CN112662659B - Universal mRNA in-vitro cyclization method - Google Patents
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Abstract
The invention discloses a universal mRNA in-vitro cyclization method, which comprises the following steps: preparing purified mRNA; performing monophosphorylation modification on the 5 'end of mRNA, and adding poly (A) to the 3' end; hybridizing the modified mRNA with DNA splint; adding T4DNA ligase into the hybridization reaction system for cyclization; digesting redundant DNA splint in a system, and then verifying a cyclization product; wherein the length of the DNA splint is 60nt, the 5' end of the DNA splint is 30nt of poly (T), and the 3' end of the DNA splint is 30nt of base which can be complementarily paired with the 5' end of the modified mRNA. The invention has the advantages that (1) poly (A) is added at the 3' end of mRNA, so that the sequence design of DNA splint is convenient to align with gaps at two ends of the mRNA, the in-vitro cyclization efficiency of the mRNA is enhanced, and the subsequent translation process is facilitated; (2) the 5' end of the mRNA is subjected to monophosphorylation modification by adopting apyrase to enhance cyclization efficiency; (3) the enzymatic method of T4DNA ligase has no substrate difference and has universality.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a universal mRNA in-vitro cyclization method.
Background
Since two research papers published in Nature by Hansen TB and Memczak S, et al, 2013 suggested that cyclic RNA in mammalian cells has a great regulating effect on cell life activities, cyclic RNA has attracted wide attention of researchers in recent years. According to related researches, the circular RNA can enable a microRNA sponge mechanism to indirectly influence mRNA by competitively combining miRNA, and plays an important role in regulation and control in the occurrence and development processes of various diseases. There are studies that have now demonstrated that some circular RNAs of internal origin, Drosophila and human, can encode proteins, i.e.have the same biological function as mRNA. Furthermore, circular RNAs lack the free ends necessary for exonuclease mediated degradation, making them resistant to several mechanisms of RNA degradation, i.e. longer life span compared to linear mRNA counterparts. Thus, circularization of mRNA can extend the half-life of the mRNA, thereby increasing the overall efficacy of the exogenous mRNA in various applications.
It is now generally accepted that circular RNA formation occurs naturally through a reverse splicing reaction, i.e., the joining of a splice site at the 5 'end of the gene to a splice site 3' of an upstream intron. Thus, the current strategy of mRNA cyclization in vitro is also a nuclease method using a similar strategy to circular RNA formation in vivo, i.e., self-splicing introns. However, the method has high requirements on gene sequences and does not have good universality.
The ligation and circularization of RNA using T4DNA ligase and DNA Splint have been reported in the literature (split ligation of RNA with T4DNA ligase. Christopher J. Kershaw and Raymond T. O 'Keefe. methods Mol biol. 2012; 941: 257-) 269), but the method only performs monophosphoric modification on the 5' end of RNA by using T4 polynucleotide kinase, which is beneficial for the ligation of T4DNA ligase, however, the ends of long-chain RNA such as mRNA are not short and long, and the gaps at the two ends of mRNA are not aligned, and the circularization efficiency of mRNA is low.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and provides an mRNA in vitro cyclization method which has universality and higher mRNA cyclization efficiency by performing monophosphorylation modification on the 5 'end of mRNA, adding poly (A) to the 3' end of the mRNA and then performing cyclization by using DNA splin and T4DNA ligase.
In order to achieve the purpose, the invention adopts the technical scheme that:
a universal mRNA in-vitro cyclization method comprises the following steps:
s1, in-vitro transcription and purification are carried out to obtain mRNA;
s2, performing monophosphorylation modification on the 5 'end of the mRNA obtained in the step S1, and adding poly (A) to the 3' end;
s3, carrying out hybridization reaction on the mRNA processed in the step S2 and the DNA spline to obtain a hybridization product;
s4, adding DNA ligase into a hybridization reaction system, and carrying out cyclization treatment on the hybridization product to obtain a cyclization product;
s5, digesting redundant DNA splin the system, and then verifying a cyclization product;
wherein the DNA splint has a length of 60nt, a 5' end of 30nt of poly (T), and a 3' end of 30nt of base which can be complementarily paired with the 5' end of the modified mRNA.
The principle of the method is as follows: the 5' end of mRNA is firstly modified by monophosphorylation so that DNA ligase catalyzes the mRNA to form phosphodiester bond with hydroxyl at the 3' end to be connected into ring, then poly (A) is added at the 3' end of the mRNA, and the condition that the DNA ligase catalyzes the RNA ring is that the RNA and the DNA are required to be hybridized, so that DNA splint is firstly added to be complementary and matched with the mRNA, and then the cyclization is carried out by using the DNA ligase. Wherein the 5' end of the DNA splint is 30nt poly (T) which can be complementarily paired with the poly (A) at the 3' end of the mRNA, and the 3' end of the DNA splint is 30nt bases which can be complementarily paired with the 5' end of the mRNA, thereby ensuring that the gaps at the two ends of the mRNA are aligned and only one nick is left, which is the key for catalyzing the successful connection of the mRNA by using the DNA splint and DNA ligase, and the poly (A) at the 3' end of the mRNA is also necessary for the subsequent translation process.
Preferably, in step S2, the monophosphorylation modification with apyrase reduces the step of dephosphorylation with alkaline phosphatase first, compared to the prior art in which monophosphorylation modification with T4 polynucleotide kinase is performed, the operation is simpler and faster, and the influence of incomplete dephosphorylation on the subsequent monophosphorylation modification and cyclization process can be avoided.
Preferably, in step S2, poly (A) is added at the 3' end using poly (A) polymerase.
Preferably, in step S3, the hybridization reaction specifically comprises: in a 10. mu.L reaction, 1.6pmol of mRNA and 0.1. mu.L of 100. mu. mol/L DNA splin were added, heated to 80 ℃ and incubated for 3min, and then gradually cooled to 25 ℃.
Preferably, the DNA ligase is T4DNA ligase and the final concentration is 20. mu. mol/L.
Preferably, after the cyclization treatment of step S4, the cyclization product is purified.
Preferably, in step S5, the digestion method is: DNase I was added to the system at a final concentration of 0.2U and incubated at 37 ℃ for 1 h.
Preferably, the verification method in step S5 is: and carrying out reverse transcription on the cyclization product to obtain cDNA, then using the cDNA as a template, designing forward and reverse primers at positions 300bp on two sides of a connection position of the cyclization product, carrying out PCR amplification reaction, carrying out gel electrophoresis on the PCR product, cutting gel and recovering, constructing the recovered product into pUC19 plasmid, and then carrying out sequencing verification.
Compared with the prior art, the invention has the beneficial effects that:
(1) the poly (A) is added at the 3' end of the mRNA, so that the sequence design of the DNA splint is facilitated, the gap alignment of the two ends of the mRNA is ensured, the T4DNA ligase catalyzes the two ends of the mRNA to be connected and form a ring, the in vitro cyclization efficiency of the mRNA is enhanced, and finally the poly (A) in the circular mRNA is also necessary for the subsequent translation process.
(2) Compared with the method adopting T4 polynucleotide kinase, the method adopts apyrase to carry out monophosphorylation modification on the 5' end of mRNA, reduces the step of carrying out dephosphorylation firstly by adopting alkaline phosphatase, has simpler and quicker operation and can avoid the influence on cyclization efficiency caused by incomplete dephosphorylation.
(3) The enzymatic method of T4DNA ligase has no substrate difference, so that the sequence, GC content and secondary structure of RNA are not dependent or critical, and the RNA can be catalyzed into a ring as long as the RNA is subjected to monophosphorylation at the 5 'end and poly (A) tail modification at the 3' end.
Drawings
FIG. 1 is an agarose gel electrophoresis picture of SOX7 mRNA obtained by in vitro transcription and purification in example 1 of the present application.
FIG. 2 is an agarose gel electrophoresis of SOX7 mRNA 3' with poly (A) added thereto in example 1 of the present application.
FIG. 3 is an electrophoretogram of cyclized SOX7 mRNA agarose gel after cyclization is completed in example 1 of the present application.
FIG. 4 is an agarose gel electrophoresis image of PCR of cDNA after reverse transcription in example 1 of the present application.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
S1, in vitro transcription and purification to obtain SOX7 mRNA:
constructing DNA corresponding to mRNA to be cyclized into a pUC19 vector containing a T7 promoter, designing primers, carrying out PCR to obtain a linear DNA template for transcription, and then carrying out in vitro transcription by using the obtained DNA template as a transcription template, wherein the 10 mu L transcription system is as follows:
after completion of transcription, 1. mu.L of 1U/. mu.l DNase I was added to the transcription system and incubated at 37 ℃ for 1 hour to digest the DNA transcription template therein. After completion of digestion, the transcribed RNA was purified using an RNA purification kit available from NEB. The obtained SOX7 mRNA was run on the gel and the results are shown in FIG. 1.
S2, performing monophosphorylation modification on the 5 'end of SOX7 mRNA and adding poly (A) to the 3' end
The mRNA obtained in step S1 was modified by 5' monophosphorylation, 48pmol of mRNA, 4. mu.L of apyrase (Apyase) produced by NEB, 0.5U/. mu.l, and 3. mu.L of 10 × Apyase buffer were added to a reaction system of 30. mu.L, and water was added to 30. mu.L, followed by incubation at 30 ℃ for 2.5 hours. After completion of the reaction, the modified RNA was purified using an RNA purification kit manufactured by NEB.
Then, modification of the 3' end with poly (A) tail was performed by adding 48pmol of mRNA, 4. mu.L of 5U/. mu.l of E.coli poly (A) polymerase, 4. mu.L of 10mM ATP and 4. mu.L of 10 Xpoly (A) polymerase buffer to a 40. mu.L reaction system, adding water to 40. mu.L, and incubating at 37 ℃ for 1 hour. After the reaction was completed, the modified RNA was purified using an RNA purification kit manufactured by NEB, and then run gel detection was performed, the results of which are shown in FIG. 2.
S3, carrying out hybridization reaction on the modified SOX7 mRNA and DNA splint
The modified mRNA obtained in step S2 is annealed for hybridization by incubating with DNA spline. In a 10. mu.L reaction system, 1.6pmol mRNA, 0.1. mu.L 100. mu. mol/L DNA splint and 1. mu.L 10 × annealing buffer were added, heated to 80 ℃ and continuously heated for 3min to open the secondary structure of RNA itself, and then gradually cooled to 25 ℃ to allow complementary pairing of RNA and DNA splint. Wherein the length of the DNA splint is 60nt, the 5' end of the DNA splint is 30nt of poly (T), and the 3' end of the DNA splint is 30nt of base which can be complementarily paired with the 5' end of the modified mRNA.
S4, adding high-concentration T4DNA ligase to carry out cyclization
To the hybridization product system obtained after completion of annealing in step S3, 2. mu.L of 600. mu.M T4DNA Ligase, i.e., T4DNA Ligase was added at a final concentration of 20. mu. mol/L, and 12. mu.L of 2 Xquick Ligase Buffer was further added, followed by incubation at 37 ℃ for 1 hour. After the reaction was completed, the reacted mRNA was purified using an RNA purification kit manufactured by NEB corporation, and then run on a gel for detection, with the results shown in FIG. 3.
S5, eliminating redundant DNA splint in the system, and verifying a cyclization product
Eliminating the redundant DNA splint in the cyclization system after the reaction in the step S4 is completed: the reaction system was 20. mu.L containing 14. mu.L of the circularization system products (including RNA and DNA splint), 2. mu.L of DNase I buffer and 2. mu.L of DEPC H 2 And O. Then incubated at 37 ℃ for 40 minutes. After completion of the reaction, the reacted RNA was purified using an RNA purification kit manufactured by NEB.
Validation of the cyclization product:reverse transcription is carried out on the obtained purified circular SOX7 mRNA, then forward and reverse primers are designed at the positions of 300bp on both sides of the 5 'end and the 3' end of the original SOX7 mRNA at the joint of the circular SOX7 mRNA by taking the obtained cDNA as a template, PCR is carried out on the obtained PCR product, the result is shown in figure 4, most of the product can be observed to be about 600bp, then the nucleic acid at the position of 600bp is recovered by gel cutting, the recovered product is constructed into a pUC19 plasmid and then sequencing is carried out, and the sequencing result shows that the sequence of the mRNA is completely matched with the sequence of the original SOX7 mRNA. Wherein the reverse transcription process is as follows: in a 10. mu.L system, 4. mu.L of RNA, 2. mu.L of 6nt long random primer, 1. mu.L of 10mmol/L dNTP, and 3. mu.L of DEPC H 2 O, immediately placed on ice after incubation for 5 minutes at 65 ℃; after incubation was complete 4. mu.L of 5 × reverse transcription buffer, 2. mu.L of 0.1mol/L DTT, 1. mu.L of 6U MMLV5 reverse transcriptase, 0.2. mu.L of RNase inhibitor and 2.8. mu.L of DEPC H were added 2 O, and then incubated at 55 ℃ for 1 hour.
Comparative example 1
This comparative example differs from example 1 in that: the step of adding poly (A) tail to the 3' end of the mRNA is omitted in step S2.
Comparative example 2
This comparative example differs from example 1 in that: the 5 'end of the mRNA is modified by monophosphorylation using T4 polynucleotide kinase instead of apyrase in step S2 by dephosphorylating the mRNA with calf intestinal alkaline phosphatase and then adding monophosphates to the 5' end of the mRNA using T4 polynucleotide kinase.
Application example
The cyclization efficiencies of example 1, comparative example 1 and comparative example 2 were compared: the cyclic SOX7 mRNA obtained in example 1, comparative example 1 and comparative example 2 was sampled in equal amounts and subjected to gel running test, and the band intensities thereof were measured by Image J software, respectively, and the band intensities measured in example 1 were taken as a reference standard value, and the band intensities measured in comparative example 1 and comparative example 2 were divided by the reference standard value to obtain the relative cyclization efficiencies of comparative example 1 and comparative example 2, respectively, the results of which are shown in table 1.
TABLE 1 relative cyclization efficiency
| Example 1 | Comparative example 1 | Comparative example 2 | |
| Relative cyclization efficiency | 100％ | 70.4％ | 75.3％ |
From the results in table 1, it can be seen that the absence of modification with poly (a) tail at the 3 'end of mRNA may result in the situation of end misalignment of mRNA, resulting in a significant decrease in mRNA cyclization efficiency, while the use of calf intestinal alkaline phosphatase and T4 polynucleotide kinase to monophosphate-modify the 5' end of mRNA may result in incomplete monophosphate modification and thus a significant decrease in mRNA cyclization efficiency when compared to the one-step method using apyrase in the present application. Therefore, the mRNA in-vitro cyclization method claimed by the application has higher cyclization efficiency.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A universal in vitro mRNA cyclization method, comprising the steps of:
s1, preparing purified mRNA;
s2, performing monophosphorylation modification on the 5 'end of the mRNA obtained in the step S1, and adding poly (A) to the 3' end;
s3, carrying out hybridization reaction on the mRNA processed in the step S2 and the DNA spline to obtain a hybridization product;
s4, adding DNA ligase into a hybridization reaction system, and carrying out cyclization treatment on the hybridization product to obtain a cyclization product;
s5, redundant DNA splint in a digestion system is digested, and then a cyclization product is verified;
wherein the DNA splint has a length of 60nt, a 5' end of 30nt of poly (T), and a 3' end of 30nt of base which can be complementarily paired with the 5' end of the modified mRNA.
2. The universal method for in vitro cyclization of mRNA of claim 1, wherein in step S2, apyrase is used for said monophosphorylation modification.
3. The method of claim 1, wherein step S2 comprises adding poly (A) to the 3' end with poly (A) polymerase.
4. The universal method for in vitro circularization of mRNA of claim 1, wherein in step S3, the hybridization reaction comprises the following steps: to a 10. mu.L reaction system, 1.6pmol of the mRNA treated in step S2 and 0.1. mu.L of 100. mu. mol/L DNA splin were added, and the mixture was heated to 80 ℃ and incubated for 3min, and then gradually cooled to 25 ℃.
5. The universal method for in vitro circularization of mRNA of claim 1, wherein in step S4, said DNA ligase is T4DNA ligase, and the final concentration is 20 μmol/L.
6. The universal in vitro circularization method of mRNA of claim 1, wherein said circularization product is purified after circularization in step S4.
7. The universal method for in vitro circularization of mRNA of claim 1, wherein in step S5, said digestion method is: DNase I was added to the system at a final concentration of 0.2U and incubated at 37 ℃ for 1 h.
8. The universal method for circularizing mRNA in vitro according to claim 1, wherein in step S5, the validation method is: and carrying out reverse transcription on the cyclization product to obtain cDNA, then using the cDNA as a template, designing forward and reverse primers at positions 300bp on two sides of a connection position of the cyclization product, carrying out PCR amplification reaction, carrying out gel electrophoresis on the PCR product, cutting and recovering gel, constructing the recovered product into pUC19 plasmid, and then carrying out sequencing verification.
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