CN116836973A - Preparation method of oligo RNA with 3-phosphate at 5' -end - Google Patents
Preparation method of oligo RNA with 3-phosphate at 5' -end Download PDFInfo
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- CN116836973A CN116836973A CN202310734755.3A CN202310734755A CN116836973A CN 116836973 A CN116836973 A CN 116836973A CN 202310734755 A CN202310734755 A CN 202310734755A CN 116836973 A CN116836973 A CN 116836973A
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- 229910019142 PO4 Inorganic materials 0.000 title claims abstract description 20
- 239000010452 phosphate Substances 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 21
- 108020004414 DNA Proteins 0.000 claims abstract description 15
- 239000012634 fragment Substances 0.000 claims abstract description 11
- 238000013518 transcription Methods 0.000 claims abstract description 11
- 230000035897 transcription Effects 0.000 claims abstract description 11
- 238000000338 in vitro Methods 0.000 claims abstract description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 5
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000003786 synthesis reaction Methods 0.000 claims description 10
- 238000000746 purification Methods 0.000 claims description 9
- 230000015572 biosynthetic process Effects 0.000 claims description 8
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 7
- 239000011324 bead Substances 0.000 claims description 4
- 239000003161 ribonuclease inhibitor Substances 0.000 claims description 4
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims description 3
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims description 3
- 102000009617 Inorganic Pyrophosphatase Human genes 0.000 claims description 3
- 108010009595 Inorganic Pyrophosphatase Proteins 0.000 claims description 3
- 101710137500 T7 RNA polymerase Proteins 0.000 claims description 3
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 claims description 3
- 239000000047 product Substances 0.000 description 17
- 238000004949 mass spectrometry Methods 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 241000282326 Felis catus Species 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 4
- 239000002777 nucleoside Substances 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000011068 loading method Methods 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- -1 phosphite triester Chemical class 0.000 description 3
- 125000006239 protecting group Chemical group 0.000 description 3
- 102000016911 Deoxyribonucleases Human genes 0.000 description 2
- 108010053770 Deoxyribonucleases Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 125000003835 nucleoside group Chemical group 0.000 description 2
- 238000005191 phase separation Methods 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 125000002652 ribonucleotide group Chemical group 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
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- 239000007864 aqueous solution Substances 0.000 description 1
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- 238000004587 chromatography analysis Methods 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
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- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
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- 238000000855 fermentation Methods 0.000 description 1
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- 239000012467 final product Substances 0.000 description 1
- 238000002509 fluorescent in situ hybridization Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000002500 ions Chemical group 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
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- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- AVBGNFCMKJOFIN-UHFFFAOYSA-N triethylammonium acetate Chemical compound CC(O)=O.CCN(CC)CC AVBGNFCMKJOFIN-UHFFFAOYSA-N 0.000 description 1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The application relates to the field of biotechnology, and particularly discloses a preparation method of oligo RNA with 3-phosphate at the 5' end, which comprises the steps of synthesizing an oligo DNA double-stranded fragment with a T7 promoter by adopting beta-acetonitrile phosphoramidite; and then, using the oligo DNA double-stranded fragment with the T7 promoter as a template, and performing in vitro transcription to synthesize oligo RNA. The application initiates the method for obtaining the oligo RNA with 3-phosphate at the 5' end, which has the advantages of rapid and simple operation, high purity and no special requirements on RNA sequence, length and the like.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a preparation method of oligo RNA with 3-phosphate at the 5' -end.
Background
The oligo RNA is short-fragment RNA designed artificially, usually consists of tens to tens of ribonucleotides, is often used as a primer or a probe, is applied to the technologies of sequencing, DNA microarrays, FISH and the like, and plays an important role in the fields of biological research and biological medicine.
In vitro synthesis of oligo RNA usually employs a phosphotriester method or a phosphite triester method, and the basic principle is that the 3' -terminal nucleoside of the oligonucleotide strand to be synthesized is immobilized on an insoluble polymer, then gradually lengthening from the terminal nucleoside, each lengthening is subjected to a cycle, finally the oligonucleotide strand is cleaved from the solid support and deprotected, and the desired product is obtained by separation and purification. Since nucleotides are multifunctional compounds, in the chemical synthesis of RNA, the groups which do not want to react must be protected, so that the 5' -position of ribonucleotides is always connected with a protecting group instead of a 3-phosphate group, thus leading to that the 5' -end of the final product does not contain the 3-phosphate group after the protecting group is removed, that is, the oligo RNA with 3-phosphate at the 5' -end cannot be synthesized by adopting a chemical synthesis mode in the prior art. Meanwhile, the traditional IVT in-vitro transcription laboratory takes supercoiled plasmid as a template after enzyme digestion linearization, and transcribes to obtain large-fragment RNA, and the template requires steps of plasmid fermentation preparation, linearization and the like, and has complicated process and high cost.
Disclosure of Invention
In order to synthesize oligo RNA with high efficiency, the application provides a preparation method of oligo with 3-phosphate at the 5' -end.
The application provides a preparation method of oligo RNA with 3-phosphate at the 5' end, which adopts the following technical scheme:
s1, synthesizing an oligo DNA double-chain fragment with a T7 promoter by adopting beta-acetonitrile phosphoramidite;
s2, using the oligo DNA double-chain fragment with the T7 promoter as a template, and performing in vitro transcription to synthesize oligo RNA.
According to the technical scheme, the template for synthesizing the short sequence is designed for IVT transcription experiments, so that the oligo RNA with the 3-phosphate at the 5' -end is directly transcribed, a large amount of raw material cost can be saved, and the process is reduced, and the method is simple, convenient and efficient.
Preferably, the oligo DNA double-stranded fragment sequence is a sequence shown as SEQ ID NO. 2 or a sequence having 80%, 90%, 95% or 98% homology with the sequence shown as SEQ ID NO. 2.
SEQ ID NO:2
ATCGAAATTAATACGACTCACTATAAGGTCTTCTGGTCCCCACAGACTCA
Preferably, the oligo RNA sequence is the sequence shown as SEQ ID NO. 1 or a sequence having 80%, 90%, 95% or 98% homology with the sequence shown as SEQ ID NO. 1.
SEQ ID NO:1
ppp-UCCAGAAGACCAGGGGUGUCUGAGU
Preferably, in step S2, T7 RNA polymerase, inorganic pyrophosphatase, RNase inhibitor and NTP are added for in vitro transcription to synthesize oligo RNA.
Preferably, DNase I is added to digest the template DNA and RNA beads are used for recovery.
Preferably, the step S2 is performed after the synthesis of the oligo RNA, and the product is purified to isolate a single length of oligo RNA product.
Preferably, the product is purified by HPLC.
Further preferably, the chromatography column for purification uses DANPacTMRP,4.6 x 50mm,4m.
Further preferred, the purification of mobile phase a:100mM TEAA in Water; mobile phase B:100mM TEAA-75% water/15% methanol/10% ethanol (v/v/v).
Further preferably, the performing purification column temperature: 80 ℃; flow rate: 0.4mL/min.
Preferably, the homogeneity and purity of the product is verified after purification.
Preferably, the uniformity and purity of the product is verified by HPLC-MS.
Further preferably, the HPLC employs ACQUITYOligonucleaotide BEH C18Colume 2.1*100mm,1.7μm。
Further preferred, the HPLC mobile phase a:8mM TEA-200mM HFIP-Water; mobile phase B: methanol.
Further preferably, the HPLC column temperature: 50 ℃; flow rate: 0.3mL/min.
In summary, the application has the following beneficial effects:
the application initiates a method for obtaining oligo RNA with 3-phosphate at the 5' end, which is a novel method for synthesizing oligo RNA, and has the advantages of rapid and simple operation, low cost, high yield, high purity, no special requirements on RNA sequence, length and the like, and wide application range.
Drawings
FIG. 1 is a diagram showing the liquid phase separation of oligo RNA;
FIG. 2 is a mass spectrum identification result of the preparation of purified products;
FIG. 3 is a mass spectrometry deconvolution profile for component 1;
FIG. 4 is a deconvolution profile of component 2 mass spectrometry identification;
FIG. 5 is a deconvolution profile of component 3 mass spectrometry identification;
FIG. 6 is a deconvolution profile of component 4 mass spectrometry identification.
Detailed Description
All the raw material components of the following examples and comparative examples are commercially available.
T7 RNA polymerase, yeasen (Shanghai next St. Biotechnology Co., ltd.), cat: 10626-A;10*Transcription buffer,Yeasen, cargo number: 10626-B;
RNase inhibitor, yeasen, cat: 10603ES05;
inorganic pyrophosphatase, yeasen, cat: 10658ES60;
4 NTPs (ATP, synthgene (Jiangsu Shenji Biotechnology Co., ltd.), product number: ATP001, GTP, synthgene, product number: GTP001, CTP, synthgene, product number: CTP001; N-me-pUTP, synthgene, product number: NMPUTP 001);
DNase i (DNase i), yeasen, cat: 10611ES84;
DEPC water, source leaf organism, cat No.: s30710-500ml;
RNA magnetic beads, yeasen, cat: 12602ES56.
(one) Synthesis of oligo DNA double-stranded fragment with T7 promoter
Oligo DNA was chemically synthesized using β -acetonitrile phosphoramidite, and the synthesis proceeds from the 3 '. Fwdarw.5 ' direction, usually with the first base at the 3' end bound to Glass support (Controlled Pore Glass, CPG). The detailed process of synthesis is as follows:
step1: removing the protecting group (DMTR) attached to the first base 5' -OH group on the CPG support to prepare a new base;
step2: activating a new base monomer (phosphoamidite) in preparation for reaction with the first base;
step3: the second base and the first base are subjected to coupling reaction;
step4: capping (Capping) the 5' -OH of the first base that is not reacted so that it does not participate any further in the reaction; step5: the nucleoside phosphites are oxidized to more stable nucleoside phosphates (i.e., trivalent phosphorus is oxidized to pentavalent phosphorus).
6. The cycle of Step1 to Step5 was repeated until the synthesis of the desired Oligo DNA sequence was completed.
7. After synthesis, oligo DNA molecules were excised from CPG and further purified.
TABLE 1 template sequences
And (II) performing in vitro transcription reaction by using the synthesized DNA double strand as a template.
An IVT reaction system was prepared as shown in the following table, and DEPC water, 10*Transcription buffer and 4 NTPs were added to a 200. Mu.l EP tube, and after vortexing and mixing, two enzymes and RNase inhibitor were added, and finally DNA template was added and mixed.
TABLE 2 in vitro transcription conditions
After the completion of the reaction, 10. Mu.l of DNase I was added to the system and mixed by vortexing, followed by a reaction at 37℃for 15 minutes in the PCR apparatus, and the mixture was recovered by RNA magnetic beads.
And (III) purifying the obtained oligo RNA by HPLC.
And (3) carrying out loading balance on a blank sample by using the set method parameters, starting loading and separating an uncap oligo crude product after the system is stable, and carrying out single collection on each chromatographic peak signal in the range of 10-20min in the chromatogram. And (3) sending the collected component solutions to LC-MS for qualitative analysis, and confirming the structure of the target uncap oligo and the corresponding components thereof. And after the structure of the target uncap oligo and the corresponding components are confirmed, continuously sampling and collecting the target fraction with the corresponding peak-exiting sequence. The fractions collected in the process are temporarily stored at the temperature of 2-8 ℃, and the fractions collected every day are stored at the temperature of-20 ℃ so as to avoid repeated freezing and thawing of the Oligo sample as much as possible.
TABLE 3 purification conditions
(IV) detection means: detection of uniformity and molecular weight of products by HPLC-MS after dilution of the products collected by HPLC purification with 10mM EDTA aqueous solution, sample injection analysis was performed according to the method parameters set forth below
Hplc method:
TABLE 4HPLC conditions
Ms conditions:
table 5 MS conditions
3. Molecular weight calculations were performed by BioPharma Finder 5.0 software.
Subjecting the in vitro transcribed oligo RNA product to liquid chromatography to obtain four interesting components (in the oligo RNA liquid phase separation spectrum of FIG. 1) with retention time of 10-20min, collecting the four components respectively as shown in FIG. 1, and identifying the results as shown in the following table,
TABLE 6 component 1 Mass Spectrometry characterization results
TABLE 7 component 2 Mass Spectrometry characterization results
TABLE 8 component 3 Mass Spectrometry characterization results
TABLE 9 component 4 Mass Spectrometry characterization results
Further analyzing the structure of the mass spectrum sample loading result of the component 2, calculating the molecular weight of the target product and the corresponding sum of the target product and the sum of the molecular weight, matching the target product with the molecular weight actually detected by the mass spectrum, determining that the reliability is high within 10ppm, and determining that the component with the mass number of 8214.060 isppp-UCCAGAAGACCAGGGGUGUCUGAGU corresponds to molecular weight, and at the same time has detected Na addition of the target product + 、K + 、Fe + And adding the ion signals. 8214.060 has a molecular weight of the non-additive component of the target sequence and a plurality of Na based on the molecular weight and having a matching error of less than 10ppm + 、K + 、Fe + The signal is detected, the matching result is fully proved to be accurate and reliable, and the reliability of the identification result is further confirmed.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Claims (9)
1. A preparation method of oligo RNA with 3-phosphate at the 5' -end is characterized by comprising the following steps:
s1, synthesizing an oligo DNA double-chain fragment with a T7 promoter by adopting beta-acetonitrile phosphoramidite;
s2, using the oligo DNA double-chain fragment with the T7 promoter as a template, and performing in vitro transcription to synthesize oligo RNA.
2. The method for preparing oligo RNA with 3-phosphate at the 5' -end according to claim 1, wherein the oligo DNA double-stranded fragment sequence is the sequence shown in SEQ ID NO. 2 or a sequence having 80%, 90%, 95% or 98% homology with the sequence shown in SEQ ID NO. 2.
3. The method for preparing oligo RNA with 3-phosphate at the 5' -end according to claim 1, wherein the oligo RNA sequence is the sequence shown in SEQ ID NO. 1 or a sequence having 80%, 90%, 95% or 98% homology with the sequence shown in SEQ ID NO. 1.
4. The method for preparing oligo RNA with 3-phosphate at the 5' -end according to claim 1, wherein T7 RNA polymerase, inorganic pyrophosphatase, RNase inhibitor and NTP are added in the step S2, and the oligo RNA is synthesized by in vitro transcription.
5. The method for preparing oligo RNA with 3-phosphate at the 5' -end according to claim 4, wherein DNase I is added to digest the template DNA and the DNA is recovered by using RNA magnetic beads.
6. The method for preparing oligo RNA with 3-phosphate at the 5' -end according to claim 1, wherein the step S2 is performed with purification of the product after the synthesis of oligo RNA, and a single length of oligo RNA product is isolated.
7. The method for preparing oligo RNA with 3-phosphate at the 5' -end according to claim 6, wherein the product is purified by HPLC.
8. The method for preparing oligo RNA with 3-phosphate at the 5' -end according to claim 7, wherein the uniformity and purity of the product are verified after purification.
9. The method for preparing oligo RNA with 3-phosphate at the 5' -end according to claim 8, wherein the uniformity and purity of the product are verified by HPLC-MS.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170240898A1 (en) * | 2014-09-01 | 2017-08-24 | Jiangsu Micromedmark Biotech Co., Ltd. | Method for inhibiting ebola virus via mirna |
| CN107805636A (en) * | 2017-10-16 | 2018-03-16 | 山东大学 | A kind of difunctional 5 ' tri phosphate siGPC 3 and preparation method and application |
| KR20220100392A (en) * | 2021-01-08 | 2022-07-15 | 한국생명공학연구원 | Method of producing 5'-triphosphate oligoadenylate |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170240898A1 (en) * | 2014-09-01 | 2017-08-24 | Jiangsu Micromedmark Biotech Co., Ltd. | Method for inhibiting ebola virus via mirna |
| CN107805636A (en) * | 2017-10-16 | 2018-03-16 | 山东大学 | A kind of difunctional 5 ' tri phosphate siGPC 3 and preparation method and application |
| KR20220100392A (en) * | 2021-01-08 | 2022-07-15 | 한국생명공학연구원 | Method of producing 5'-triphosphate oligoadenylate |
Non-Patent Citations (1)
| Title |
|---|
| BARONTI, L.,: "A guide to large-scale RNA sample preparation", 《 ANALYTICAL AND BIOANALYTICAL CHEMISTRY》, vol. 410, no. 14, pages 3239 - 3252, XP036509412, DOI: 10.1007/s00216-018-0943-8 * |
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