CN114317677A - Method for generating alternative splicing molecular tool for RNA through in vitro screening - Google Patents
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Abstract
The invention discloses a method for generating an alternative splicing molecular tool for RNA through in vitro screening. Belongs to the field of biological medicine, and comprises the following steps: performing alternative splicing on the precursor mRNA by using deoxyribozyme to generate two fragments to be connected; connecting the fragments by using the deoxyribozyme with RNA connecting activity to form mature RNA, and verifying the feasibility of in vitro splicing of the deoxyribozyme; cleaving an intron of a precursor messenger RNA of interest using a dnazyme having RNA cleavage activity to generate two RNA fragments to be ligated; obtaining threose nuclease with RNA ligase activity by a technical means of in vitro screening; using threose nuclease to connect two exons to produce a functional messenger RNA; threose nuclease has the advantages of good stability, difficult degradation, safety, low toxicity and the like compared with natural nuclease. The invention effectively realizes the splicing of RNA and provides a molecular tool for treating diseases caused by RNA splicing errors.
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to a method for generating an alternative splicing molecular tool for RNA through in vitro screening.
Background
RNA splicing (RNA splicing) is an important process for eukaryotic gene expression, and refers to the process of removing introns from the transcribed precursor messenger RNA and joining exons to form a continuous RNA. In vivo RNA splicing is realized by a spliceosome mechanism, and the error of the splicing mechanism can cause diseases, such as galactosialiosis, because the A of the 7 th intron of cathepsin A is mutated into G, so that the 7 th exon skipping occurs in the splicing process, and active protein cannot be generated. With the development of non-native nucleic acids, it is desirable to utilize non-native nucleases to achieve the correct splicing process of RNA in vitro, providing a molecular tool for the treatment of diseases caused by mis-splicing of RNA.
Disclosure of Invention
The purpose of the invention is as follows: the object of the present invention is to provide a method for generating a means for alternatively splicing molecules of RNA by in vitro screening.
The technical scheme is as follows: the invention relates to a method for generating an alternative splicing molecular tool for RNA by in vitro screening, which comprises the following steps:
(1) performing alternative splicing on precursor mRNA by using the existing deoxyribozyme with RNA cleaving enzyme activity and RNA ligase activity to verify the feasibility of the experiment;
(2) screening TNA enzyme (threose nuclease) with RNA ligase activity by an in vitro screening technical means;
(3) firstly, incubating the precursor mRNA of the prepared target gene with deoxyribozyme to generate two exon fragments;
and then, incubating with the TNA enzyme obtained by screening to generate a connection reaction, generating mature RNA, and detecting by using denaturing polyacrylamide gel electrophoresis.
Further, in step (1), the alternative splicing method for the precursor mRNA specifically comprises: the precursor mRNA is cleaved by DNAzyme having RNA cleavage activity, the intron is excised, two exon fragments having 2 ', 3 ' -cyclic phosphate termini and 5 ' -hydroxyl termini are obtained, and the two exon fragments are ligated by DNAzyme having RNA ligation activity to form a mature RNA.
Further, in the step (2), the technical means of in vitro screening comprises the following steps: in one containing about 1016Isolating and enriching TNA molecules with connecting activity in the TNA library by applying screening pressure;
the construction method of the TNA library specifically comprises the following steps: firstly, a DNA library is synthesized in a solid phase, and a TNA library is obtained by an extension reaction by utilizing KOD-RI polymerase;
the expression method of the KOD-RI polymerase specifically comprises the following steps: constructing a PQE-80-KOD-RI recombinant plasmid, transforming the recombinant plasmid into escherichia coli BL21, performing induced expression by IPTG, then breaking cells, extracting and purifying polymerase KOD-RI by column chromatography;
the separation method specifically comprises the following steps: modifying biotin at the 5' end of the left binding arm of the RNA so that the sequence with the biotin label binds to streptavidin magnetic beads, and then washing out the complementary pairing with 100mM NaOH, but not the active TNA sequence attached; the RNA that has completed ligation is then disrupted with 200mM NaOH at 37 ℃ leaving the TNA molecule with ligation activity;
the enrichment method specifically comprises the following steps: carrying out reverse transcription reaction on the TNA library with activity obtained by separation by Bst 2.0 polymerase to obtain a complementary DNA library, and then enriching the DNA library by PCR technology index;
specifically, the method comprises the following steps: firstly, synthesizing an initial DNA molecule random library and a primer, and then screening; in each round of screening, firstly, preparing a left RNA binding arm which is labeled with biotin at the 5' end and is provided with a cyclic phosphate tail end by adopting an enzyme digestion method; then using DNA primer with right RNA binding arm to obtain non-natural nucleic acid library by primer extension method, and constructing non-natural nucleic acid library containing left binding arm and right binding arm and Mg2+A reaction system of a reaction buffer solution with the concentration of 40 mM; after 6-20 h of incubation, the reaction system is added into magnetic beads modified with streptavidin, and the magnetic beads are hung on the magnetic beads through strong affinity of the streptavidin and the biotinOn the magnetic beads, sequentially treating the magnetic beads with 100mM NaOH solution and 200mM NaOH solution, and then amplifying the magnetic beads by reverse transcription and PCR technology to obtain an enriched DNA library for the next round of screening; repeatedly screening for multiple rounds, sequencing the enriched DNA molecules, deducing sequence information of the non-natural nucleic acid molecules according to a sequencing result, then preparing single-chain non-natural nucleic acid molecules, adding a left binding arm with a cyclic phosphate terminal and a right binding arm with a hydroxyl terminal into a reaction system, and obtaining TNA molecules which can catalyze RNA connection, namely TNA ligase;
the method for screening the non-natural nuclease in vitro specifically comprises the following steps:
(1) preparing a left RNA binding arm which is labeled with biotin at the 5' end and has a cyclic phosphate end through the enzyme digestion reaction of deoxyribozyme 8-17;
(2) solid-phase synthesis of an initial DNA random library and upstream and downstream primers;
(3) taking a DNA random library, using polymerase KOD-RI and tA, tT, tC and tG as substrates, performing primer extension by using a primer with a right binding arm, and then performing strand separation to obtain a non-natural nucleic acid library with the right binding arm;
(3) construction of a library of non-natural nucleic acids comprising a left binding arm with a right binding arm and 40mM Mg2+The reaction buffer of (1);
(4) combining the nucleic acid mixture with biotin and streptavidin, washing off TNA molecules which are complementary to each other and have no RNA connection activity by using a 100mM NaOH solution, and then destroying the RNA molecules subjected to the connection reaction by using a 200mM NaOH solution to obtain TNA molecules with catalytic RNA connection activity;
(5) carrying out reverse transcription reaction on the separated nucleic acid solution by Bst 2.0 polymerase;
(6) taking the reverse transcription product as a template, carrying out PCR amplification for 10-18 cycles, and separating single chains to obtain an enrichment library obtained by the screening in the current round, wherein the enrichment library can be used for the next screening round;
(7) repeating the steps (2) to (6);
(8) after 5-8 rounds of screening, connecting PCR amplification products obtained in the last round of screening to a carrier, and sequencing colonies containing successfully connected carriers;
(9) deducing sequence information of the TNA molecule according to a sequencing result, and preparing a single-chain TNA molecule;
(10) adding a left binding arm with a cyclic phosphate end and a right binding arm with a hydroxyl end into a reaction system, and testing the activity of the TNA molecules in catalyzing RNA connection;
(11) and performing secondary structure prediction and truncation optimization on the TNA ligase with higher catalytic efficiency to obtain the TNA ligase.
Further, in the step (3), the incubation manner with the TNA enzyme obtained by screening is specifically: two exons with 2 ', 3 ' -cyclic phosphate end and 5 ' hydroxyl end are subjected to ligation reaction under the action of TNA enzyme; specifically, in the splicing experiment of target gene precursor messenger RNA, precursor messenger RNA is firstly obtained through transcription reaction, deoxyribozyme 8-17 with RNA cleavage enzyme activity is added into the obtained RNA for incubation, two exon fragments are recovered through gel recovery, TNA molecules with RNA ligase activity obtained through screening are added, and the connection effect is detected through modified polyacrylamide gel electrophoresis.
Has the advantages that: compared with the prior art, the invention has the characteristics that: the invention screens threose nuclease which can catalyze RNA ligation reaction, and is combined with deoxyribozyme which catalyzes RNA cutting reaction to realize RNA splicing, thereby providing a molecular tool for treating diseases caused by RNA splicing errors; obtaining threose nuclease with RNA ligase activity by a technical means of in vitro screening; the two exons are ligated using threose nuclease to create a functional messenger RNA. Threose nuclease has the advantages of good stability, difficult degradation, safety, low toxicity and the like compared with natural nuclease. The method can effectively realize the splicing of RNA and provides a molecular tool for treating diseases caused by RNA splicing errors.
Drawings
FIG. 1 is a flow chart of the operation of the present invention;
FIG. 2 is a schematic diagram showing the splicing of a precursor RNA by a deoxyribozyme in the present invention;
FIG. 3 is a schematic diagram of a non-natural nuclease screen used in the present invention;
FIG. 4 is a schematic chemical formula of a non-natural ribonucleotide TNA monomer used in the present invention;
FIG. 5 is a 7 th intron cleavage pattern diagram for cathepsin A in the present invention;
FIG. 6 is a schematic diagram showing the ligation pattern of exons 6 and 7 of cathepsin A according to the present invention.
Detailed Description
The present invention will be further described with reference to the following examples.
Example splicing of exons 6 to 7 of the precursor messenger RNA of cathepsin a:
1. the synthesis of precursor RNA sequence, binding arm different two 8-17 cutting enzyme and 7CX10 ligase RNA precursor nucleic acid sequence, as follows:
5’-GGAGCAGCAUCAGACAGCUAGGAAGAGAAGGAGAUAUAUAGGCAUAGGAAGAGAUCGCGACGG-3’；
the 8-17 cleavage enzyme 1 sequence is shown below:
5’-TTCTCTTCTGTCAGCGACTCGAAAGCTGTCTG-3’；
the 8-17 cleavage enzyme 2 sequence is shown below:
5’-ATCTCTTCTGTCAGCGACTCGAAATGCCTATA-3’；
the 7CX10 ligase sequence is shown below:
5’-CCGTCGCGATCTCTTCGTGAGAGCAATACGGAAGTTTCGCGCCTAGCTGTCTGATGCTGC-3’；
the cleavage reaction system contained 100nM of the RNA substrate, 1uM of 8-17 deoxyribozyme, annealed in a buffer system containing 70mM Tris, 150mM NaCl, 2mM KCl, pH 7.5 (annealing conditions: 95 ℃ for 2 minutes at room temperature for 15 minutes) and then ZnCl was added2Storage of the solution to Zn2+Reaction at a concentration of 1mM for half an hour at a temperature of 23 ℃ and verified by electrophoresis on a 20% denaturing polyacrylamide gel;
the ligation reaction system contained 100nM of the RNA substrate with 2 ', 3 ' cyclic phosphate on the left, the RNA substrate with 5 ' hydroxyl on the right, in the presence of 1mM ZnCl2In a reaction buffer of pH 7.5 at a temperature of 23 deg.CThe reaction was continued for 3 hours and verified by electrophoresis on a 20% denaturing polyacrylic gel.
2. Synthesis of initial random libraries of DNA and primers:
random library nucleic acid sequences of DNA molecules, as shown below:
5’-biotin-ATGTCGCTTACTCGTGAACA-N40-CTTTGATAACGACTGTGACACTGATTCGAGTAG-3’；
n40 represents a random nucleic acid sequence, with the four deoxyribonucleotide monomer ratios shown below:
A:T:C:G＝2:2:2:1；
the primer sequences were extended as shown below (underlined RNA):
5’-GCUUUGAUCGAATTTTTTTTTTTTspacer18CTACTCGAATCAGTGTCACAGT
the upstream primer nucleic acid sequence is shown as follows:
5’-biotin-ATGTCGCTTACTCGTGAACA-3’；
the downstream primer nucleic acid sequence is shown as follows:
5’-CTACTCGAATCAGTGTCACAGT-3’。
3. procedure for preparation of the cyclophosphate modified RNA left binding arm:
the full-length sequence of the RNA substrate is as follows:
5’-biotin-GCACUUAUUACUCGUGAACAGGCUUUGAUCGAA-3’；
the sequences of 8-17DNA cleaving enzymes are reported in the literature as follows:
5’-TTCGATCAAAGTCCGAGCCGGACGATGTTCACGAGTAATAAGTGC-3’；
1uM of RNA substrate was added to 10uM of DNase and 250mM Mg was added2+The reaction buffer solution of (1) under the reaction condition of 37 ℃ for 6 hours;
carrying out gel recovery and desalination on the obtained cleavage reaction product solution to obtain a 2 ', 3' cyclic phosphate modified RNA left side binding arm; the method comprises the following specific steps:
desalting the cut product by using a desalting column, reducing the volume and removing ions in the cut product, then carrying out electrophoresis separation by using 20% polyacrylamide gel, cutting a target strip, soaking the cut strip by using ultrapure water at 37 ℃ for 8-10 hours, sucking supernatant and desalting by using the desalting column;
4. in vitro screening for non-natural nucleases procedure:
4.1, taking an initial DNA molecule random library to carry out primer extension to obtain a non-natural nucleic acid library; as shown in the figure, the specific steps are as follows:
adding 3.6nmol DNA random library and 3.6nmol primer into 1 × Thermopol reaction buffer solution, gradient annealing (annealing condition is 90 deg.C, 5min, gradient cooling, 10 deg.C/min), and 0.2mM MnCl2Incubating with 1mg/mL KOD-RI for 15min at the same volume, adding into reaction system, adding 0.1mM tNTPs, and mixing at 55 deg.C for 4 hr;
4.2, adding a solution of the single-strand non-natural nucleic acid library into the RNA left binding arm with the cyclic phosphate modification; and 40mM Mg was added2+Placing the buffer solution at 37 ℃ and incubating for 8-18 h to obtain a nucleic acid mixture;
4.3, separating active non-natural nucleic acid sequences from the nucleic acid mixture in a mode of combining modified streptavidin magnetic beads and biotin, and specifically comprising the following steps:
washing the magnetic beads carrying the streptavidin by using a washing buffer for 3 times, adding the magnetic beads into a reaction system for incubation for 30min, washing the magnetic beads by using the washing buffer for 3 times, washing the magnetic beads by using 100mM NaOH for 30s at room temperature for one time, repeating the washing for 4-5 times, eluting by using the washing buffer for 3 times, heating 200mM NaOH solution at 37 ℃ for 15min, degrading RNA which is connected, repeating the washing for 4-5 times until the concentration of the solution measured by the nanodrop is lower than 3ng/uL, and collecting 200mM NaOH eluate;
4.4, reverse transcription is carried out on the nucleic acid mixture solution by Bst 2.0 polymerase, and the specific steps are as shown in the figure:
add the NaOH eluent in reverse transcription volume 3/10, add 100nM forward primer, 0.25mM dNTPs, 1mM MnCl21.2U Bst 2.0 polymerase, and the reaction conditions are as follows: 4h at 55 ℃;
4.5, PCR amplification step: taking a reverse transcription product with 1/10 volumes of a PCR reaction volume as a template, adding the reverse transcription product into a 1 XTaq polymerase mixture, adding 0.5uM of upstream and downstream primers, amplifying for 10-18 cycles, separating single strands to obtain an enrichment library of the round of screening, wherein the enrichment library can be used for the next round of screening, and the PCR amplification conditions are as follows: 95 ℃ for 5 min; (95 ℃, 30 s; 50 ℃, 30 s; 72 ℃, 30s) x 10-18 rounds; 72 ℃ for 5 min;
carrying out chain separation and desalination on the obtained PCR product to obtain a single-chain natural nucleic acid library; the method comprises the following specific steps:
washing the resin loaded with streptavidin by using a binding buffer for 3 times, adding a PCR product for incubation for 30min, washing by using the binding buffer for 3 times, eluting by using 200mM NaOH for 4-5 times until the concentration of the solution measured by the nanodrop is lower than 3ng/uL, eluting by using the binding buffer for 1 time, eluting by using ultrapure water for 1 time, eluting by using hot water at 95 ℃ for 4-5 times until the concentration of the solution measured by the nanodrop is lower than 3ng/uL, collecting the hot water eluent, desalting by using a desalting column and reducing the volume;
4.6, repeating the steps of 4.1-4.5;
4.7, after 5-8 rounds of screening, connecting the PCR amplification product obtained by screening to a pEASY-T1 vector, and sequencing the bacterial colony successfully connected with the vector;
4.8, deducing TNA sequence information according to a sequencing result, synthesizing a primer extension template, carrying out primer extension reaction, and separating a single strand to obtain a TNA sequence;
4.9, testing the activity of the TNA molecules for catalyzing RNA connection through a three-molecule connection experiment to obtain the TNA enzyme with the highest RNA connection activity, and specifically comprising the following steps:
the sequence of the left binding arm of RNA is as follows:
5’-Cy5.5-GCACUUAUUACUCGUGAACAG-3’；
the sequence of the right binding arm of RNA is as follows:
5’-GCUUUGAUCGAA-3’；
RNA left binding arm, right binding arm, and non-native nuclease were ligated in a 1: 20: 5 is added to the reaction system, and 40mM Mg is added2+Placing the buffer solution at 37 ℃, incubating for 6h, detecting the connection effect through 20% modified polyacrylamide gel electrophoresis, performing truncation optimization and secondary structure prediction on the TNA sequence with high connection yield, and determining the final sequence of the TNA ligase;
5. RNA splicing experiments:
5.1, construction of CTSA Exon6-Exon7 gene:
exon6-Exon7 Gene sequences are as follows (the underline is the intron sequence):
5’-TCTGGAGAGAAGACCAAAGCCTGTAGGAGATATCCCACACCTGTTCCCCAGAAGGCCATG-3’；
exon6-Exon7 Gene messenger RNA sequences are as follows (the underlines are intron sequences):
5’-CAUGGCCUUCUGGGGAACAGGUGUGGGAUAUCUCCUACAGGCUUUGGUCUUCUCUCCAGA-3’；
CTSA is in NCBI database Access number: NG _008291.1, constructing a gene fragment from exon6 to exon7, adding 200NG/10uL of template, 1 × transcription buffer solution, 50NG/uL of bovine serum albumin, 1/50 reaction volume of pyrophosphatase, 1/100 reaction volume of nuclease inhibitor, 0.5mM of ribonucleotide monomer mixture, 0.5uM of T7 RNA pol mutation 639 and 10mM of dithiothreitol into the transcription reaction system, and reacting at 37 ℃ for 2 h;
adding 3 times of anhydrous ethanol, 1/10 reaction volume NaAC solution and 1/50 reaction volume glycogen into a system after the reaction is finished, freezing for 30min at-20 ℃, removing supernatant, dissolving precipitate again by using a stop buffer, performing electrophoresis separation by using 20% deformed polyacrylamide gel, cutting a target strip, soaking for 8-10 hours at 37 ℃ by using ultrapure water, sucking supernatant, and desalting by using a desalting column;
5.2 cleavage of messenger RNA before CTSA Exon6-Exon 7: the transcript of CTSA was mixed with 8-17DNA cleaving enzyme at a ratio of 1: 10, adding 250mM Mg2+The reaction buffer of (1), reacting at 37 ℃ for 5 h;
desalting the cut product by using a desalting column, reducing the volume, then carrying out electrophoresis separation by using 20% polyacrylamide gel, cutting a target strip, soaking for 8-10 hours at 37 ℃ by using ultrapure water, sucking supernatant, desalting by using the desalting column, and recovering two sections of exons;
5.3 ligation of CTSA Exon6-Exon7 pre-messenger RNA:
exon6 and Exon7 after cleavage reactionAnd the TNA enzyme obtained by screening is mixed with the TNA enzyme obtained by screening in a proportion of 1: 1: 10, adding 40mM Mg2+The reaction buffer of (1), reacting at 37 ℃ for 18 h;
desalting the connection product after the reaction is finished by using a desalting column, reducing the volume, then carrying out electrophoresis separation by using 12% polyacrylamide gel, cutting a target strip, soaking the cut target strip in ultrapure water at 37 ℃ for 8-10 hours, sucking supernatant, desalting by using the desalting column, recovering messenger RNA before Exon6-Exon7 from which a seventh intron is removed, and identifying the connection product by using mass spectrum.
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention.
Claims (8)
1. A method for generating a molecular tool for alternative splicing of RNA by in vitro screening, which is characterized by comprising the following specific operation steps:
(1) alternatively splicing the precursor mRNA by means of a deoxyribozyme;
(2) TNA enzyme with catalytic RNA connection activity is obtained by an in vitro screening method;
(3) firstly, incubating the prepared precursor mRNA and deoxyribozyme;
and then, incubating with the TNA enzyme obtained by screening to generate a connection reaction, generating mature RNA, and detecting by using denaturing polyacrylamide gel electrophoresis.
2. A method for generating a means for alternatively splicing molecules of RNA by in vitro screening according to claim 1,
in step (1), the alternative splicing of the precursor mRNA by the deoxyribozyme is specifically:
first, a precursor mRNA is cleaved using a deoxyribozyme having an RNA cleavage activity to excise an intron, thereby obtaining two exon fragments having a 2 ', 3 ' -cyclic phosphate terminus and a 5 ' -hydroxyl terminus;
then, the two exon fragments are ligated by using a deoxyribozyme having RNA ligation activity, thereby forming a mature RNA.
3. A method for generating a means for alternatively splicing molecules of RNA by in vitro screening according to claim 1,
in step (2), the in vitro screening method specifically comprises: in one containing about 1016The TNA library is used to isolate and enrich for active nucleic acid molecules by applying a screening pressure.
4. A method for generating a means for alternatively splicing molecules of RNA by in vitro screening according to claim 3,
the construction method of the TNA library specifically comprises the following steps:
firstly, a DNA library is synthesized by a solid phase;
then, a TNA library was obtained by extension reaction using KOD-RI polymerase.
5. A method for generating a means for alternatively splicing molecules of RNA by in vitro screening according to claim 4,
the expression method of the KOD-RI polymerase specifically comprises the following steps:
firstly, constructing a PQE-80-KOD-RI recombinant plasmid, transforming the PQE-80-KOD-RI recombinant plasmid into escherichia coli BL21, and performing induced expression by IPTG;
then, the polymerase KOD-RI was purified by disrupting the cells, extracting and using column chromatography.
6. A method for generating a means for alternatively splicing molecules of RNA by in vitro screening according to claim 3,
the separation method specifically comprises the following steps: firstly, modifying biotin at the 5' end of the left binding arm of RNA to enable a sequence with a biotin label to be bound with a streptavidin magnetic bead, and then washing out complementary pairing by using 100mM NaOH, but not connecting active TNA sequence; the ligated RNA was then disrupted with 200mM NaOH at 37 ℃ to leave a TNA molecule with ligation activity.
7. A method for generating a means for alternatively splicing molecules of RNA by in vitro screening according to claim 3,
the enrichment method specifically comprises the following steps: firstly, carrying out reverse transcription reaction on a TNA pool with activity obtained by separation by using Bst 2.0 polymerase to obtain a complementary DNA library; the DNA library is then exponentially enriched by PCR techniques.
8. A method for generating a means for alternatively splicing molecules of RNA by in vitro screening according to claim 1,
in the step (3), the incubation with the TNA enzyme obtained by screening is specifically: two exons with 2 ', 3 ' -cyclic phosphate ends and 5 ' hydroxyl ends undergo ligation under the action of TNA ligase.
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| CN114317684B (en) * | 2021-12-15 | 2023-12-26 | 南京大学 | Intracellular magnesium ion imaging method based on TNA molecules |
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