRNA in sequence. The RNA in each fraction was collected and the yield was calculated. The RNA in the eluate and the dsRNA residue in the starting input RNA were analyzed by dot blot.

[0189] The elution curve of the RNA chromatogram (UV absorbance at 260 nm) showed that compared with 1000 nt, >2000 nt RNA was not eluted at 16% (v / v) organic solvent concentration. When the isopropanol concentration of the eluent was reduced to 8% (v / v), most of the RNA was eluted, and the yield of this part of RNA was 52% ( Figure 9 When the isopropanol concentration of the eluate reached 6% (v / v), the RNA yield was 13%. The dsRNA residual test showed ( Figure 10 ), compared to the input RNA, an 8% isopropanol concentration in the eluate resulted in 80% dsRNA removal. A 6% isopropanol concentration in the eluate revealed a higher concentration of dsRNA. This experiment confirms that the removal of dsRNA contaminants from cellulose-based RNA purification is determined by both the organic solvent concentration in the eluate and the RNA length. Release of RNA >2000 nt from the cellulose column requires a lower organic solvent concentration in the eluate.

[0190] Example 6: Screening for Optimal Conditions for Pretreatment of Cellulose-Purified RNA Samples

[0191] Enzymatically produced mRNA transcripts contain large amounts of bioengineered enzyme proteins and RNA. Cellulose-based RNA purification requires organic solvents such as ethanol, isopropanol, or n-propanol in the mobile phase. These two factors present a natural conflict. Organic solvents not only affect protein solubility, potentially causing precipitation, but also partially precipitate RNA, preventing direct downstream chromatographic purification. Therefore, all reported cellulosic RNA purification processes utilize LiCl and / or alcohol precipitation to pre-precipitate and re-dissolve the RNA sample.

[0192] Adding a small amount of neutral salt, such as ammonium sulfate, sodium sulfate, or sodium chloride, to an aqueous protein solution increases the surface charge of the protein molecules, enhancing the interaction between the protein and water molecules, thereby increasing the protein's solubility in the aqueous solution. This phenomenon is known as salt dissolution. Dilute concentrations can also promote protein dissolution, a phenomenon known as salt dissolution. Furthermore, dilute salt solutions have the advantage of protecting proteins from denaturation because salt ions partially bind to the protein.

[0193] In view of the above analysis, we innovatively used the salt dissolution principle to screen out an optimal condition for sample pretreatment. Under this optimal condition, the transcribed RNA product can be directly purified by cellulose chromatography in one step without the pretreatment step of precipitation and centrifugation.

[0194] To avoid the need for a LiCl precipitation step before RNA purification, the cellulose RNA purification process was simplified and the RNA purification method was further optimized. The goal was to perform a simple pretreatment of the RNA before loading to ensure that the RNA mixture was not precipitated and could be loaded directly. During the loading process, the RNA would bind to the cellulose column, while the rest of the mixture would not. The target RNA would be collected in the eluate, ultimately achieving the goal of purifying RNA.

[0195] Select the post-transcribed RNA mixture for testing. First, prepare the dilution buffer. The buffer composition is shown in Table 4. Then, take 400 μL of the post-transcribed RNA mixture and mix it with the dilution buffer in a 1:1 ratio. The mixed RNA mixture contains isopropanol concentrations of 20%, 25%, 30%, and 35%, and salt concentrations of 15.625mM, 31.25mM, 62.5mM, 125mM, 250mM, and 500mM, respectively. The mixed sample was allowed to stand at room temperature for 30 minutes and observed with the naked eye to see if there was turbidity. After 30 minutes, the mixture was placed in a centrifuge and centrifuged at 12,000 rpm for 2 minutes to remove the precipitated RNA and protein mixture. The RNA supernatant was taken and the RNA was recovered using a spin column. The recovered RNA was dissolved in water, the RNA concentration was measured, and the sample yield was calculated. The yield calculation results are shown in Table 5.

[0196] Table 4 Dilution buffer preparation composition table

[0197]

[0198]

[0199] When the salt concentration is ≤60mM, the RNA yield in the transcription mixture is not significantly affected by the increase of the isopropanol concentration in the equilibrium buffer, and no precipitation occurs in the mixture when the salt concentration is ≤60mM. Figure 11). When the salt concentration is >125mM, the yield of RNA in the transcription mixture can be significantly reduced as the isopropanol concentration in the equilibration buffer increases, and precipitation of the mixture can be observed with the naked eye. The transcription mixture is greatly affected by the isopropanol concentration. When the isopropanol concentration is 20%-30% (v / v), the best salt concentration is ≤125mM. When the isopropanol concentration is 35%, the increase in salt concentration can significantly reduce the yield of RNA in the transcription mixture. Based on the yield data, the organic solvent concentration of 20-30% (v / v) and the salt concentration of ≤125mM are selected for post-transcription sample pretreatment.

[0200] Based on the screened isopropanol concentrations of 20-30% (v / v) and salt concentrations ≤125mM for sample pretreatment, and a fixed buffer salt concentration of 125mM, the isopropanol concentration for sample pretreatment on the cellulose column was further optimized from the 20-30% (v / v) isopropanol concentration. The transcribed RNA mixture was diluted with dilution buffer containing 40%-60% isopropanol. The diluted transcription mixtures contained 20% (v / v) isopropanol and 125mM salt, 25% (v / v) isopropanol and 125mM salt, and 30% (v / v) isopropanol and 125mM salt, respectively. When the salt concentration was 125mM, RNA flow-through and the final RNA yield in the eluted product were verified during cellulose purification of the transcription mixture containing 20-30% (v / v) isopropanol.

[0201] A 25mL XK16 / 20 cellulose column was loaded and equilibrated with equilibration buffer containing 20%, 25%, or 30% isopropanol. The transcribed mixture, containing 20mg of RNA, was diluted with dilution buffer (20mM HEPES, 250mM NaCl, 0.2mM EDTA, 40% (v / v), 50% (v / v), or 60% (v / v) isopropanol, pH 7.2) and purified using the cellulose column. RNA was released by reducing the isopropanol concentration in the eluate by 10% (v / v). RNA samples were collected from each elution peak, and the RNA concentration was measured using a nucleic acid concentration analyzer. The yield was then calculated.

[0202] The elution curve of the RNA chromatogram (UV absorbance at 260nm) showed that a flow-through peak appeared when the sample was loaded. The flow-through sample was collected and LiCl precipitation was used to verify whether there was RNA that was not bound to the filler. The results showed that when the final concentration of isopropanol in the diluted transcription mixture was 20% and 25%, RNA was detected in the collected flow-through peak, and when the final concentration of isopropanol in the diluted transcription mixture was 30%, no RNA was detected in the collected flow-through peak. This experiment determined that when the final concentration of isopropanol in the transcription mixture was 30%, RNA could be completely bound to the cellulose column filler when the sample was loaded, and impurities flowed through ( Figure 12 ). When the isopropanol concentration of the eluate was reduced to 10% (v / v), a single RNA peak appeared, and the RNA yield of this component was over 90%. In order to further improve the cellulose RNA purification method, the capped RNA mixture and the tailed RNA mixture were tested in turn. The results showed that when the capped RNA mixture was loaded onto the cellulose column, the sample needed to be diluted to a buffer solution containing a final concentration of 30% (v / v) isopropanol. This ensures that when the RNA mixture is loaded, the RNA does not precipitate and the RNA is completely bound to the cellulose. The RNA was washed out in 10% (v / v) isopropanol, and the yield was over 80%. When the tailed RNA mixture was loaded onto the cellulose column, the sample needed to be diluted to a buffer solution containing a final concentration of 25% (v / v) isopropanol. This ensures that when the RNA mixture is loaded, the RNA does not precipitate and the mixture flows through completely, and the RNA is completely bound to the cellulose. The RNA was washed out in 10% (v / v) isopropanol, and the yield was over 80%.

[0203] Table 5 Effects of different salt concentrations and isopropanol concentrations on post-transcription RNA samples

[0204]

[0205]

[0206] Example 7: RNA loading capacity test on cellulose filler under process amplification conditions

[0207] An XK 16 / 20 40mL cellulose column (Sigma C6288) was used. 40mg, 70mg, and 100mg of a mixture of transcribed PB RNA (>2000nt in length) were diluted with dilution buffer (20mM HEPES, 250mM NaCl, 0.2mM EDTA, 60% (v / v) isopropanol, pH 7.2) to a 30% (v / v) isopropanol concentration. The diluted sample was directly applied to the cellulose column with a retention time of 50min. Elution of RNA bound to the cellulose was performed with elution buffer (10mM HEPES, 125mM NaCl, 0.1mM EDTA, 10% (v / v) isopropanol, pH 7.2). The column was then washed with injection water for regeneration. Finally, the amount of RNA in the flowthrough, the amount of RNA in the eluate, and the amount of RNA sample during column regeneration were calculated. The cellulose column capacity was calculated based on the amount of sample flowthrough during loading and the amount of sample eluted. When the load was increased from 40 mg to 100 mg of RNA sample, no target RNA flowthrough occurred in the flowthrough, indicating that the RNA was completely eluted by the elution buffer. Based on this calculation, for an RNA mixture >2000 nt, a cellulose capacity of 2.5 mg / mL corresponds to a binding capacity of 6.25 mg of RNA per 1 g of cellulose.

[0208] Example 8, Screening of Chromatographic Separation Conditions for dsRNA and ssRNA in Isopropanol Mobile Phase

[0209] Experiments conducted in this study demonstrate that the majority of dsRNA is a nonspecific secondary structure between ssRNA molecules, which does not affect the cellular function of ssRNA, or mRNA. In capillary electrophoresis, this dsRNA can be largely converted back to ssRNA after denaturation. Cells transfected with dsRNA and collected from the peaks can achieve translation efficiencies comparable to those of pure ssRNA.

[0210] In order to verify whether the residual dsRNA in the eluate RNA will affect cell transfection. Select PB mRNA with a length of >2000nt after tailing, and equilibrate the cellulose column with 30% isopropanol buffer. Load the RNA onto the cellulose column, reduce the isopropanol concentration of the eluate to 12% (v / v), 10% (v / v), 6% (v / v), and 0% (v / v), and release the RNA in sequence to collect the RNA of each component. Recover the RNA using a spin column column, replace the RNA in an aqueous solution, and calculate the RNA yield of each part. Dot blot analysis of the RNA in the eluate and the DsRNA content in the starting input RNA. Analyze the RNA integrity using a labchip detector.

[0211] The elution curve of the RNA chromatogram (UV absorbance at 260 nm) showed that when the isopropanol concentration of the eluate was reduced to 12% (v / v), 10% (v / v), 6% (v / v), and 0% (v / v), a single RNA peak appeared ( Figure 13 ), the total RNA yield of each component was above 60%. The RNA integrity analysis results showed that ( Figure 14 ), the integrity of the sample RNA after cellulose purification is not affected, and the target RNA is also contained when the column is regenerated. Compared with the initial input RNA sample, the dsRNA content after purification is greatly reduced ( Figure 15 The results of the cell function test of RNA samples washed with different concentrations of isopropanol and RNA samples after column regeneration showed that there was no significant difference in the cell function results between the RNA samples washed with cellulose column and the RNA samples before loading ( Figure 16 ).

[0212] Analysis of cell function data suggests that to further improve sample yield and reduce costs for process scale-up, the isopropanol concentration in the elution buffer can be reduced by 6-10% (v / v) to increase yield. When the isopropanol concentration in the elution buffer is 6%, the yield can be increased by 18%. Furthermore, small amounts of residual dsRNA do not affect cell function.

[0213] Example 9: Correlation between Cellulose Chromatographic Filler Particle Size and RNA Adsorption Capacity

[0214] Different cellulose chromatographic filler particle sizes should have different RNA adsorption capacities. The main purpose of optimizing the filler particle size is to increase the loading capacity and / or separation efficiency of the production filler, and to allow for the separation of proteins and NTPs rather than being limited to the separation of ssRNA and dsRNA using existing patented technologies.

[0215] To test the RNA binding capacity of different particle size packings, we screened the RNA binding capacity of packings with different particle sizes ranging from 25 to 200 μm from different manufacturers, using Sigma (C6288) as a control (Table 5). Weigh 0.1 g of cellulose packing of different particle sizes and add 400 μL of 0.1 M / L NaOH to the packing. After incubation in a metal bath at 1000 rpm for 45 minutes, centrifuge at 12000 rpm for 2 minutes, remove the supernatant, and resuspend the packing in 400 μL of equilibration buffer using a pipette. Repeat this process six or more times until the pH matches that of the buffer solution, at which point the solution is no longer needed. The transcribed RNA solution is diluted with dilution buffer, ensuring that the isopropanol concentration in the diluted RNA buffer is consistent with the isopropanol concentration in the equilibration buffer, both at 30%. The diluted RNA concentration is 1.25 mg / mL. 0.6 mg of the diluted RNA sample was incubated with the filler. The mixture was placed in a metal bath at 1000 rpm for 30 minutes and then centrifuged at 12000 rpm for 5 minutes. The supernatant was collected and the RNA not bound to the filler was recovered using a spin column from NEB's Monarch RNA Purification Kit. The recovered RNA was then exchanged in aqueous solution and its concentration was determined. The amount of RNA bound to fillers of different particle sizes was calculated (Table 5).

[0216] The results showed that the amount of RNA bound to cellulose of different particle sizes for cellulose fillers from three different manufacturers varied, while the RNA loading capacity of Sigma fillers with different particle sizes did not differ significantly. For Aladdin fillers, the smaller the particle size (≤250 μm), the more RNA bound and the higher the filler loading capacity. The larger the particle size, the less RNA bound to the filler and the lower the loading capacity. For Maclean's cellulose fillers, the loading capacity of fillers with a particle size of 65 μm was slightly higher than that of fillers with a particle size of 100 μm.

[0217] Table 5 RNA loading capacity of different particle size fillers

[0218]

[0219]

[0220] *The RNA adsorption capacity under chromatographic column conditions will be different from that under test tube mixed adsorption conditions. This experiment mainly analyzes the change trend of RNA adsorption capacity with particle size change.

[0221] Example 10: Scale-up of the large-scale production process for mRNA purification using cellulose chromatography

[0222] To verify whether cellulose columns can be used to amplify RNA production, the large-scale transcribed PB mRNA mixture was purified using a cellulose column and loaded onto an XK50 / 18 350 mL cellulose column. 350 mg of the RNA mixture was diluted 1:1 with water and then diluted with dilution buffer (20 mM HEPES, 250 mM NaCl, 0.2 mM EDTA, 60% (v / v) isopropanol pH 7.2). The sample was filtered using a 0.22 μm vacuum filtration system and loaded with the sample at a flow rate of 21 cm / h. The elution buffer used was (10 mM HEPES, 125 mM NaCl, 0.1 mM EDTA, 8% (v / v) isopropanol pH 7.2). The eluted sample was collected, the RNA concentration was measured using a nucleic acid concentration detector, and the sample yield was calculated.

[0223] The elution curve of the RNA chromatogram (UV absorbance at 260 nm) shows that a flow-through peak appears when the sample is loaded. The flow-through sample is collected and precipitated with LiCl to verify whether there is RNA that has not bound to the filler ( Figure 17 The results showed that when the final isopropanol concentration in the diluted transcription mixture reached 30%, no RNA was detected in the collected flowthrough peak. When the isopropanol concentration in the eluate reached 8% (v / v), a single RNA peak emerged, and the RNA yield from this fraction exceeded 90%, yielding a total of 300 mg of RNA. This experiment demonstrates the utility of cellulose columns for RNA amplification and production. Sequence Listing <110> Shanghai Cell Therapy Group Co., Ltd. <120> Method for RNA purification using cellulose chromatography <130> 210139 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1179 <212> DNA <213> Artificial Sequence <400> 1 tctagataat acgactcact atagggagaa ttcgccacca tggtgagcaa gggcgaggag 60 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 120 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc 180 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 240 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 300 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 360 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 420 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 480 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag 540 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 600 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc 660 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 720 gccgggatca ctctcggcat ggacgagctg tacaagtaag gatcctgcac tagtgctgtc 780 gacgctcgct ttcttgctgt ccaatttcta ttaaaggttc ctttgttccc taagtccaac 840 tactaaactg ggggatatta tgaagggcct tgagcatctg gattctgcct aataaaaaac 900 atttattttc attgcgctcg ctttcttgct gtccaatttc tattaaaggt tcctttgttc 960 cctaagtcca actactaaac tgggggatat tatgaagggc cttgagcatc tggattctgc 1020 ctaataaaaa acatttattt tcattgcaaa aaaaaaaaaa aaaaaaaaa aaaaaaaaaa 1080 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1140 aaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaggtacc 1179 <210> 2 <211> 2198 <212> DNA <213> Artificial Sequence <400> 2 tctagataat acgactcact atagggagaa ttcgccacca tgggctctag cctggacgac 60 gagcacatcc tgagcgccct gctgcagagc gacgacgaac tggtgggcga ggacagcgac 120 agcgaggtca gcgaccacgt gtccgaggac gacgtgcagt ccgacaccga ggaagccttc 180 atcgacgagg tgcacgaagt gcagcctacc agcagcggct ccgagatcct ggacgagcag 240 aacgtgatcg agcagcctgg cagctccctg gccagcaaca gaatcctgac cctgccccag 300 360 agagtgtccg ccctgaacat cgtgcggagc cagaggggcc ccaccagaat gtgcagaaac 420 atctacgacc ccctgctgtg cttcaagctg ttcttcaccg acgagatcat cagcgagatc 480 gtgaagtgga ccaacgccga gatcagcctg aagaggcggg agagcatgac cagcgccacc 540 ttcagagaca ccaacgaga cgagatctac gccttcttcg gcatcctggt gatgaccgcc 600 gtgagaagg aaaccacat gagcaccgac gacctgttcg acagatccct gagcatggtg 660 720 gacaagagca tcagacccac cctgcgggag aacgacgtgt tcaccccgt gcggaagaatc 780 tgggacctgt tcatccacca gtgcatccag aactacaccc ctggcgccca cctgaccatc 840 gatgagcagc tgctggggctt cagaggcaga tgccccttca gagtgtacat ccccaacaag 900 cccagcaagt acggcatcaa gatcctgatg atgtgcgaca gcggcaccaa gtacatgatc 960 aacggcatgc cctacctggg cagaggcacc cagaaacg gcgtgcccct gggcgagtac 1020 tacgtgaaag aactgagcaa gcctgtgcat ggcagctgca ggaaccac ctgcgacac 1080 tggttcacca gcatccccct ggccagac ctgctgcagg aaccctaca gctgaccac 1140 gtgggcaccg tgcggagca caagcgggag atcccagagg tgctgagaa cagcagatcc 1200 agacctgtgg gaacaagcat gttctgctc gacggcccc tgaccctgt gtcctacaag 1260 cccaagcccg ccagatggt gtacctgctg tccagctgcg acgaggacgc cagcatcac 1320 gagagcaccg gcaagcccca gatggtgatg tactacaacc agaccaaggg cggcgtggac 1380 accctggacc agatgtgcag cgtgatgacc tgcagcagaa agaccacag atggcccatg 1440 gccctgctgt acggcatgat caatcgcc tgcatcaca gctcatcat ctacagccac 1500 aacgtgtcca gcaagggcga gaggtgcag agccggaag attcatgcg gaacctgtac 1560 atgagcctga cctccagctt catgagaaag agactggaag cccccaccct gaagagatac 1620 ctgcgggaca acatcagca catcctgccc aaggaagtgc caggaacaag cgacgacagc 1680 accgaggaac ccgtgatgaa gagaggacc tactgcacct actgtcccag caatcaga 1740 agaaaggcca acgccagctg caagaaatgc aaaaaagtga tctgccggga gcacaacatc 1800 gacatgtgcc agagctgttt ctgaggatcc tgcactagtg ctgtcgacgc tcgctttctt 1860 gctgtccaat ttctattaaa ggttcctttg ttccctaagt ccaactacta aactggggga 1920 tattatgaag ggccttgagc atctggattc tgcctaataa aaaacattta ttttcattgc 1980 gctcgctttc ttgctgtcca atttctatta aaggttcctt tgttccctaa gtccaactac 2040 taaactgggg gatattatga agggccttga gcatctggat tctgcctaat aaaaacatt 2100 tatttcatt gcaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 2160 aaaaaaaaa aaaaaaaaaa aaaaaaaaa aaggtacc 2198

Claims

1. A method for enriching single-stranded RNA, comprising: (1) contacting a mixture containing single-stranded RNA, 20%-35% (v / v) isopropanol and 60-125 mM salt with a cellulose material, (2) A liquid containing single-stranded RNA is obtained from the cellulose material using an eluent containing 6%-16% isopropanol and 60-125 mM sodium chloride, potassium chloride or lithium chloride.

2. The method according to claim 1, wherein The single-stranded RNA is a single-stranded RNA produced in vivo and extracted, and / or The single-stranded RNA is a single-stranded RNA produced by in vitro biosynthesis or modification, and / or The length of the single-stranded RNA is at least 200 bp, and / or The mixture comprises single-stranded RNA, 20%-35% (v / v) isopropanol, and 60-125 mM salt selected from sodium chloride, potassium chloride, and lithium chloride.

3. The method according to claim 1, wherein Prior to step (1), the single-stranded RNA or the mixture containing the single-stranded RNA is not subjected to pre-purification treatment.

4. The method according to claim 1, wherein The contacting of step (1) comprises mixing the mixture with a cellulosic material or loading the mixture onto a column packed with a cellulosic material, and / or The contact time of step (1) is 30-150 min, and / or Step (2) includes: (2a) eluting the liquid containing the single-stranded RNA from the cellulose material using an eluent, or (2b) obtaining the single-stranded RNA from the cellulose material using an eluent and obtaining the liquid containing the single-stranded RNA by solid-liquid separation.

5. The method according to claim 4, wherein The elution rate in step (2a) is 30-100 cm / h.

6. The method according to any one of claims 1 to 5, wherein The eluent contains 4-18% isopropanol, and / or The eluent further comprises EDTA, and / or The eluent may also contain HEPES or Tris.

7. The method according to claim 6, wherein The EDTA concentration is 0.01-0.5 mM.

8. The method according to claim 6, wherein The concentration of HEPES or Tris is 1-20 mM.

9. The method according to claim 6, wherein The eluent contains 6%-16% isopropanol, 60-125 mM sodium chloride, potassium chloride or lithium chloride, 0.05-0.3 mM EDTA and 6-15 mM HEPES or Tris.

10. The method according to any one of claims 1 to 5, characterized in that The particle size of the cellulosic material is 20 microns to 250 microns, and / or The RNA loading capacity of the cellulose material is 2.5-6.25 mg / g, and / or The cellulose material is pretreated before contacting with the mixture, wherein the pretreatment solution comprises 20-35% organic alcohol, 50-150 mM salt, and optionally further comprises 0.01-0.5 mM EDTA and / or 1-20 mM HEPES or Tris.

11. A method for enriching single-stranded RNA, comprising: (1) mixing a composition with a sample containing single-stranded RNA at a volume ratio of 1:1 and contacting the mixture with a cellulose material, wherein the composition comprises 50%-60% isopropanol, 120-250 mM sodium chloride, potassium chloride or lithium chloride, 0.1-0.5 mM EDTA, and 10-30 mM HEPES or Tris, (2) A liquid containing single-stranded RNA is obtained from the cellulose material using an eluent comprising 6%-16% isopropanol, 60-125 mM sodium chloride, potassium chloride or lithium chloride, 0.05-0.3 mM EDTA and 6-15 mM HEPES or Tris.

12. The method according to claim 11, wherein The sample containing single-stranded RNA is a sample containing single-stranded RNA produced in vivo and extracted from RNA, and / or The sample containing single-stranded RNA is a sample containing single-stranded RNA produced by in vitro biosynthesis of RNA or modification of RNA, and / or The single-stranded RNA is at least 200 bp in length.

13. The method according to claim 11, wherein Prior to step (1), the sample containing single-stranded RNA is not pre-purified.

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