RNA connection method based on dsDNA splint

Abstract

The invention belongs to the technical field of nucleic acid, particularly relates to an RNA (Ribonucleic Acid) connection method based on a dsDNA (Deoxyribose Nucleic Acid) splint, and mainly solves the problem of low connection efficiency caused by a complex structure of RNA in the connection and connection looping processes. Comprising the following steps: preparing a dsDNA splint, preparing an RNA fragment, mixing the RNA fragment and the dsDNA splint, sequentially performing denaturation and annealing to form an RNA / DNA hybrid, performing RNA connection and DNA removal treatment, and finally obtaining the multi-fragment connected RNA or circular RNA. According to the invention, double-stranded DNA instead of a traditional single-stranded DNA splint is used for the first time, and an RNA / DNA hybrid structure is obtained by direct denaturation annealing in a DNA and RNA mixed system and is used as a connection precursor. The dsDNA splint has good availability, the length of the dsDNA splint is not limited by a synthesis method, the influence of the complex structure of RNA can be overcome through pairing competition of DNA and the structure of an RNA substrate, finally, the connection efficiency is improved, particularly, the connection efficiency of a structured fragment is improved, and the connection efficiency is 100 times higher than that of a traditional method. In addition, the method can effectively inhibit non-specific wrong connection. In a word, the method can be used for quickly, simply, flexibly, accurately and efficiently connecting multiple fragments of RNA or connecting RNA to form a ring in a one-pot system.

Description

Technical Field

[0001] The invention belongs to the field of nucleic acid technology, and in particular relates to an RNA connection method based on a dsDNA splint. Background Art

[0002] RNA (ribonucleic acid) has many important functions in organisms and has extensive and important applications in biotechnology. The research and application of RNA requires RNA synthesis in vitro, such as mRNA drug research (Signal Transduct. Target Ther. 2022, 7 (1), 166). In addition to short-chain RNA that can be chemically synthesized, longer RNA is generally synthesized by in vitro transcription, and longer or regionally modified RNA can be achieved through RNA ligation. For example, in order to avoid the risk of ribosomal frameshift caused by m1ψ modification in the coding region (CDS) of drug mRNA (Nature 2024, 625 (7993), 189-194), RNA ligation can be used to synthesize mRNA without m1ψ modification in the coding region to reduce the risk of frameshift. After all, transcription will cause overall modification of mRNA.

[0003] However, since RNA (especially long RNA) has complex secondary and tertiary structures (Proc. Natl. Acad. Sci. USA 2019, 116 (48), 24075-24083), existing methods for their ligation based on short single-stranded DNA (ssDNA) splints ( Figure 1 ) is generally less efficient (Nucleic Acids Res. 2022, 50(20), e115.). This is mainly because ssDNA is difficult to compete with these structures, hindering the formation of DNA / RNA hybrids, resulting in low efficiency, large sequence differences, and low versatility of traditional ligation methods. Especially in RNA ligation with complex structures and multi-fragment RNA ligation, the ligation efficiency is even lower, even approaching 0%. Therefore, it is urgent to establish a method that can efficiently, accurately, conveniently and versatility connect single-stranded RNA. Summary of the invention

[0004] Given that short ssDNA is not competitive enough to bind to substrate RNA during annealing, we speculate that by lengthening ssDNA, its competitiveness can be improved and better paired with the RNA to be connected to form a connection-compatible RNA / DNA hybrid structure with a nick. However, the technology for obtaining longer ssDNA is either very difficult (chemical synthesis) or the production process is cumbersome and the product is complex and difficult to purify (biosynthesis, such as ssDNA preparation based on in vitro rolling circle amplification and phage-based synthesis) (Chem 2021, 7 (4), 959-981). In order to solve the above technical problems, the present invention provides an RNA connection method based on a dsDNA splint, referred to as a long dsDNA splint-assisted RNA connection method (DALI; Figure 2 ). This method uses double-stranded DNA (dsDNA) instead of traditional single-stranded DNA (ssDNA) as a splint for the first time. In a mixed system of DNA and RNA, RNA / DNA hybrid structures are directly denatured and annealed to obtain them as connection precursors. The dsDNA splint is easy to obtain, and its length is not limited by the synthesis method. It can effectively compete with the complex structure of RNA itself during the annealing process, ultimately improving the connection efficiency, especially in the connection of structured fragments, the efficiency can be increased by up to 100 times compared with traditional methods. This method can quickly, simply, flexibly and efficiently connect multiple RNA fragments or connect RNA into rings in a one-pot system.

[0005] A dsDNA splint-based RNA connection method for solving the above technical problems comprises the following steps:

[0006] (1) preparing a dsDNA splint;

[0007] (2) preparing RNA fragments;

[0008] (3) mixing RNA fragments with dsDNA splints in annealing buffer;

[0009] (4) denaturation and annealing are performed sequentially to form RNA / DNA hybrids;

[0010] (5) mixing the annealing product obtained in step (4) with a ligase and a ligation buffer, and performing ligation;

[0011] (6) performing DNA removal treatment on the ligation product;

[0012] (7) Purify the RNA obtained in step (6).

[0013] Furthermore, the dsDNA splint in step (1) can be obtained by amplification techniques such as PCR or plasmid enzyme digestion, etc. The above method is known and will not be described in detail here.

[0014] Furthermore, in the step (1), the length of the dsDNA splint is ≥89 bp; further, it is optimized to be equal to the total length of the connecting RNA and have the same sequence.

[0015] Furthermore, the RNA fragment in step (2) can be obtained by chemical synthesis or in vitro transcription. The above method is known and will not be described in detail here.

[0016] Furthermore, in the step (3), the concentration of the substrate RNA to be connected added to the annealing buffer is 0.1 pM-10 μM, and the concentration of the corresponding splint dsDNA is 0.1 pM-10 μM; further, it is optimized to an equimolar ratio.

[0017] Furthermore, in step (4), when the annealing buffer is 1×, its composition is 10 mM Tris-HCl (pH 8.0), 20 mM NaCl, and annealing additive.

[0018] Further, the annealing additive can help DNA and RNA hybridize better, and can be any one of the following reagents: polyethylene glycol 8000 (PEG8000; 5%), polyethylene glycol 4000 (PEG400; 20%), trehalose (0.2M), betaine (1M), tetramethylammonium chloride (TMAC; 40mM), urea (1M), dimethyl sulfoxide (DMSO; 1%-40%) or dimethylformamide (DMF; 10%); further, the DMSO concentration is optimized to 20%.

[0019] Furthermore, in step (4), the denaturation treatment conditions are 85°C-100°C for 1 sec-10 min; the annealing treatment conditions are gradient cooling to 24-26°C at -0.02°C / s; and further, the denaturation temperature is optimized to 90°C and the denaturation time is 1 s.

[0020] Furthermore, the ligation reaction conditions in step (5) are 10°C-42°C for 1 min-5 h, and the ligation buffer is 50 mM Tris-HCl (pH=7.5), 2 mM MgCl2, 1 mM DTT and 0.4 mM ATP.

[0021] Furthermore, in the step (5), the ligase is T4 RNA ligase 2, and the ligation additive is RppH enzyme; further, the concentration of T4 RNA ligase 2 is 0.1-10U / μL, and the amount of the additive RppH enzyme used is 0.1-10U per microgram of RNA; further, the concentration of T4 RNA ligase 2 is optimized to 0.4U / μL, and the amount of RppH enzyme used is optimized to 1U per microgram of RNA.

[0022] Furthermore, in the step (6), the amount of DNase I used is 0.1-10U per microgram of DNA, and the DNA digestion treatment is carried out at 30°C-42°C for 1-60min; further, it is optimized to use 2U of DNase I per microgram of DNA, digestion treatment at 37°C for 30min, and then at 42°C for 30min.

[0023] Furthermore, in step (7), the RNA ligation product is purified by size exclusion high performance liquid chromatography (SEC-HPLC), and the purification column is a Suzhou Saifen SEC column (4.6×300 mm, particle size 5 μm, pore size ), the mobile phase uses TE buffer (10mM Tris-HCl, 1mM EDTA, pH6); further, the specific process is: first, the RNA connection product is precipitated with lithium chloride; after redissolution, it is denatured at 75°C for 5 minutes, and then loaded, the flow rate is 0.2mL / min, and detection is performed by ultraviolet absorption at a wavelength of 260nm, and the target sample is collected; the collected samples are precipitated with 5M ammonium acetate, and analyzed by urea-PAGE after redissolution.

[0024] Beneficial effects achieved in the present invention:

[0025] (1) High ligation efficiency: Through the competitive advantage of long-chain DNA, the spontaneous folding structure of long-fragment RNA is inhibited, achieving efficient annealing and ligation.

[0026] (2) Strong versatility: This method can be used to connect long fragments, RNAs with different terminal connecting nucleotides, or RNAs with complex structures relatively efficiently. Therefore, this method has better versatility in RNA sequences and lengths.

[0027] (3) The splint and RNA substrate can be obtained from the same plasmid, which is simple and economical: the optimized splint dsDNA and RNA substrate have the same sequence and total length, and the splint dsDNA can be obtained by amplification / enzyme digestion of the same plasmid or the substrate RNA can be obtained by in vitro transcription.

[0028] (4) It can realize synchronous connection of multiple fragments: splint dsDNA can pair with multiple RNA fragments at one time and mediate synchronous connection.

[0029] (5) It can achieve efficient connection and ring formation without introducing irrelevant sequences: the splint dsDNA with inverted pairing region can be paired with multiple RNA fragments at one time, mediating the ring formation of a single RNA substrate or multiple RNA substrates.

[0030] (6) One-pot ligation, simple and fast: the dsDNA splint and RNA substrate are denatured, annealed, ligated and DNA removed in the same pot, and there is no need to purify and separate single-stranded ssDNA from dsDNA separately.

[0031] (7) High ligation specificity: It can inhibit non-specific fragment ligation and increase ligation accuracy.

[0032] The method of the present invention can improve the synthesis efficiency of regionally modified linear RNA and circular RNA, and is expected to promote RNA research and therapeutic applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the method of using short ssDNA to assist RNA ligation and ligation into circles.

[0034] (A, linear RNA fragments connected; B, linear RNA connected into a ring)

[0035] Figure 2 This is a schematic diagram of the patented method using short dsDNA to assist RNA connection and linking into a ring.

[0036] (A, linear RNA fragments connected; B, linear RNA connected into a ring)

[0037] Figure 3 Example 1, verification of one-step hybridization of dsDNA and ssRNA.

[0038] Figure 4 Example 2: dsDNA-assisted RNA ligation with different numbers of fragments.

[0039] (A, single-fragment RNA connection, i.e. single-fragment connection into a ring. B, analysis of the ring formation of the cyclized product. C, double-fragment RNA connection, D, triple-fragment RNA connection. E, quadruple-fragment RNA connection)

[0040] Figure 5 Example 3, complex structure RNA connection.

[0041] (A, mFold predicts the structure of the substrate RNA, with the ligation ends shown in red. B, ligation product analysis.)

[0042] Figure 6 Example 4, condition optimization.

[0043] (A, ligation time optimization. B, ligase concentration optimization. C, annealing buffer optimization. D, denaturation stability optimization. E, splint type optimization. F, additive optimization. The additive concentrations used were: polyethylene glycol 8000 (PEG8000;

[0044] 5%), polyethylene glycol 4000 (PEG400; 20%), trehalose (trehalose; 0.2M), betaine (betaine;

[0045] 1M), tetramethylammonium chloride (TMAC; 40mM), urea (urea; 1M), dimethyl sulfoxide (DMSO; 1%-40%), dimethylformamide (DMF; 10%), RppH enzyme (1U / μg RNA). G. Optimization of annealing additive DMSO concentration. )

[0046] Figure 7 Example 5, optimized ligation efficiency and the effect of dsDNA splint length on ligation efficiency.

[0047] Figure 8 Example 6, versatility of ligation substrate RNA.

[0048] (A. Effect of RNA length on ligation efficiency. B. Effect of RNA terminal nucleotide type on ligation efficiency.)

[0049] Fig. 9 Example 7, purification of ligation product RNA.

[0050] (A, SEC-HPLC purified RNA ligation crude product spectrum. B, urea-PAGE analysis of recovered components. C, ligation yield and total yield.) DETAILED DESCRIPTION

[0051] The present invention is further described in detail below with reference to specific embodiments and accompanying drawings.

[0052] The ssDNA splints used in the following examples were all synthesized by chemical methods and were synthesized by Sangon (Shanghai) Technology Co., Ltd.

[0053] The splint dsDNA and transcription template dsNDA used in the following examples were prepared from plasmids by PCR. Briefly, 0.01 ng / μL DNA plasmid, 1× PrimeSTAR Max Premix (Takara Bio) and 0.8 μM primers were used to synthesize transcription templates and ligation fragments by PCR, and the obtained products were analyzed and purified by agarose electrophoresis and Cycle Pure Kit (OMEGA), respectively.

[0054] The RNA substrates used in the following examples were prepared by in vitro transcription (IVT) using plasmid PCR fragments as templates. Briefly, the in vitro transcription reaction was carried out at 37°C for 2 hours with 1 μg DNA transcription template, NTPs (5 mM each), IVT buffer [40 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES; pH 8.5), 40 mM dithiothreitol (DTT), 10 mM NaOAc, 75 mM Mg(OAc)2 and 0.2 mM spermidine], and T7 RNA polymerase (0.1 mg / mL). For synthetic 5'-monophosphorylated RNA transcription, GMP (4 mM) was added and the standard GTP was reduced to 0.8 mM. The crude transcripts were treated with DNase I (2 U per microgram of DNA; NEB) at 37°C for 15 min to digest the DNA template, and then the RNA was precipitated with lithium chloride (LiCl, 2.5 M) to obtain pure RNA substrate for ligation.

[0055] The following examples all used urea denaturation PAGE (urea-PAGE) analysis for the analysis of the ligated RNA products, specifically: prepare 5% urea-PAGE, whose composition is: 5% polyacrylamide, 1×TBE buffer, 0.1% APS, 0.01% TEMED, 8M urea; the sample is mixed with 2×RNA loading buffer [0.02% (w / v) xylene cyanol, 20% glycerol, 8M urea] in a ratio of 1:1, denatured at 95°C for 1 min and then loaded, and RiboRuler High Range RNA Ladder (ThermoFisher; RM) or DL2,000 DNA Marker (Takara; DM) is also loaded as a marker; electrophoresis is performed in a 55°C oven at 200V for 60min; after electrophoresis, the gel is stained with Ultra GelRed (Vazyme) and visualized on a ChemiDoc XRS+ system (BioRad).

[0056] The following examples calculate the ligation yield in the same manner, specifically: the calculation of the ligation yield depends on the relative molar amount measured on PAGE; after determining the bands of the product and the by-product according to the molecular size and the mobility on PAGE, the complete ligation product (P, the desired product) and the incomplete ligation product (by-product: P1 ... P2) of the selected substrate (S; the molar amount of all RNA substrates is equal, and the longest one is selected in the present invention) are measured using ImageLab software (BioRad). n The intensity of the gel band is expressed as "I". S " indicates that the gel band intensity of P is expressed as "I P " indicates that P1 to P n The gel band intensity is expressed as "IP1 "to"I Pn " indicates. Since the band intensity is linearly related to the RNA length, the band intensity can be used as the relative mass of the corresponding RNA, and its ratio to the molar mass (M) can be regarded as the relative molar amount. The ligation yield is calculated based on the ratio of the actual ligation product amount (equal to the relative molar amount of the complete ligation product) to the theoretical ligation product amount (equal to the sum of the relative molar amounts of the substrate and its derivative products), and the calculation formula is as follows:

[0057]

[0058] The nucleotide sequences involved in the present invention are shown in Table 1

[0059] Table 1.

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] Example 1

[0067] RNA, dsDNA one-step hybridization

[0068] 1) Raw materials

[0069] The two hybridization substrates are SEQ-1 and SEQ-2.

[0070] 2) Hybridization (denaturation, annealing)

[0071] Equimolar amounts of RNA substrate and corresponding dsDNA splint were mixed in annealing buffer (Biyuntian). The hybridization reaction was denatured at 90°C for 1 second and then slowly annealed to 25°C at a rate of -0.02°C per second.

[0072] 3) Nuclease digestion

[0073] The hybridization products were digested with RNase H (0.5 U / μL; NEB) or DNase I (RNase free; 0.2 U / μL; NEB) to confirm the formation of hybridization.

[0074] 4) Non-denaturing PAGE electrophoresis

[0075] All samples were analyzed by non-denaturing PAGE electrophoresis, specifically: first, a non-denaturing PAGE gel (5% polyacrylamide, 1×TBE buffer, 0.1% APS, 0.01% TEMED) was prepared; the sample was mixed with 2×RNA non-denaturing loading buffer [0.02% (w / v) xylene cyanol, 20% glycerol] and electrophoresed in an ice-water bath at 200V for 60min. The gel was stained with Ultra GelRed (Vazyme) and visualized on a ChemiDoc XRS+ system (BioRad). RiboRuler High Range RNA Ladder (ThermoFisher) and DL2,000 DNA Marker (Takara) were used as electrophoresis markers.

[0076] Figure 3 The results are shown in Figure 2. The results show that the corresponding dsDNA and ssRNA can be competitively paired by direct denaturation annealing to efficiently and quickly produce stable DNA / RNA hybrids, and denaturation annealing is a necessary condition for hybrid formation. The formation of this hybrid is both the basis of RNA connection and the difficulty of RNA connection.

[0077] Example 2

[0078] Ligation of different numbers of RNA

[0079] 1) Raw materials

[0080] In a fragment ligation experiment, i.e., a ligation ring experiment: the ligation substrate includes an RNA, which is SEQ-3; the ligation splint is different in different reaction systems, using SEQ-4 (20nt ssDNA), SEQ-5 (59nt ssDNA) or SEQ-6 (1431bpdsDNA).

[0081] In the two-fragment ligation experiment: the ligation substrate includes two RNAs, SEQ-1 and SEQ-7 respectively; the ligation splint is different in different reaction systems, using SEQ-8 ​​(20nt ssDNA), SEQ-9 (59nt ssDNA) or SEQ-10 (967bpdsDNA).

[0082] In the three-fragment ligation experiment: the ligation substrate includes three RNAs, namely SEQ-11, SEQ-1 and SEQ-7; the ligation splint is different in different reaction systems. The single-stranded splint-assisted ligation system includes three ssDNAs, namely SEQ-12 and SEQ-8 ​​(both 20nt), or SEQ-13 and SEQ-9 (both 59nt), and the double-stranded splint-assisted ligation system includes one dsDNA, namely SEQ-2 (1146bp).

[0083] In the four-fragment ligation experiment: the ligation substrate includes four RNAs, namely SEQ-11, SEQ-14, SEQ-15 and SEQ-7; the ligation splint is different in different reaction systems. The single-stranded splint-assisted ligation system includes two ssDNAs, namely SEQ-12, SEQ-16 and SEQ-8 ​​(all 20nt), or SEQ-13, SEQ-16 and SEQ-9 (all 59nt), and the double-stranded splint-assisted ligation system includes one dsDNA, namely SEQ-2 (1146bp).

[0084] 2) Hybridization (denaturation, annealing)

[0085] Equimolar amounts of RNA substrate and corresponding dsDNA splint were mixed in annealing buffer (Biyuntian). The hybridization reaction was denatured at 90°C for 1 second and then slowly annealed to 25°C at a rate of -0.02°C per second.

[0086] 3) RNA ligation and DNA removal

[0087] The annealed product was mixed with T4 RNA ligase 2 (final concentration 0.4 U / μL, Bio-Tech) and ligation buffer (50 mM Tris-HCl, pH 7.5; 2 mM MgCl2, 1 mM DTT; 0.4 mM ATP) and the ligation reaction was carried out at 25°C for 2 hours. Subsequently, DNA was digested with DNase I (2 U per microgram of DNA; NEB) and incubated twice at 37°C and 55°C for 30 minutes each time.

[0088] 4) Perform urea-PAGE electrophoresis to calculate the ligation yield.

[0089] The method is as described above and will not be described again here.

[0090] 5) Confirm that the RNA in a fragment RNA ligation is successfully circularized.

[0091] RNA circularization was verified by reverse transcription-PCR and first-generation sequencing. Specifically, circularization verification primers (sequences: 5'-CGGAAACCTGGCCCTGTC-3' and 5'-GTTCCGCTGCCTGCAAAG-3') were designed across the circularization junction site and reverse transcription kit was used. III 1st Strand cDNA Synthesis Kit reverse transcription was followed by PCR amplification, and the amplified products were verified by agarose gel electrophoresis and first-generation sequencing.

[0092] Figure 4The results are shown in Figure 2. The results show that dsDNA can be used as a splint to assist RNA ligation, and even without optimized conditions, its ligation efficiency is higher than that of the traditional ssDNA-assisted method. Specifically, a fragment of RNA can be ligated into a ring using dsDNA as a splint, and its ligation efficiency is significantly better than that of the traditional ligation ring method using ssDNA (20, 59 nt) as a splint ( Figure 4 A); dsDNA splints can assist RNA in connecting two fragments, and the connection efficiency is higher than the traditional ssDNA (20, 59nt) as the auxiliary connection method, and the efficiency is 81 times higher than the most commonly used 20nt ssDNA splint; dsDNA splints can also assist RNA in connecting three fragments, and the connection efficiency is higher than the traditional ssDNA (2 20nt or 2 59nt) as the auxiliary connection method, which is 17 times higher than the 20nt splint and 2.8 times higher than the 59nt splint; dsDNA splints can also assist RNA in connecting four fragments, and the connection efficiency is significantly higher than the traditional ssDNA (3 20nt or 3 59nt) as the auxiliary connection method, which is 46 times higher than the 20nt splint and 8.3 times higher than the 59nt splint. From the above results, it can be seen that RNA connection can be achieved very easily through the easily available long dsDNA, and the connection efficiency is also significantly improved compared with the traditional method.

[0093] Example 3

[0094] Complex RNA structures

[0095] 1) Raw materials

[0096] The ligation substrate includes two RNAs, namely SEQ-18 and SEQ-19; the ligation splint is different in different reaction systems, using ssDNA splint SEQ-20 (20nt), SEQ-21 (59nt) or SEQ-22 (89nt), or using dsDNA splint SEQ-28 (1463bp).

[0097] 2) Predict secondary structure

[0098] mFold was used to predict the secondary structures of the selected RNAs SEQ-18 and SEQ-19 (Nucleic Acids Res. 2003, 31(13), 3406-3415), and the secondary structures of the regions adjacent to the junction ends were displayed.

[0099] 3) Hybridization (denaturation, annealing)

[0100] Equimolar RNA substrates and corresponding dsDNA splints were mixed in annealing buffer (Biyuntian) containing DMSO as an annealing additive. The hybridization reaction was denatured at 90°C for 1 second and then slowly annealed to 25°C at a rate of -0.02°C per second.

[0101] 4) RNA ligation and DNA removal

[0102] The annealed product was mixed with T4 RNA ligase 2 (final concentration 0.4 U / μL, Bio-Tech) and ligation buffer (50 mM Tris-HCl, pH 7.5; 2 mM MgCl2, 1 mM DTT; 0.4 mM ATP) and the ligation reaction was carried out at 25°C for 2 hours. Subsequently, DNA was digested with DNase I (2 U per microgram of DNA; NEB) and incubated twice at 37°C and 55°C for 30 minutes each time.

[0103] 5) Perform urea-PAGE electrophoresis and calculate the ligation yield.

[0104] The method is as described above and will not be described again here.

[0105] Figure 5 The results show that RNA is very easy to form secondary and tertiary structures. The selected two RNAs, SEQ-18 and SEQ-19, have obvious secondary structures at the ends of the connection ( Figure 5 A). In subsequent studies, we found that the secondary structure at the ligation end significantly inhibited RNA ligation in the traditional ssDNA splint-dependent method (yield less than 2%; Figure 5 B), not only that, the ligation reaction also showed obvious mis-ligation (R6-R6). Even without optimized conditions, RNA ligation assisted by equal-length dsDNA can achieve a high efficiency, which is 34 times, 20 times and 6.7 times higher than the efficiency of 20, 59 and 89nt ssDNA-assisted ligation, respectively. In addition, dsDNA-assisted RNA ligation does not produce non-specific ligation. From the above results, it can be seen that long-chain dsDNA with the same total length as the ligated RNA can efficiently assist the high-precision ligation of RNA, especially in RNA with a more complex ligation end structure.

[0106] Example 4

[0107] Optimizing connection conditions

[0108] 1) Raw materials

[0109] In the annealing buffer optimization experiment: the ligation substrate includes two RNAs, SEQ-1 and SEQ-7 respectively; the ligation splint SEQ-10; the annealing buffer is a commercially available RNA annealing buffer (Biyuntian), Buffer1 (10mM Tris-HCl (pH 8.0), 20mM NaCl), Buffer2 (10mM Tris-HCl (pH 8.0), 20mM NaCl, 1mM EDTA), Buffer3 (100mMKOAc, 30mM HEPES-KOH, 2mM Mg(OAc)2) or Buffer4 (50mM sodium citrate).

[0110] In the ligation time optimization experiment, ligase concentration optimization experiment and denaturation temperature optimization experiment: the ligation substrate includes three RNAs, namely SEQ-11, SEQ-1 and SEQ-7; and the ligation splint is SEQ-2.

[0111] In the optimization experiment of splint type and annealing additive: the ligation substrate includes two RNAs, SEQ-18 and SEQ-19 respectively; the ligation splint is SEQ-28.

[0112] 2) Hybridization (denaturation, annealing)

[0113] Equimolar RNA substrates and corresponding dsDNA splints were mixed in Beyotime annealing buffer (excluding buffer optimization experiments) (additives were also included in the optimization experiments). The hybridization reaction was denatured at 90°C (excluding denaturation temperature optimization experiments) for 1 second and then slowly annealed to 25°C at a rate of -0.02°C per second.

[0114] 3) RNA ligation and DNA removal

[0115] The annealed product was mixed with T4 RNA ligase 2 (final concentration 0.4U / μL, Bio-Tech, excluding ligase concentration optimization experiments) and ligation buffer (50mM Tris-HCl, pH 7.5; 2mM MgCl2, 1mM DTT; 0.4mM ATP) (RppH enzyme was also added in the ligation additive experiment), and the ligation reaction was carried out at 25°C for 2 hours (excluding ligation time optimization experiments). Subsequently, DNA was digested with DNase I (2U per microgram of DNA; NEB), and incubated twice at 37°C and 55°C, each for 30 minutes.

[0116] 4) Perform urea-PAGE electrophoresis to calculate the ligation yield.

[0117] The method is as described above and will not be described again here.

[0118] Figure 6The results are shown in Figure 2. The results show that the maximum reaction yield can be achieved when the ligation reaction time is 30min-120min ( Figure 6 A). When T4 RNA ligase 2 (Biyuntian) was at 0.27-0.4U / μL, the ligation yield reached the maximum ( Figure 6 B). A commercial buffer (Biyuntian) and two buffers with known composition (Buffer1 and Buffer2) can achieve efficient ligation ( Figure 6 C). In addition, the denaturation-annealing process also has a great influence on the connection yield ( Figure 6 D). Specifically, at lower denaturation temperatures (60-85°C), no product is formed by ligation ( Figure 6 D), but a longer misligation product (R3+R3) was formed instead. The desired ligation product was generated at both 90°C (optimal temperature) and 95°C. In addition, the results of the splint type experiment showed that both linearized and supercoiled plasmid DNA (4292 bp, longer than the length of the desired RNA product 1463 nt) can also serve as ligation splints, although their yields are slightly lower than that of the dsDNA concatemer of the same length (1463 bp) ( Figure 6 E). To further improve the efficiency of dsDNA-splint ligation to mRNA, we tested various ligation additives. Macromolecular crowders (PEG8000 and PEG400), small molecule crowders (trehalose and betaine), and chaotropes (TMAC, urea, DMSO, and DMF) were added during the annealing process to promote hybrid formation, while the ligation additive RppH enzyme was added during the ligation process to convert the triphosphorylated mRNA produced during GMP-cotranscription into a ligation-compatible monophosphorylated form. Ligation yield analysis showed that most annealing additives significantly improved ligation efficiency; for example, DMSO produced relatively high yields of the desired product, while nonspecific products (R6-6; Figure 6 F) is very small. After concentration optimization ( Figure 6 G), 20% DMSO and RppH enzyme were selected as additives for annealing and ligation, respectively, and their combined use could obtain the best ligation efficiency. Based on these optimized conditions, we established an optimized dsDNA splint-based ligation method, called the DALI strategy: First, equimolar RNA fragments and corresponding dsDNA fragments (whose size is equal to the total length of all combined RNA fragments) were mixed in an annealing buffer (Biyuntian buffer or Buffer-1) containing 20% ​​DMSO; then, the mixture was denatured and annealed, heated to 90℃ for 1 second, and then gradually cooled to 25℃ at a rate of -0.02℃ / sec; finally, 0.4U / μL T4 RNA ligase 2 was added to initiate the ligation reaction, and incubated at 25℃ for 2 hours with RppH enzyme.

[0119] Example 5

[0120] Optimized ligation efficiency and the effect of dsDNA splint length on ligation efficiency

[0121] 1) Raw materials

[0122] In the two-fragment ligation experiment: the ligation substrate includes two RNAs, namely SEQ-18 and SEQ-19; the ligation splint is different in different reaction systems, using ssDNA splint SEQ-20 (20nt), SEQ-21 (59nt) or SEQ-22 (89nt), or using dsDNA splint SEQ-23 (20bp), SEQ-24 (59bp), SEQ-25 (89bp), SEQ-26 (200bp), SEQ-27 (500bp) or SEQ-28 (1463bp).

[0123] 2) Hybridization (denaturation, annealing)

[0124] Equimolar RNA substrates and corresponding dsDNA splints were mixed in annealing buffer (Biyuntian) and a mixture supplemented with 20% DMSO. The hybridization reaction was denatured at 90°C for 1 second and then slowly annealed to 25°C at a rate of -0.02°C per second.

[0125] 3) RNA ligation and DNA removal

[0126] The annealed product was mixed with T4 RNA ligase 2 (final concentration 0.4 U / μL, Bio-Tech), ligation buffer (50 mM Tris-HCl, pH 7.5; 2 mM MgCl2, 1 mM DTT; 0.4 mM ATP) and additive RppH enzyme (1 U / μg RNA) and the ligation reaction was carried out at 25°C for 2 hours. Subsequently, DNA was digested with DNase I (2 U per microgram of DNA; NEB) and incubated twice at 37°C and 55°C for 30 minutes each time.

[0127] 4) Perform urea-PAGE electrophoresis to calculate the ligation yield.

[0128] The method is as described above and will not be described again here.

[0129] Figure 7The result is shown in Figure 2. The results show that the secondary structure at the end of the connection significantly inhibits the RNA connection in the traditional ssDNA splint-dependent method (the yield is less than 2%). Not only that, the connection reaction also shows obvious misconnection (R6-R6). By using the optimized system, the connection efficiency of the dsDNA splint connection method (DALI) was increased to 31.8% (10.1% before optimization). In addition, by increasing the length of dsDNA, the complementary pairing region between DNA and substrate RNA is increased, the efficiency of hybrid formation is improved, and the connection efficiency is rapidly increased. Among them, the dsDNA splint with the same length as the total length of the substrate RNA has the highest auxiliary connection efficiency, which is 106 times, 21 times and 19 times higher than the connection efficiency of 20, 59, and 89nt ssDNA-assisted connection, respectively. Moreover, RNA connection assisted by equal-length dsDNA is not only efficient, but also does not produce non-specific connection byproducts, which provides the possibility for efficient purification of connection products. From the above results, it can be seen that long-chain dsDNA with the same total length as the connecting RNA can efficiently assist in the high-precision connection of RNA, especially in RNA with more complex connecting end structures, its efficiency can be improved by hundreds of times.

[0130] Example 6

[0131] RNA substrate universality experiment

[0132] 1) Raw materials

[0133] In the experiment on the effect of RNA length on ligation efficiency, the ligation substrate included two upstream and downstream ssRNAs, the downstream RNA was fixed to SEQ-32 (Rd, 268nt), and the upstream RNA was SEQ-29 (Ra, 100nt), SEQ-30 (Rb, 300nt) or SEQ-31 (Rc, 500nt); the splints in the RNA ligation involving these three upstream RNAs corresponded to: SEQ-33, SEQ-34 or SEQ-35.

[0134] In the experiment of the influence of the 3' terminal nucleotide of RNA on the ligation efficiency: the ligation substrate includes two upstream and downstream ssRNAs, the downstream RNA is fixed to SEQ-7 (R4), and the upstream RNA is SEQ-36 (R3_A), SEQ-37 (R3_C), SEQ-38 (R3_G) or SEQ-36 (R3_U); the splints in the RNA ligation in which these four upstream RNAs participate correspond to: SEQ-40, SEQ-41, SEQ-42 or SEQ-43.

[0135] 3) Hybridization (denaturation, annealing)

[0136] Equimolar RNA substrates and corresponding dsDNA splints were mixed in annealing buffer (Biyuntian) containing DMSO (20%) as an annealing additive. The hybridization reaction was denatured at 90°C for 1 second and then slowly annealed to 25°C at a rate of -0.02°C per second.

[0137] 4) RNA ligation and DNA removal

[0138] The annealed product was mixed with T4 RNA ligase 2 (final concentration 0.4 U / μL, Bio-Tech), ligation buffer (50 mM Tris-HCl, pH 7.5; 2 mM MgCl2, 1 mM DTT; 0.4 mM ATP) and additive RppH enzyme (1 U / μg RNA) and the ligation reaction was carried out at 25°C for 2 hours. Subsequently, DNA was digested with DNase I (2 U per microgram of DNA; NEB) and incubated twice at 37°C and 55°C for 30 minutes each time.

[0139] 4) Perform urea-PAGE electrophoresis to calculate the ligation yield.

[0140] The method is as described above and will not be described again here.

[0141] Figure 8 The results are shown in Figure 2. The results show that the dsDNA splint-assisted RNA ligation method can efficiently connect RNA fragments of different lengths and RNAs with different terminal sequences without showing obvious preference. This proves that this method is a simple, efficient and universal RNA ligation method.

[0142] Example 7

[0143] RNA ligation product purification

[0144] 1) Raw materials

[0145] The connection substrate includes two RNAs, namely SEQ-44 and SEQ-45; the connection splint is SEQ-46.

[0146] 3) Hybridization (denaturation, annealing)

[0147] Equimolar RNA substrates and corresponding dsDNA splints were mixed in annealing buffer (Biyuntian) containing DMSO (20%) as an annealing additive. The hybridization reaction was denatured at 90°C for 1 second and then slowly annealed to 25°C at a rate of -0.02°C per second.

[0148] 4) RNA ligation and DNA removal

[0149] The annealed product was mixed with T4 RNA ligase 2 (final concentration 0.4 U / μL, Bio-Tech), ligation buffer (50 mM Tris-HCl, pH 7.5; 2 mM MgCl2, 1 mM DTT; 0.4 mM ATP) and additive RppH enzyme (1 U / μg RNA) and the ligation reaction was carried out at 25°C for 2 hours. Subsequently, DNA was digested with DNase I (2 U per microgram of DNA; NEB) and incubated twice at 37°C and 55°C for 30 minutes each time.

[0150] 5) SEC-HPLC purification of RNA

[0151] The RNA ligation product was purified by size exclusion high performance liquid chromatography (SEC-HPLC), and the specific process was as follows: the separation column used was a Sepax Technologies SEC column (4.6×300 mm, particle size 5 μm, pore size ), TE buffer (10 mM Tris, 1 mM EDTA, pH 6) was used as the mobile phase; the RNA ligation product was first precipitated with lithium chloride; after redissolution, it was denatured at 75°C for 5 minutes and then loaded; the HPLC flow rate was 0.2 mL / min, detection was performed by ultraviolet absorption at a wavelength of 260 nm, and the target sample was collected.

[0152] 6) urea-PAGE electrophoresis, calculate the ligation yield and analyze the purity of the samples before and after purification

[0153] The method is as described above and will not be described again here.

[0154] 7) Detect the sample concentration and calculate the total yield after purification.

[0155] After purification, the sample concentration was detected at a wavelength of 260 nm in an ultraviolet spectrophotometer, and the sample concentration was calculated according to the absorption coefficient, and the yield and total yield were calculated.

[0156] Fig. 9 The results show that the mRNA obtained by the patented method can be purified by SEC-HPLC to obtain a high-purity sample conveniently and quickly ( Fig. 9 A and 9B), the ligation efficiency (calculated based on urea-PAGE bands) reached 60%, and the final total yield (after SEC-HPLC purification) reached 29%. The above results prove that this method has high ligation efficiency and the product is easy to purify. It is a simple, efficient and universal method for preparing ligated RNA.

Claims

1. A dsDNA splint-based RNA ligation method, characterized in that: The following steps are involved: (1) preparing a dsDNA splint; (2) preparing RNA fragments; (3) mixing RNA fragments with dsDNA splints in annealing buffer; (4) denaturation and annealing are performed sequentially to form RNA / DNA hybrids; (5) mixing the annealing product obtained in step (4) with a ligase, a ligation buffer and a ligation additive, and performing ligation; (6) performing DNA removal treatment on the ligation product; (7) Purify the RNA obtained in step (6).

2. The RNA ligation method based on dsDNA splint according to claim 1, characterized in that: In the step (1), the length of the dsDNA splint is ≥89 bp.

3. The RNA ligation method based on dsDNA splint according to claim 1, characterized in that: In the step (3), the substrate RNA to be connected and the corresponding splint dsDNA are mixed in an annealing buffer at a molar ratio of 1:

1.

4. The RNA ligation method based on dsDNA splint according to claim 3, characterized in that: When the annealing buffer is 1×, its composition is 10 mM Tris-HCl (pH 8.0), 20 mM NaCl, and annealing additives.

5. A dsDNA splint-based RNA ligation method according to claim 4, characterized in that: The annealing additive can be any one or more of the following reagents: polyethylene glycol 8000 (PEG8000; 5%), polyethylene glycol 4000 (PEG400; 20%), trehalose (0.2M), betaine (1M), tetramethylammonium chloride (TMAC; 40mM), urea (1M), dimethyl sulfoxide (DMSO; 1%-40%) or dimethylformamide (DMF; 10%).

6. The RNA ligation method based on dsDNA splint according to claim 1, characterized in that: In the step (4), the denaturation treatment condition is 75°C-100°C for 1 sec-10 min; the annealing treatment condition is to cool down to 24-26°C at a gradient rate of -0.02°C / s.

7. The RNA ligation method based on dsDNA splint according to claim 1, characterized in that: In the step (5), the ligase is T4 RNA ligase 2, and the ligation additive is RppH enzyme.

8. A dsDNA splint-based RNA ligation method according to claims 1 and 7, characterized in that: In the step (5), after the RNA is denatured and annealed to form a hybrid with the DNA, the RNA is ligated using an annealing mixture, T4 RNA ligase 2, a ligation buffer and a ligation additive, and a ligation reaction is performed at 10° C. to 42° C. for 1 min to 5 h.

9. A dsDNA splint-based RNA ligation method according to claims 7 and 8, characterized in that: The concentration of T4 RNA ligase 2 is 0.4U / μL; when the connection buffer is 1×, its composition is 50mM Tris-HCl (pH=7.5), 2mM MgCl2, 1mM DTT and 0.4mM ATP; wherein the usage amount of the connection additive RppH enzyme is 1U per microgram of RNA.

10. The RNA ligation method based on dsDNA splint according to claim 1, characterized in that: In the step (6), the amount of DNase I used is 0.1-10 U per microgram of DNA, and the DNA digestion treatment is performed at 30° C.-42° C. for 1-60 min.

11. The RNA ligation method based on dsDNA splint according to claim 1, characterized in that: In the step (7), the RNA ligation product is purified by size exclusion high performance liquid chromatography (SEC-HPLC), and the purification column is a Suzhou Saifen SEC column (4.6×300 mm, particle size 5 μm, pore size ), and the mobile phase was TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 6).

12. A dsDNA splint-based RNA ligation method according to claim 11, characterized in that: The RNA ligation product was first precipitated with lithium chloride (LiCl, 2.5M), and then denatured at 75°C for 5 minutes after redissolution. The sample was then loaded at a flow rate of 0.2mL / min, detected by ultraviolet absorption at a wavelength of 260nm, and the target sample was collected. The collected sample was precipitated with 5M ammonium acetate, redissolved, and analyzed by urea-PAGE.

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