CN116497019A - Transcription buffer solution capable of improving in-vitro synthesized RNA yield and transcription reaction system - Google Patents

Transcription buffer solution capable of improving in-vitro synthesized RNA yield and transcription reaction system Download PDF

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CN116497019A
CN116497019A CN202310205612.3A CN202310205612A CN116497019A CN 116497019 A CN116497019 A CN 116497019A CN 202310205612 A CN202310205612 A CN 202310205612A CN 116497019 A CN116497019 A CN 116497019A
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rna
transcription
tris
yield
concentration
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肖潇
尹丹丹
华成品
鲁笑笑
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Jiangsu Shenji Biotechnology Co ltd
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    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a transcription buffer solution capable of improving the output of in vitro synthesized RNA, which comprises the following components in final concentration: 300-500 mM of Tris-HCl salt; 300-460 mM of magnesium acetate; spermidine 10-30 mM; 50-200 mM dithiothreitol; the magnesium ion salt type adopted in the transcription buffer solution is magnesium acetate, the concentration of magnesium ions in the magnesium acetate is low, the generation of transcription byproducts and reaction system precipitation can be reduced, and the magnesium acetate with the optimal concentration can be screened out through experiments, so that the yield of RNA can be greatly improved.

Description

Transcription buffer solution capable of improving in-vitro synthesized RNA yield and transcription reaction system
Technical Field
The invention relates to the field of biological detection, in particular to a transcription buffer solution and a transcription reaction system capable of improving the yield of in-vitro synthesized RNA.
Background
mRNA vaccines are where mRNA encoding a disease-specific antigen is introduced into the body via a delivery system, and the antigen is produced by the protein synthesis machinery of the host cell, thereby triggering an immune response. In addition to vaccines, mRNA technology acts as a versatile platform technology, and in theory mRNA has the potential to synthesize either protein, potentially replacing and supplementing protein therapies to some extent. The successful market marker mRNA technology of the first mRNA vaccine worldwide in 2020 entered the commercialized era, and with the urgent need for early technology accumulation, mRNA technology entered the rapid development period. Nevertheless, great development of mRNA therapies remains to be broken through by further techniques.
The production of mRNA vaccines mainly includes: designing and preparing a DNA template sequence, preparing an mRNA stock solution, purifying the mRNA stock solution, wrapping lipid nano particles and the like. The mRNA raw liquid preparation process directly affects the quality and production cost of mRNA, wherein the yield and quality of in vitro transcription synthesis mRNA are key factors affecting the mRNA raw liquid preparation process.
In vitro transcription refers to the process of simulating in vivo transcription into RNA in an in vitro cell-free system by using linear DNA containing a promoter region as a template and adding RNA polymerase, transcription buffer, nucleotide and other raw materials. In vitro transcription reaction systems require: (1) a linearized plasmid template comprising a T7 promoter; (2) 4 free nucleotides; (3) An RNase inhibitor for inhibiting RNase which may be present in an in vitro transcription reaction system; (4) Inorganic pyrophosphatase, degrading pyrophosphoric acid accumulated in IVT reaction; (5) Transcription buffer containing magnesium ions, antioxidants and polyamines.
In the transcription buffer, magnesium ions are used as cofactors of T7 RNA polymerase, and are critical for the activity of T7 enzyme. However, too high a concentration of magnesium ions can result in the production of large amounts of transcription byproduct dsRNA that can activate innate immunity through the RIG-I/MDA-5-MAVS pathway, thereby impairing mRNA translation; meanwhile, a large amount of magnesium pyrophosphate is produced during transcription, and the magnesium pyrophosphate has poor solubility and precipitates product RNA, so that the reaction system is precipitated, and the reaction yield is greatly reduced.
Disclosure of Invention
Based on the technical problems, the application provides a transcription buffer solution and a transcription reaction system capable of improving the yield of in vitro synthesized RNA.
The first eyesight improving aspect of the present application is to provide a transcription buffer solution capable of improving the yield of in vitro synthesized RNA, which adopts the following technical scheme:
a transcription buffer capable of increasing the yield of in vitro synthesized RNA, comprising the following components in final concentration: 300-500 mM Tris-HCl salt;
300-460 mM of magnesium acetate;
spermidine 10-30 mM;
50-200 mM dithiothreitol.
The magnesium ion salt type adopted in the transcription buffer solution is magnesium acetate, the concentration of magnesium ions in the magnesium acetate is low, the generation of transcription byproducts and reaction system precipitation can be reduced, and the magnesium acetate with the optimal concentration can be screened out through experiments, so that the yield of RNA can be greatly improved.
Preferably, the transcription buffer comprises the following components in final concentration:
350-450 mM Tris-HCl salt;
300-460 mM of magnesium acetate;
15-25 mM of spermidine;
80-150 mM dithiothreitol.
Preferably, the transcription buffer comprises the following components in final concentration:
350-450 mM Tris-HCl salt;
400-460 mM of magnesium acetate;
15-25 mM of spermidine;
80-150 mM dithiothreitol.
Preferably, the transcription buffer comprises the following components in final concentration:
350-450 mM Tris-HCl salt;
300-350 mM of magnesium acetate;
15-25 mM of spermidine;
80-150 mM dithiothreitol.
Preferably, the transcription buffer comprises the following components in final concentration:
Tris-HCl salt 400mM;
400-460 mM of magnesium acetate;
spermidine 20mM;
dithiothreitol 100mM.
Preferably, the transcription buffer comprises the following components in final concentration:
Tris-HCl salt 400mM;
magnesium acetate 460mM;
spermidine 20mM;
dithiothreitol 100mM.
The concentration of magnesium acetate in the transcription buffer of the present application is 400-460 mM, and the RNA yield and the RNA integrity are relatively high.
The concentration of magnesium acetate in the transcription buffer of the present application is 300-350 mM, and the RNA yield and RNA integrity are higher than those of the same concentration of magnesium chloride.
The reduction in RNA yield and RNA integrity is small when the concentration of magnesium acetate in the transcription buffer of the present application is reduced from 400-460 mM to 300-350 mM, and when the concentration of magnesium chloride is reduced from 400-460 mM to 300-350 mM.
Preferably, when the concentration of magnesium acetate is 300-350 mM, the RNA yield can be improved by 120-325% compared with the magnesium chloride with the same concentration.
Preferably, when the concentration of magnesium acetate is 350mM, the RNA yield can be improved by 120% compared with the same concentration of magnesium chloride.
Preferably, the RNA yield is increased by 325% when the concentration of magnesium acetate is 300mM, as compared to the equivalent concentration of magnesium chloride.
By adopting the technical scheme, compared with magnesium chloride (MgCl) 2 ) For example, magnesium acetate (MgAc) 2 ) The method has more advantages in ensuring the concentration and the yield of in vitro co-transcription reaction under the condition of lower magnesium ion concentration.
Preferably, the RNA integrity is improved by 0.5 to 2% when the concentration of magnesium acetate is 300 to 350mM, compared with the equivalent concentration of magnesium chloride.
Preferably, the RNA integrity is improved by 2% when compared to the equivalent concentration of magnesium chloride at a concentration of 350mM magnesium acetate under Tris-salt cap analogue and Tris-salt NTP conditions.
Preferably, the RNA integrity is improved by 1.5% when compared to the equivalent concentration of magnesium chloride at a concentration of 300mM for Tris-salt cap analogues and Tris-salt NTP.
Preferably, the RNA integrity is improved by 0.5% when compared to the equivalent concentration of magnesium chloride at a magnesium acetate concentration of 350mM under the conditions of the ammonium salt type cap analogue and the sodium salt type NTPs.
Preferably, the RNA integrity is improved by 2% when the concentration of magnesium acetate is 300mM under the conditions of the ammonium salt type cap analogue and the sodium salt type NTPs, compared with the magnesium chloride with the same concentration.
Compared with magnesium chloride (MgCl) 2 ) For example, magnesium acetate (MgAc) 2 ) It is more advantageous to ensure the integrity of the in vitro co-transcription reaction product at lower magnesium ion concentration.
The second object of the present invention is to provide a transcription reaction system comprising the above transcription buffer, T7 RNA Polymerase Mix, and Tris-salt type cap analogues and Tris-salt type NTPs.
Sodium ions and ammonium ions are common cations in a co-transcription reaction system, and related researches show that the excessive concentration of sodium ions can inhibit the activity of T7 enzyme, thereby influencing the yield of in vitro transcription. The sodium ions and ammonium ions in the co-transcription system are often introduced by cap analogues and free nucleotide NTPs, so that the selection of proper cap analogues and NTP salt forms to participate in the co-transcription reaction is important. Through a large number of experiments, the inventor discovers that the cap analogue and NTP of the Tris salt type have obvious advantages in the aspects of co-transcription yield, product quality, dsRNA control and the like compared with the sodium salt type.
Preferably, the T7 RNA Polymerase Mix comprises T7 RNA Polymerase, a murine rnase inhibitor, and inorganic magnesium pyrophosphate.
In the T7 RNA polymerase mixed system, a murine RNase inhibitor is additionally added besides T7 RNA polymerase, and the murine RNase inhibitor is used for inhibiting the generation of magnesium ions, so that the influence on the yield of transcribed RNA is reduced; inorganic magnesium pyrophosphate is added to generate soluble magnesium phosphate after transcription reaction, so that the generation of transcription reaction precipitation is reduced.
Preferably, the Tris salt-type NTPs comprise the following components in final concentration: ATP10mM of Tris salt;
GTP of Tris salt 10mM;
CTP10mM of Tris salt;
tris salt N1-Me-pUTP10mM.
By adopting the technical scheme, the NTPs of the application adopt the NTPs of Tris salt, compared with the NTPs of sodium ions, the NTPs are favorable for improving the transcribed RNA yield, and the dsRNA yield can be reduced, so that the reaction quality is improved.
A kit comprising a buffer as described above or a transcription reaction system as described above and an acceptable adjuvant or carrier.
The transcription reaction system can be industrially applied to a kit, and RNA obtained by transcription reaction of the kit has high yield and good RNA integrity.
In summary, the present application has at least the following technical effects:
1) The magnesium ion salt type adopted in the transcription buffer solution is magnesium acetate, the concentration of magnesium ions in the magnesium acetate is low, the generation of transcription byproducts and reaction system precipitation can be reduced, and the magnesium acetate with the optimal concentration can be screened out through experiments, so that the yield of RNA can be greatly improved;
2) The concentration of magnesium acetate in the transcription buffer solution is 350-460 mM, so that the RNA yield and the integrity are higher, and further, when the concentration of magnesium acetate is 400-460 mM, the RNA yield is higher, and the RNA integrity is better;
3) In the transcription buffer of the present application, magnesium acetate (MgAc 2 ) The method has the advantages of ensuring the concentration, the yield and the integrity of the reaction products of the in-vitro co-transcription reaction under the condition of lower magnesium ion concentration;
4) The Tris salt type cap analogue and NTPs transcription reaction system is adopted, so that the RNA yield obtained by the transcription reaction is improved, and the RNA integrity is improved;
5) The kit comprises transcription buffer solution containing magnesium acetate, tris salt type cap analogues, tris salt type NTPs and acceptable auxiliary agents or carriers, and has the advantages of high RNA yield and high RNA integrity.
Drawings
FIG. 1 is a schematic diagram showing the dsRNA content of co-transcript of ammonium salt-type cap analogues and sodium salt-type NTPs and Tris salt-type cap analogues and NTPs.
Detailed Description
The present application will be described in further detail with reference to the accompanying drawings, examples, and comparative examples. All raw material components referred to in examples and comparative examples of the present application are commercially available.
At present, magnesium ion salt type of transcription buffer solution used in T7 RNA in vitro transcription kits of a plurality of companies on the market is magnesium chloride, the inventor changes magnesium chloride in the transcription buffer solution into magnesium acetate, and by screening the magnesium acetate with the optimal concentration, the RNA yield is obviously improved, and the cost of subsequent RNA synthesis is reduced.
Example 1:
a transcription buffer capable of increasing the yield of in vitro synthesized RNA, comprising the following components in final concentration: tris-HCl salt 400mM, magnesium acetate 460mM, spermidine 20mM, dithiothreitol 100mM.
Example 2:
a transcription buffer capable of increasing the yield of in vitro synthesized RNA, comprising the following concentrations of each component: tris-HCl salt 400mM, magnesium acetate 400mM, spermidine 20mM, dithiothreitol 100mM.
Example 3:
a transcription buffer capable of increasing the yield of in vitro synthesized RNA, comprising the following concentrations of each component: tris-HCl salt 400mM, magnesium acetate 350mM, spermidine 20mM, dithiothreitol 100mM.
Example 4:
a transcription buffer capable of increasing the yield of in vitro synthesized RNA, comprising the following concentrations of each component: 400mM Tris-HCl salt, 300mM magnesium acetate, 20mM spermidine and 100mM dithiothreitol.
Comparative example 1:
a transcription buffer capable of increasing the yield of in vitro synthesized RNA, comprising the following concentrations of each component: 400mM Tris-HCl salt, 280mM magnesium acetate, 20mM spermidine and 100mM dithiothreitol.
Comparative example 2:
a transcription buffer capable of increasing the yield of in vitro synthesized RNA, comprising the following concentrations of each component: tris-HCl salt 400mM, magnesium acetate 520mM, spermidine 20mM, dithiothreitol 100mM.
Comparative example 3:
a transcription buffer capable of increasing the yield of in vitro synthesized RNA, comprising the following concentrations of each component: tris-HCl salt 400mM, magnesium chloride 460mM, spermidine 20mM, dithiothreitol 100mM.
Comparative example 4:
a transcription buffer capable of increasing the yield of in vitro synthesized RNA, comprising the following concentrations of each component: tris-HCl salt 400mM, magnesium chloride 400mM, spermidine 20mM, dithiothreitol 100mM.
Comparative example 5:
a transcription buffer capable of increasing the yield of in vitro synthesized RNA, comprising the following concentrations of each component: tris-HCl salt 400mM, magnesium chloride 350mM, spermidine 20mM, dithiothreitol 100mM.
Comparative example 6:
a transcription buffer capable of increasing the yield of in vitro synthesized RNA, comprising the following concentrations of each component: 400mM Tris-HCl salt, 300mM magnesium chloride, 20mM spermidine and 100mM dithiothreitol.
The transcription buffers prepared in examples and comparative examples were subjected to in vitro transcription reactions according to the following experimental procedures:
(1) Preparation of DNA templates
The template with the through length is linearization plasmid, PCR amplified product or synthesized DNA fragment. For the preparation of linearized plasmid templates, the target fragment is inserted into the multiple cloning site of the plasmid by using methods such as enzyme digestion or recombination, the plasmid generally has a corresponding RNA polymerase starting site, such as T7, SP6 or T3, and then the plasmid is amplified in a biological factory such as E.coli, and the extracted and purified modified plasmid can be used as a DNA template for in vitro transcription of mRNA after being cut and purified by using restriction enzymes. For the preparation of PCR amplified products, the target DNA fragments are usually prepared by using DNA polymerase, specific primers and related buffers as systems, the amplified products usually comprise starting sites of corresponding RNA polymerase, such as T7, SP6 or T3, in this embodiment, T7 RNA polymerase is selected, and the amplified products can be used as DNA templates for in vitro transcription of mRNA after purification.
(2) In vitro transcription system
In the RNA in vitro synthesis system of Table 1, T7 RNA Polymerase Mix contains T7 RNA polymerase, inorganic pyrophosphatase (Inorganic Pyrophosphatase) and a murine RNase inhibitor (Murine RNase Inhibitor). The reaction system was then sequentially loaded according to Table 2, i.e.the linearized DNA template was finally added to prevent precipitation of the DNA template by spermidine in 10X Transcription Buffer, mixed well, centrifuged briefly to the bottom of the tube and incubated at 37℃for 2.5 hours. DNaseI treatment, i.e., 2. Mu.L DNaseI (RNase-free) was added to each tube and incubated at 37℃for 15 minutes to remove the template DNA.
Table 1 T7 RNA Polymerase Mix (2. Mu.L) composition and volume
Component (A) Volume of
T7 RNA Polymerase(50U/μL) 1μL
Murine RNase inhibitors (Murine RNase Inhibitor, 40U/. Mu.L) 0.96μL
Inorganic pyrophosphatase (Inorganic Pyrophosphatase, 1U/. Mu.L) 0.04μL
TABLE 2 in vitro RNA synthesis System of Tris salt-type cap analogues and NTPs
(3) And (3) purifying a product:
the RNA after in vitro transcription can be purified by using RNA Cleaner magnetic beads to remove proteins, free nucleotides and the like.
RNA clearer bead purification method:
RNA clean bead in advances was removed from the 4℃cabinet, equilibrated to room temperature (about 30 minutes) and dried with RNase-free H 2 O dilutes the transcript to 50. Mu.L.
(1) The beads were thoroughly mixed by inversion or vortexing, 6 Xbeads (300. Mu.L) were pipetted into the RNA sample (50. Mu.L), and the mixture was thoroughly mixed by pipetting 6 strokes. The RNA was bound to the beads by incubation at room temperature for 5 min.
(2) The sample was placed on a magnetic rack for 5 minutes and after the solution was clear, the supernatant was carefully removed.
(3) The sample was kept on a magnetic rack, 200. Mu.L of 80% ethanol (with RNase-free H 2 O configuration, as-prepared) rinse beads, incubate at room temperature for 30s, carefully remove supernatant. This operation was repeated once.
(4) The sample is kept on the magnetic rack all the time, and the magnetic beads are air-dried for 5 minutes after being uncapped.
(5) The sample was removed from the magnet holder and 200. Mu.L of RNase-free H was added 2 O, blow with a pipette for 6 times to mix well, and stand at room temperature for 5 minutes.
(6) Samples were placed on a magnetic rack for 5 minutes, after which the solution was clarified, 200. Mu.L of supernatant was carefully transferred to a new RNase-free centrifuge tube.
The detection means comprises:
(1) RNA yield
And (3) detecting the concentration of the purified RNA by an ultraviolet absorption method. The yield of RNA was determined by measuring A260 readings of the product. Specifically, selecting an option of RNA concentration measurement by using a NanoDrop One of a Simer femto ultra-micro ultraviolet spectrophotometer, calibrating the instrument three times by using 1.5 mu L RNase-free H2O, and taking 1.5 mu L RNA sample for concentration measurement to obtain the concentration of each group of RNA; based on the concentration of each group, the volume of 200. Mu.L was multiplied, and the yield of RNA of each group was calculated.
(2) RNA integrity
RNA integrity was determined by the BiopticQsep100Bio-Fragment Analyzer assay.
BiopticQsep100Bio-Fragment Analyzer can be used to evaluate RNA integrity, which requires only a small amount of RNA for analysis.
The method comprises the following specific steps:
a. reagent preparation:
the W (Wash) disk and the C (Clean) disk of the RNA card slot are added with 5mLDEPC water; 4mL DEPC water was added at the P (Park) position, and 4mL 1X Separation Buffer was added on an S (Separation) disk;
b. preparing a solution:
preparing a Marker: placing 30 mu L of RNAMarker stock solution in the MC1 position;
preparing 0.1×volume Buffer: 10 Xdilution Buffer was diluted to 0.1 Xwith DEPC water for use.
c. Sample processing:
sample Dilution was performed using a 0.1 Xdilution Buffer at a concentration of 10 ng/. Mu.L and a volume of about 50. Mu.L, based on the concentration of the mRNA product detected; the diluted mRNA product was denatured at 70℃for 5 minutes in a PCR instrument.
d. Instrument setting:
(3) dsRNA detection
The enzyme method immunoblotting method is used for detecting the dsRNA, and comprises the following specific steps:
(1) mRNA samples (200 ng) were blotted onto positively charged nylon blotting membranes and dried for 30 minutes.
(2) The membranes were incubated with blocking buffer for 1 hour.
(3) The membrane was rinsed twice with TBS-T buffer.
(4) Membranes were incubated with anti-dsRNA antibodies for 1 hour at room temperature.
(5) The membrane was rinsed 4 times with TBS-T buffer and 6 more times for 5 minutes each.
(6) Detection antibody solution was added and the membrane incubated at room temperature for 1 hour.
(7) The membrane was rinsed 4 times with TBS-T buffer and 6 more times for 5 minutes each. ECL chemiluminescent reagents were added and the signal captured using a suitable imaging system to compare spot color.
(4) Cell transfection of Co-transcripts
(1) The nucleotide sequence was calculated to give the desired concentration for each sample based on the concentration of the sample.
(2) Cell culture (six well plate): the day (18-24 hours) before transfection, about 20-70 ten thousand cells per well were seeded into six well plates for culture to achieve a cell density of about 70-90% the next day.
(3) Before the transfection step, the six well plate with cells cultured was replaced with 2mL of fresh culture medium (containing serum, no antibiotics) per well.
(4) For the cells of each well in the six-well plate to be transfected, two clean sterile RNase-free centrifuge tubes are taken, 125 mu L of Opti-MEM culture medium without antibiotics and serum is respectively added, 100pmol of RNA is added into one tube, and a gun is used for gently blowing and mixing; another tube was added with 5. Mu.LLipo 6000 TM The transfection reagent is gently beaten and mixed by a gun, and after standing for 5 minutes at room temperature, the culture solution containing RNA is gently added with Lipo6000 by the gun TM In the culture solution of the transfection reagent, the centrifuge tube is gently inverted or the mixture is gently beaten and mixed by a gun, and the mixture is kept stand for 5 minutes at room temperature.
(5) 250. Mu.L Lipo6000 per well in a six well plate TM The amount of transfection reagent-RNA mixture was added dropwise to the whole well uniformly, followed by gentle mixing.
(6) For maximum transfection efficiency, the cells should be replaced with fresh complete medium after 4-6 hours of culture following transfection.
(7) After further culturing for 12-18 hours, photographs can be taken with a fluorescence microscope.
(1) The results of the RNA yield detection are shown below:
a) The results of the detection of RNA yield in Tris-salt cap analogues and Tris-salt NTPs are shown in Table 3:
TABLE 3 Table 3
As is clear from Table 3, when the transcription buffer described in the present application is applied to the RNA in vitro transcription reaction of Tris-salt type cap analogues and NTPs, the RNA yield obtained by transcription is 170 to 220. Mu.g/1. Mu.g of template when the concentration of magnesium acetate is 300 to 460mM; as is clear from comparative examples 1 and 2, the RNA yield by transcription reaction was significantly decreased at 280mM of magnesium acetate, and the RNA yield by transcription reaction was significantly decreased at 520mM of magnesium acetate, indicating that the RNA yield by transcription reaction was only 80. Mu.g/1. Mu.g of template 2+ Concentration is too highWhen the amount is high, transcription byproducts are increased, and the yield of transcription reaction RNA is further affected; therefore, the concentration of magnesium acetate is in the range of 300-460 mM, the RNA yield is high, and the RNA yield is greatly reduced beyond the range of 300-460 mM, so that the RNA yield is remarkably improved when the concentration of magnesium acetate is in the range of 300-460 mM.
As is clear from Table 3, in the transcription buffer of the present application, the concentration of magnesium acetate is preferably 350 to 460mM, and the RNA obtained by the transcription reaction is 200 to 220. Mu.g/1. Mu.g of template; further preferably, the RNA obtained by the transcription reaction is 216 to 220. Mu.g/1. Mu.g template when the concentration of magnesium acetate is 400 to 460mM; as is clear from comparative examples 3 to 6, the RNA yield reached 180 to 200. Mu.g/1. Mu.g of template at a concentration of 400 to 460mM, and 90. Mu.g/1. Mu.g of template at a concentration of 350mM, and 40. Mu.g/1. Mu.g of template at a concentration of 300mM, whereby it was found that the RNA yield was significantly decreased and the yield was lower under the condition of low magnesium ion concentration by using the transcription buffer containing magnesium chloride.
If the transcription reaction system is replaced by Table 4, samples are sequentially added according to the sequence of Table 4, in-vitro transcription reaction is performed to prepare a kit, and similarly, RNA after in-vitro reaction is purified and then the concentration and yield of RNA are detected.
TABLE 4 RNA in vitro Synthesis System of ammonium salt type cap analogues and sodium salt type NTPs
b) The results of the detection of RNA yield in the ammonium salt type cap analogue and sodium salt type NTPs are shown in Table 5:
TABLE 5
As is clear from Table 5, in the ammonium salt type cap analogue and sodium salt type NTPs system, when the concentration of magnesium acetate used is 350-460 mM, the RNA yield obtained by transcription reaction is 184-200 mug/1 mug template, the concentration of magnesium acetate is below 350mM, and the decrease of RNA yield is obvious; the concentration of the magnesium chloride is 400 to the maximumAt 460mM, the RNA yield is 180-200. Mu.g/1. Mu.g of template, and at magnesium chloride concentrations below 400mM, the RNA yield is greatly reduced, for example, at magnesium chloride concentrations of 350mM, the RNA yield is only 60. Mu.g/1. Mu.g of template, and at magnesium chloride concentrations of 300mM, the RNA yield is 20. Mu.g/1. Mu.g of template; it can be seen that magnesium acetate (MgAc) 2 ) The method has more advantages in ensuring the concentration and the yield of in vitro co-transcription reaction under the condition of lower magnesium ion concentration.
As can be seen from tables 3 and 5, the RNA yield obtained in the Tris-salt cap analogue and NTPs system was higher than that obtained in the ammonium-salt cap analogue and sodium-salt NTPs system, as compared with the magnesium acetate at the same concentration, and the Tris-salt cap analogue and NTPs system were more advantageous for RNA transcription at a low magnesium ion concentration.
(2) RNA integrity
a) The results of the detection of RNA integrity in Tris-salt cap analogues and Tris-salt NTPs are shown in Table 6:
TABLE 6
Sample of MgCl 2 /MgAc 2 Concentration (mM) RNA integrity (%)
Example 1 MgAc 2 460 96.5
Example 2 MgAc 2 400 96
Example 3 MgAc 2 350 95
Example 4 MgAc 2 300 93.5
Comparative example 3 MgCl 2 460 95
Comparative example 4 MgCl 2 400 94
Comparative example 5 MgCl 2 350 93
Comparative example 6 MgCl 2 300 92
b) The results of the detection of RNA integrity in the ammonium salt type cap analogue and sodium salt type NTPs are shown in Table 7:
TABLE 7
Sample of MgCl 2 /MgAc 2 Concentration (mM) Completion of RNAIntegrity (%)
Example 1 MgAc 2 460 95
Example 2 MgAc 2 400 94
Example 3 MgAc 2 350 93.5
Example 4 MgAc 2 300 93
Comparative example 3 MgCl 2 460 94.8
Comparative example 4 MgCl 2 400 93
Comparative example 5 MgCl 2 350 93
Comparative example 6 MgCl 2 300 91
From tables 6 and 7It is known that, in the case of the ammonium salt type cap analogue and the sodium salt type NTPs, or the Tris salt type cap analogue and the Tris salt type NTPs, the magnesium chloride (MgCl) 2 ) For example, magnesium acetate (MgAc) 2 ) It is more advantageous to ensure the integrity of the in vitro co-transcription reaction product at lower magnesium ion concentration. Specifically, under the conditions of Tris salt type cap analogues and Tris salt type NTPs, the RNA integrity after transcription can reach 96.5% when the concentration of magnesium acetate is 460mM, and is maintained to be more than 95% when the concentration of magnesium acetate is 350-460 mM, and 93% when the concentration of magnesium acetate is 300 mM; in the conditions of the ammonium salt type cap analogue and the sodium salt type NTPs, the RNA integrity is up to 95% when the concentration of magnesium acetate is 460mM, and is only 93% when the concentration of magnesium acetate is 300 mM; RNA integrity was as low as 91% at a magnesium chloride concentration of 300 mM.
(3) Production of by-product dsRNA
Referring to FIG. 1, the left side of the vertical line is a schematic region of dsRNA content under the conditions of Tris salt type cap analogue and Tris salt type NTPs, the right side of the vertical line is a schematic region of dsRNA content under the conditions of ammonium salt type cap analogue and sodium salt type NTPs, and as can be seen from the data of the enzyme immunoblotting, the byproduct dsRNA content under the conditions of Tris salt type cap analogue and Tris salt type NTPs is very small, and the byproduct dsRNA content under the conditions of ammonium salt type cap analogue and sodium salt type NTPs is remarkably increased, and compared with the conditions of ammonium salt type cap analogue and sodium salt type NTPs, the byproduct dsRNA can be reduced under the conditions of 10X Transcription Buffer of 460mM magnesium acetate.
(4) Translation Effect of Co-transcript
TABLE 8 relative intensities of fluorescence from cells of each group under conditions of ammonium salt cap analogue and sodium salt NTPs
Group of Relative intensity of cell fluorescence (relative to 460mM MgCl) 2 )
460mM MgCl 2 1
400mM MgCl 2 0.9
350mM MgCl 2 0.3
300mM MgCl 2 0.1
460mM MgAc 2 1
400mM MgAc 2 1
350mM MgAc 2 1
300mM MgAc 2 0.4
TABLE 9 relative intensities of cell fluorescence for each group under Tris-salt cap analogue and Tris-salt NTPs conditions
Group of Relative intensity of cell fluorescence (relative to 460mM MgCl) 2 )
460mM MgCl 2 1
400mM MgCl 2 1
350mM MgCl 2 0.4
300mM MgCl 2 0.2
460mM MgAc 2 1
400mM MgAc 2 1
350mM MgAc 2 0.9
300mM MgAc 2 0.4
After GFP RNA obtained by co-transcription is transfected into cells for 24 hours, by observing fluorescence conditions of GFP in the cells, the data in tables 8 and 9 show that the relative intensity of fluorescence of cells is higher, basically more than 0.9, no matter whether magnesium chloride or magnesium acetate is adopted, the concentration is between 400 and 460mM, and no matter whether Tris salt type cap analogues and Tris salt type NTPs conditions or ammonium salt type cap analogues and sodium salt type NTPs conditions are adopted; at lower magnesium ion concentrations, magnesium acetate (MgAc) 2 ) The fluorescence of RNA, which is the product of the in vitro co-transcription reaction of the system, in cells is stronger, namely the translation effect is better than that of magnesium chloride (MgCl) 2 ) The method comprises the steps of carrying out a first treatment on the surface of the In addition, anotherIn addition, the conditions of Tris salt type cap analogues and Tris salt type NTPs have better in-vitro transcription and translation effects than the conditions of ammonium salt type cap analogues and sodium salt type NTPs.

Claims (10)

1. A transcription buffer capable of increasing the yield of in vitro synthesized RNA, comprising the following components in final concentration:
300-500 mM of Tris-HCl salt;
300-460 mM of magnesium acetate;
spermidine 10-30 mM;
dithiothreitol 50-200 mM.
2. A transcription buffer for increasing the yield of in vitro synthesized RNA according to claim 1, comprising the following final concentrations of each component:
350-450 mM of Tris-HCl salt;
300-460 mM of magnesium acetate;
15-25 mM of spermidine;
80-150 mM dithiothreitol.
3. A transcription buffer for increasing the yield of in vitro synthesized RNA according to claim 1, comprising the following final concentrations of each component:
350-450 mM of Tris-HCl salt;
400-460 mM of magnesium acetate;
15-25 mM of spermidine;
80-150 mM dithiothreitol.
4. A transcription buffer for increasing the yield of in vitro synthesized RNA according to claim 1, comprising the following final concentrations of each component:
350-450 mM of Tris-HCl salt;
300-350 mM of magnesium acetate;
15-25 mM of spermidine;
80-150 mM dithiothreitol.
5. A transcription buffer for increasing the yield of in vitro synthesized RNA according to claim 1, wherein: when the concentration of magnesium acetate is 300-350 mM, the RNA yield can be improved by 120-325% compared with the magnesium chloride with the same concentration.
6. A transcription buffer for increasing the yield of in vitro synthesized RNA according to claim 1, wherein: when the concentration of magnesium acetate is 300-350 mM, compared with the magnesium chloride with the same concentration, the RNA integrity is improved by 0.5-2%.
7. A transcription reaction system, characterized in that: a transcription buffer according to any one of claims 1 to 4, T7 RNA Polymerase Mix, tris-salt-type cap analogues and Tris-salt-type NTPs.
8. The transcription reaction system according to claim 7, wherein: the T7 RNA Polymerase Mix includes T7 RNA Polymerase, a murine RNase inhibitor and inorganic magnesium pyrophosphate.
9. A transcription reaction system according to claim 7, wherein the Tris salt type NTPs comprise the following final concentrations of components:
ATP10mM of Tris salt;
GTP of Tris salt 10mM;
CTP10mM of Tris salt;
tris salt N1-Me-pUTP10mM.
10. A kit, characterized in that: comprising a buffer according to any one of claims 1 to 6 or a transcription reaction system according to any one of claims 7 to 9 and an acceptable adjuvant or carrier.
CN202310205612.3A 2023-07-01 2023-07-01 Transcription buffer solution capable of improving in-vitro synthesized RNA yield and transcription reaction system Pending CN116497019A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117004671A (en) * 2023-09-26 2023-11-07 中国医学科学院医学生物学研究所 In vitro transcription system for reducing byproduct dsRNA yield

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117004671A (en) * 2023-09-26 2023-11-07 中国医学科学院医学生物学研究所 In vitro transcription system for reducing byproduct dsRNA yield

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