CN116286916B

CN116286916B - Method and application of an improved group I intron ribozyme sequence for constructing circular RNA

Info

Publication number: CN116286916B
Application number: CN202310162747.6A
Authority: CN
Inventors: 邱满堂; 李浩然; 刘政; 蔡景升
Original assignee: Peking University Peoples Hospital
Current assignee: Peking University Peoples Hospital
Priority date: 2023-02-24
Filing date: 2023-02-24
Publication date: 2025-08-22
Anticipated expiration: 2043-02-24
Also published as: CN116286916A

Abstract

The invention discloses a method for constructing circular RNA by using an improved I-type intron ribozyme sequence and application thereof. The invention provides a I-type intron ribozyme sequence skeleton fragment, which is obtained by splitting and simulating E1 and E2 sequences of a CVB3_IRES sequence of an internal ribosome entry site of a Coxsackie virus on the basis of an anabaena I-type intron sequence. The invention creatively proposes a mode for synthesizing the circRNA in vitro by solving the problem of introducing redundant sequences by utilizing the I-type intron ribozyme sequence based on anabaena (Anabaena) tRNA for the first time, optimizes a sequence design mode, simplifies the structural components of the sequence on the premise of not influencing cyclization, and reserves a key cyclization sequence.

Description

Method for constructing circular RNA by using improved I-type intron ribozyme sequence and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a method for constructing circular RNA by using an improved I-type intron ribozyme sequence and application thereof.

Background

Type I intronic ribozymes are the earliest discovered RNA catalysts that have a highly conserved secondary structure that can undergo two transesterification reactions in the presence of magnesium ions and exogenous guanylic acid resulting in the self-splicing of introns and ligation of exons. Numerous scholars have used rearranged type I intron ribozyme sequences to synthesize circRNA. Puttaraju M et al demonstrated that circRNA (Nucleic Acids Res,1992,20,5357-5364) could be synthesized using the type I intron ribozyme sequence and the exon sequence from anabaena (Anabaena) tRNA. In 2018 Anderson et al report on Nature Communications that the use of the anabaena (Anabaena) type I intron in vitro transcribed circulations into circular RNA and the efficient expression of the corresponding proteins was achieved, the mode of operation of which is shown in FIG. 1A. However, the sequences reported by Anderson et al have the inherent disadvantage of introducing unwanted redundant sequences (E2, 5 'internal homology sequences, 5' spacer sequences, 3 'internal homology sequences, 3' spacer sequences, E1, etc sequences (also known as "scar" sequences) in FIG. 1A).

Disclosure of Invention

The invention aims to provide an improved anabaena (Anabaena) I type intron sequence which utilizes a split coxsackievirus internal ribosome entry site (CVB3_IRES) sequence to simulate E1 and E2 sequences of the anabaena intron sequence, and the improved I type intron sequence can effectively complete cyclization of a target sequence, has no residue of the anabaena exon sequence and can realize successful expression of the target sequence.

In a first aspect, the present invention provides a type I intron ribozyme sequence backbone fragment, which is obtained by splitting and optimizing a cvb3_ires sequence based on an anabaena type I intron sequence to simulate E1 and E2 sequences, thereby obtaining a type I intron ribozyme sequence backbone fragment having a self-splicing property.

The I-type intron ribozyme sequence skeleton fragment sequentially comprises a5 'homologous arm, a 3' end of an anabaena T4 intron, a CVB3_IRES_1, an exogenous gene, a CVB3_IRES_2, a5 'end of the anabaena T4 intron and a 3' homologous arm;

the nucleotide sequence of CVB3_IRES_1 is 185 th to 934 th of the sequence 1;

the nucleotide sequence of CVB3_IRES_2 is 1655-1697 of the sequence 1.

In the type I intron ribozyme sequence backbone fragments described above,

The nucleotide sequence of the 5' homology arm is the 24 th-53 th position of the sequence 1;

The nucleotide sequence of the 3' -end of the anabaena T4 intron is 54 th to 184 th positions of the sequence 1;

The nucleotide sequence of the exogenous gene replaces the position of 935 th to 1654 th positions of the sequence 1;

the nucleotide sequence of the 5' end of the anabaena T4 intron is 1698 th-1813 rd position of the sequence 1;

the nucleotide sequence of the 3' homology arm is 1814-1848 of the sequence 1.

The type I intron ribozyme sequence backbone fragment described above (designated as a type I intron ribozyme sequence backbone fragment with a transcriptional promoter) also includes a transcriptional promoter located upstream of the 5' homology arm.

In the type I intron ribozyme sequence backbone fragments described above,

The transcription promoter is a T7 promoter;

the nucleotide sequence of the T7 promoter is 1 st-23 rd positions of the sequence 1.

Further, in the embodiment of the invention, the exogenous gene takes EGFP coding gene as an example, and the nucleotide sequence of the I-type intron ribozyme sequence skeleton fragment is the sequence 1.

In embodiments of the invention, the type I intron ribozyme sequence backbone fragments may be synthesized directly.

In a second aspect, the invention provides a plasmid for use in the preparation of circular RNA.

The plasmid provided by the invention is any one of the following:

1) A plasmid comprising a backbone fragment of a type I intron ribozyme sequence with a transcription promoter according to the first aspect, said backbone fragment of a type I intron ribozyme sequence being transcribed using its own transcription promoter;

Specifically entrusting company to synthesize a plasmid containing I-type intron ribozyme sequence skeleton fragment with transcription promoter;

2) In order to insert the I-type intron ribozyme sequence backbone fragment without transcription promoter in the first aspect into the downstream of the promoter in the expression vector, the I-type intron ribozyme sequence backbone fragment is transcribed by using the transcription promoter in the expression vector.

In the examples of the present invention, the plasmid was constructed in the first mode, and the expression vector was specifically pUC57, and the above-mentioned plasmid containing the backbone fragment of the type I intron ribozyme sequence with the transcription promoter of the first aspect was specifically plasmid pUC57-cEGFP.

In a third aspect, the present invention provides a method of preparing a circular RNA comprising the steps of:

1) Linearizing the plasmid of the second aspect to obtain a linearized plasmid;

the restriction recognition site for the enzyme used for linearization is present in the plasmid of the second aspect in a region other than the backbone fragment of the type I intron ribozyme sequence, that is, the restriction recognition site described above is not present on the backbone fragment;

Further, in the embodiment of the present invention, the expression vector is pUC57, and the cleavage recognition site selected in pUC57 in the embodiment is NdeI.

2) Amplifying the I-type intron ribozyme sequence skeleton fragment from the linearization plasmid to obtain an I-type intron ribozyme sequence skeleton fragment amplification product;

the primers used for the amplification described above are as follows:

Forward primer:TGCATCTAGATTAATACGACTCACT

Reverse primer:CTAGATATGCTGTTATCCGTCGATT。

3) In vitro transcribing the amplification product of the I-type intron ribozyme sequence skeleton fragment to obtain a transcription product;

In an embodiment of the invention, the transcription is performed using an RNA synthesis kit (E2040S, NEB, USA).

4) And adding GTP into the transcription product for incubation, and realizing cyclization to obtain the cyclized RNA.

In the examples of the present invention, the final concentration of GTP in the incubation system was 2mM, and the incubation conditions were 55℃for 15min.

In a fourth aspect, the invention provides a kit for preparing a circular RNA comprising:

1) A type I intron ribozyme sequence backbone fragment and an expression vector according to the first aspect, or a plasmid according to the second aspect;

2) An enzyme for linearization in the third aspect;

3) Primers required for amplification in the third aspect;

4) Reagents and/or instrumentation required for in vitro transcription in the third aspect;

5)GTP。

the invention creatively proposes a mode for solving the in-vitro synthesis of circRNA by introducing redundant sequences by utilizing the I-type intron ribozyme sequence based on anabaena (Anabaena) tRNA for the first time, and has the following advantages:

(1) Optimizing a sequence design mode, simplifying the structural components of the sequence on the premise of not influencing the looping, and reserving a key looping sequence;

(2) The IRES original and the coding region for promoting translation are not introduced with redundant sequences, so that rejection reaction of organism innate immunity stimulated by the introduction of exogenous redundant sequences can be reduced theoretically, stable translation of the coding sequence is promoted, and the aim of treatment is fulfilled;

(3) The invention constructs a basic frame of the sequence, selects an IRES original with good translation effect, namely a CVB3_IRES original, and only needs to replace a coding region if a new target point is needed on the basis, so that the operation is simple and easy.

Drawings

FIG. 1 is a schematic diagram of a ring formation strategy of a type I catalytic ribozyme, a schematic diagram of a current common ring formation strategy, and a schematic diagram of a modified ring formation system.

FIG. 2 shows the identification of the loop formation, the RNA gel electrophoresis, the PCR gel electrophoresis, the linker site, the western detection of EGFP expression, wherein IVT represents in vitro transcribed product, circRNA represents purified product, and circRNA+RNase R represents RNase R enzyme treated product.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 preparation of plasmids expressing circular RNAs

1. Construction of I-type intron ribozyme sequence skeleton and cEGFP circularization template plasmid

The present invention employs an improved strategy for constructing cEGFP circularized template plasmids based on the I-type intron ribozyme of anabaena (Anabaena) tRNA.

The improved anabaena (Anabaena) tRNA based type I intron ribozyme is based upon the previously reported sequence, and the invention fully contemplates and redesigns the reported inherent deficiencies of type I intron synthase for self-splicing. The present inventors studied the importance of exon E1 and exon E2 in type I intron synthase self-splicing based on the existing sequences, and accurately split and optimize IRES elements (CVB 3 is used in the present invention) that promote translation to mimic the sequences of E1 and E2 (as shown in FIG. 1B). The present invention will not introduce additional exogenous sequences (i.e., (E2, 5' internal homologous sequence, 5' spacer sequence, 3' internal homologous sequence, 3' spacer sequence, E1, etc. (also referred to as "scar" sequences)) while not altering the nature of the type I intron ribozyme's self-splicing) so that the body's cell's innate immune response to exogenous RNA is reduced.

The I-type intron ribozyme sequence skeleton containing exogenous gene based on anabaena (Anabaena) tRNA sequentially comprises the following elements from 5' to 3', wherein the 5' homology arm, anabaena T4 intron 3' end, CVB3_IRES_1, exogenous gene, CVB3_IRES_2, anabaena T4 intron 5' end and 3' homology arm, and a transcription promoter such as T7 promoter can be connected at the upstream of the 5' homology arm.

When the exogenous gene is an EGFP coding gene, the nucleotide sequence of the I-type intron ribozyme sequence skeleton of the EGFP coding gene is a sequence 1.

Wherein, the 1 st to 23 rd positions of the sequence 1 are T7 promoters, the 24 th to 53 th positions of the sequence 1 are 5 'homology arms, the 54 th to 184 th positions of the sequence 1 are 3' ends of anabaena T4 introns, the 185 th to 934 th positions of the sequence 1 are CVB3_IRES_1, the 935 th to 1654 th positions of the sequence 1 are positions of exogenous genes (EGFP coding genes in the present case), the 1655 th to 1697 th positions of the sequence 1 are CVB3_IRES_2, the 1698 th to 1813 th positions of the sequence 1 are 5 'ends of anabaena T4 introns, and the 1814 th to 1848 th positions of the sequence 1 are 3' homology arms.

The plasmid pUC57-cEGFP containing the I-type intron ribozyme sequence skeleton of EGFP-encoding gene is synthesized by Nanjing Jinsri technology Co., ltd, specifically, the I-type intron ribozyme sequence skeleton of EGFP-encoding gene is cloned into pUC57 expression vector, and the I-type intron ribozyme sequence skeleton fragment is transcribed by using its own transcription promoter.

Then the pUC57-cEGFP plasmid is introduced into escherichia coli to obtain recombinant bacteria, and after culturing, the plasmid of the recombinant bacteria is extracted to obtain cEGFP cyclization template plasmid.

2. Template amplification and purification recovery

1) Linearization

The cEGFP circularized template plasmid was cut with the endonuclease NdeI (which is not present in the type I intron ribozyme sequence backbone of the EGFP-encoding gene, but is located on the pUC57 plasmid) into a linearized plasmid.

2) Amplification of type I intron ribozyme sequence backbone of EGFP encoding Gene

Primers for amplifying the I-type intron ribozyme sequence backbone of EGFP-encoding gene were designed, and KOD-Plus-Neo (KOD-401, TOYOBO, JAPAN) was used for high-fidelity PCR amplification using the linearized plasmid as a template.

Forward primer:TGCATCTAGATTAATACGACTCACT

Reverse primer:CTAGATATGCTGTTATCCGTCGATT

After the PCR reaction, the amplified product was passed through a DNA template purification recovery column (DP 214, TIANGEN, china) for use to obtain purified template DNA (with T7 promoter).

3. In vitro transcription and cyclization

The above purified template DNA was subjected to in vitro transcription using an RNA synthesis kit (E2040S, NEB, USA) with a T7 promoter sequence primer, and the excess template DNA was digested with DNase I and recovered by purification (T2030S, NEB, USA) to give a purified linear RNA precursor (in vitro transcription product IVT, at a concentration of about 2000 ng/ul).

GTP is added into the purified linear RNA precursor, the final concentration of the GTP is 2mM, the GTP is incubated for 15min at 55 ℃, in vitro cyclization is carried out, an incubated RNA product is obtained, and the incubated RNA product is subjected to column purification again, so that a cyclized product (marked as circRNA) is obtained.

4. Identification of the ring formation

According to the characteristic of tolerance of the circRNA to RNase R, the cyclized product obtained in the above 3 is treated with RNase R to remove the linear RNA precursor remaining in the cyclizing reaction, followed by further column-passing purification to obtain an RNase R enzyme-treated product (designated as circRNA+RNase R).

1) Agarose gel electrophoresis

The cyclized product obtained in the above 3, the in vitro transcribed product obtained in the above 3 and the RNase R enzyme-treated product were subjected to agarose gel electrophoresis.

As shown in FIG. 2A, IVT is in vitro transcribed product, circRNA is cyclized product, circRNA+RNase R is RNase R enzyme treated product, linear control is linear RNA obtained by in vitro transcription of DNA molecule consisting of CVB3_IRES sequence and EGFP coding gene sequence (935-1654 positions of sequence 1) (the 2 partial sequences are spliced closely adjacent to each other), and PolyA tail is added;

The CVB3_IRES sequence is an original undivided sequence, and is described in reference ：Wesselhoeft RA,Kowalski PS,Anderson DG.Engineering circular RNA for potent and stable translation in eukaryotic cells.Nat Commun.2018;9(1):2629.Published 2018Jul 6.doi:10.1038/s41467-018-05096-6).

As can be seen from the figure, both the in vitro transcribed product and the product after the RNase R enzyme treatment of the circularized product form loops compared to the linear control, indicating that the RNA forms a distinct loop.

2) Post reverse transcription validation

The in vitro transcription product (IVT) obtained in the above 3 and the RNase R enzyme-treated product (referred to as circRNA+RNase R in the figure) were subjected to reverse transcription in accordance with the following system and procedure to obtain a reverse transcription product.

The reverse transcription system is shown in Table 1 (PRIMESCRIPT ^TM RT Master Mix, takara, RR 036A) below.

TABLE 1

The procedure for reverse transcription was as follows:

37 ℃ for 15min (reverse transcription reaction)

85 ℃ 5Sec (reverse transcriptase inactivation reaction)

4°C

And then taking each reverse transcription product as a template, and carrying out PCR amplification by designing primers of the joint sites to obtain PCR amplification products.

The primers for the above-mentioned linker sites were as follows:

Forward primer:GGATCACTCTCGGCATGGAC

Reverse primer:GCTAGCGCCCAATGGTAAGA

as a result, as shown in FIG. 2B, it can be seen that a distinct single band PCR product was obtained.

The above PCR products were subjected to Sanger sequencing, and the result is shown in FIG. 2C, in which the loop formation site (the linker site indicated by the arrow, where they are connected end to end) was visible.

3) Exogenous gene expression protein

The DNA molecule (2 parts of the sequence are spliced closely adjacent) consisting of CVB3_IRES sequence and EGFP coding gene sequence (935-1654) is subjected to in vitro transcription to obtain linear RNA, and Poly A tail is added), the in vitro transcription product IVT obtained in the step 3, the cyclization product (circRNA) obtained in the step 3 and the product (circRNA+RNase R) obtained after RNase R enzyme treatment are respectively transferred into lung adenocarcinoma cell line H1299 cells, and the cells are collected after 24 hours of transfection. Western and IP lysates (P0013, beyotime, china) were used for ice lysis for 30min, centrifuged at 12000g for 20 min, and the supernatant was collected.

The extracted protein is detected by western analysis.

The results are shown in FIG. 2D, and it can be seen that EGFP is significantly expressed in IVT, circRNA and circRNA+RNase R groups.

Based on the evidence, the invention can creatively solve the problem of introducing exogenous redundant sequences, and can still detect the expression of obvious EGFP proteins.

Claims

1. A group I intron ribozyme sequence backbone fragment, which is obtained by splitting and optimizing the CVB3_IRES sequence based on the group I intron sequence of Anabaena to mimic the E1 and E2 sequences, thereby obtaining a group I intron ribozyme sequence backbone fragment with self-splicing properties;

The type I intron ribozyme sequence skeleton fragment includes: 5' homology arm, 3' end of Anabaena T4 intron, CVB3_IRES_1, foreign gene, CVB3_IRES_2, 5' end of Anabaena T4 intron and 3' homology arm;

The nucleotide sequence of CVB3_IRES_1 is positions 185-934 of sequence 1;

The nucleotide sequence of CVB3_IRES_2 is positions 1655-1697 of sequence 1.

2. The group I intron ribozyme sequence backbone fragment according to claim 1, characterized in that:

The nucleotide sequence of the 5' homology arm is positions 24-53 of SEQ ID NO: 1;

The nucleotide sequence at the 3' end of the Anabaena T4 intron is positions 54-184 of SEQ ID NO: 1;

The nucleotide sequence of the exogenous gene replaces the positions 935-1654 of sequence 1;

The nucleotide sequence at the 5' end of the Anabaena T4 intron is positions 1698-1813 of SEQ ID NO: 1;

The nucleotide sequence of the 3' homology arm is positions 1814-1848 of sequence 1.

3. The group I intron ribozyme sequence backbone fragment according to claim 1 or 2, characterized in that:

The type I intron ribozyme sequence backbone fragment further includes a transcription promoter located upstream of the 5' homology arm.

4. The group I intron ribozyme sequence backbone fragment according to claim 3, wherein the transcription promoter is a T7 promoter;

The T7 promoter is positions 1-23 of sequence 1.

5. A plasmid for preparing circularized RNA, any of the following:

1) A plasmid containing the type I intron ribozyme sequence backbone fragment according to claim 3 or 4; the type I intron ribozyme sequence backbone fragment is transcribed using its own transcription promoter;

2) A plasmid obtained by inserting the type I intron ribozyme sequence backbone fragment according to claim 1 or 2 into the downstream of the promoter in an expression vector; the type I intron ribozyme sequence backbone fragment is transcribed using the transcription promoter in the expression vector.

6. A method for preparing circularized RNA, comprising the following steps:

1) linearizing the plasmid according to claim 5 to obtain a linearized plasmid;

The restriction enzyme cleavage site of the linearization enzyme is present in the region excluding the backbone fragment of the type I intron ribozyme sequence in the plasmid of claim 5;

2) amplifying the group I intron ribozyme sequence backbone fragment from the linearized plasmid to obtain a group I intron ribozyme sequence backbone fragment amplification product;

3) in vitro transcribing the amplified product of the group I intron ribozyme sequence backbone fragment to obtain a transcription product;

4) Adding GTP to the transcription product for incubation to achieve cyclization and obtain circularized RNA.

7. A kit for preparing circularized RNA, comprising:

1) The group I intron ribozyme sequence backbone fragment according to any one of claims 1 to 4 and the expression vector according to claim 5; or the plasmid according to claim 5;

2) The enzyme used for linearization according to claim 6;

3) primers required for amplification according to claim 6;

4) Reagents and/or instruments required for in vitro transcription;

5)GTP.