Method for efficiently synthesizing long-chain RNA (Ribonucleic Acid) with fluorescent label at specified site and application of long-chain RNA

Abstract

The invention discloses a method for efficiently synthesizing long-chain RNA (Ribonucleic Acid) with a fluorescent label at a specified site and application of the long-chain RNA. According to the invention, the efficiency of synthesizing the site-specific fluorescently-labeled long-chain RNA is obviously improved, so that the large-scale synthesis of the fluorescently-labeled target RNA becomes possible, and the in-depth study on the intracellular and extracellular dynamic structural characteristics and the regulation function of the target RNA by using richer fluorescence detection means is greatly promoted. The synthesis efficiency is improved, so that the method can be further applied to fluorescence labeling and synthesis of RNA drugs and RNA vaccines with longer chains, and has huge potential in research and development of RNA drugs, application research of RNA as a diagnostic tool and the like.

Description

Technical Field

[0001] The invention relates to a method for efficiently synthesizing a long-chain RNA with a fluorescent label at a designated site and application thereof, and belongs to the field of fluorescent label long-chain RNA synthesis. Background Art

[0002] Fluorescent labeling of RNA can facilitate RNA visualization, making it possible to use a variety of fluorescent detection methods to conduct in-depth studies on the structure and function of target RNA. Among them, precise labeling of single structural regions or specified nucleotide sites of long-chain RNA can greatly improve the resolution and specificity of target RNA structure detection. It is a long-sought modified RNA synthesis technology that has important value in basic and applied research on RNA structure, function, and its use as a diagnostic tool.

[0003] Solid-phase chemical synthesis is the most widely used method for RNA in vitro synthesis and modification, but the efficiency of solid-phase synthesis gradually decreases as the length of the synthetic chain increases, so it is more suitable for the synthesis of short-chain modified RNA. Site-specific labeling of RNA (PLOR) can achieve fluorescent labeling and synthesis of long-chain RNA. It uses a special solid-liquid mixed phase in vitro transcription mode. By controlling the type of NTPs in the system, T7 RNA polymerase transcribes in a transcription pause-restart mode, thereby introducing NTPs modified with fluorescent groups such as Cy3 and Cy5 into the designated sites of long-chain RNA, and finally synthesizing long-chain RNA with fluorescent labels such as Cy3 and Cy5 at the designated sites.

[0004] However, compared with the four unmodified NTPs, due to the influence of steric hindrance, the efficiency of NTPs modified with large fluorescent groups such as Cy3 and Cy5 being recognized and incorporated into RNA by T7 RNA polymerase is significantly reduced, making it extremely difficult to use PLOR to synthesize large amounts of long-chain RNA with fluorescent labels at designated sites. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for efficiently synthesizing long-chain RNA with a fluorescent label at a specified site, thereby solving the inefficiency of the PLOR method in fluorescent labeling; and applying it to the target RNA to prove its efficiency and practicality, in order to provide a method reference for the fluorescent labeling and synthesis of more long-chain RNAs with important biological functions.

[0006] Technical solution: To solve the above technical problems, the present invention provides a method for synthesizing site-selective azide-labeled long-chain RNA (azide-RNA), comprising the following steps:

[0007] (1) Incubate the target DNA with biotin modification at the 5' end of the coding strand with agarose beads coated with streptavidin to obtain solid-phase bead-DNA, which serves as a DNA template for the transcription system;

[0008] (2) Based on the PLOR technology, the bead-DNA described in step (1) is used to synthesize the azide-RNA. The solid-phase bead-DNA is used as a template for transcription in the PLOR system, and transcription can be initiated by adding liquid-phase T7 RNA polymerase, NTPs, etc. The added NTPs are controlled to be only three or fewer types, and the T7 RNA polymerase will be forced to pause transcription at a specific site due to the lack of the corresponding type of NTP. Through solid-liquid phase separation and solid phase cleaning, a new NTP combination (still only three or fewer types) is re-added to the transcription system to restart transcription. After multiple rounds of transcription pause-restart, until the T7 RNA polymerase transcription reaches the upstream of the marking site, azide-NTP is added to the transcription system to replace the corresponding unmodified NTP, thereby introducing azide into the corresponding site of the target RNA; after the labeling is completed, all four types of NTPs are added to the transcription system, so that the transcription enters the termination stage and the synthesis of the full-length RNA is completed;

[0009] Wherein, the target DNA with biotin modification at the 5' end of the coding chain in step (1) is obtained by PCR amplification using a plasmid containing the target DNA as a template; the 5' end of the forward primer of the PCR amplification is modified with biotin.

[0010] Wherein, the plasmid containing the target DNA also contains a linker sequence and a promoter sequence recognized and bound by T7 RNA polymerase.

[0011] Wherein, the streptavidin-coated agarose beads in step (1) include streptavidin-coated agarose beads (streptavidin-agarose beads) and neutral avidin-coated agarose beads (neutral avidin-agarose beads).

[0012] Its synthetic route is as follows:

[0013]

[0014] The detailed steps include:

[0015] 1) Design the corresponding DNA template sequence according to the sequence of the target RNA to be synthesized, and insert the promoter sequence and linker sequence that are specifically recognized and bound by T7 RNA polymerase (T7 RNA polymerase, T7 RNAP) upstream of the transcription start point. Obtain a plasmid containing the target DNA sequence (linker + promoter sequence + template DNA sequence) through plasmid construction as a template for the PCR system;

[0016] 2) Design corresponding primers to amplify the target DNA fragment. The 5' end of the forward primer is modified with biotin, and the target DNA fragment (biotin-DNA) with biotin modification on the 5' end of the target DNA coding chain is obtained through PCR amplification and product purification.

[0017] 3) Incubate the biotin-DNA with streptavidin-agarose beads to obtain solid-phase bead-DNA for PLOR reaction.

[0018] 4) Design the corresponding PLOR synthesis strategy according to the labeling site on the target RNA, and introduce the azide group into the designated site. Among them, add transcription buffer, T7RNAP, and NTPs to the solid phase bead-DNA, and transcription will begin (transcription initiation, Step 1). At this time, the added NTP types are controlled to be less than 4, and T7RNAP will pause at the missing NTP site. After multiple washings to remove the excess NTPs in the system, the transcription enters the extension stage (Step 2), and continues to add less than 4 types of NTPs to restart the transcription, and T7RNAP will pause again at the missing NTP site. After multiple rounds of transcription pause-restart, until T7RNAP transcription reaches the upstream of the labeling site, azide-NTP is added to the transcription system to replace the corresponding unmodified NTP, so as to introduce azide into the corresponding site of the target RNA (Step X). After the labeling is completed, all 4 NTPs are added to the transcription system, so that the transcription enters the termination stage (Step X+1), and the synthesis of the full-length RNA is completed, and finally the target RNA (azide-RNA) with azide modification at the designated site is obtained.

[0019] Furthermore, the biotin-DNA templates mentioned in steps 1) and 2) can be directly synthesized by a biological company.

[0020] The present invention also provides an azide-RNA synthesized by the method.

[0021] The present invention also provides the use of the azide-RNA in synthesizing a long-chain RNA with a fluorescent marker at a designated site.

[0022] The present invention also provides a method for synthesizing a long-chain RNA with a fluorescent label at a designated site, wherein azide-RNA and a fluorescent group modified with an orthogonal reagent are subjected to a copper-free catalyzed SPAAC click chemistry reaction to generate the fluorescently labeled long-chain RNA.

[0023] Wherein, the fluorescent groups include Cy3 and Cy5.

[0024] Wherein, the molar ratio of the fluorescent group modified by the orthogonal reagent to azide-RNA is 500-1000:1.

[0025] Furthermore, the molar ratio of the fluorescent group modified by the orthogonal reagent to azide-RNA is 500-750:1.

[0026] Wherein, the click chemistry reaction time is 2-90min.

[0027] Wherein, the temperature of the click chemistry reaction is 25-37°C.

[0028] The designated sites include designated sites of four different structural regions: bulge loop, inner loop, double helix, and single strand.

[0029] The specific synthetic route is as follows:

[0030]

[0031] The specific reaction process is as follows:

[0032] 1) Add DBCO-Cy3 to the azide-RNA synthesized by PLOR and react at room temperature for 90 minutes to allow the azide-RNA to react to generate Cy3-RNA.

[0033] 2) The excess DBCO-Cy3 small molecules in the reaction product of 1) are removed by concentration and ultrafiltration to obtain Cy3-RNA.

[0034] Wherein, the reaction temperature in step 1) can be increased to 37°C.

[0035] Among them, the reaction time of SPAAC occurring in different structural regions of RNA in step 1) varies, ranging from 2 to 90 minutes.

[0036] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0037] Compared with existing methods, the efficiency of synthesizing site-specific fluorescently labeled long-chain RNA in the present invention is significantly improved, making it possible to synthesize large amounts of fluorescently labeled target RNA, and greatly promoting the use of more abundant fluorescent detection methods to deeply study the dynamic structural characteristics and regulatory functions of target RNA inside and outside the cell.

[0038] The improvement in synthesis efficiency enables this invention to be further applied to the fluorescent labeling and synthesis of longer-chain RNA drugs and RNA vaccines, showing great potential in the application research of RNA drug development and RNA as a diagnostic tool. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is the urea-PAGE detection picture of the synthesized U43azide-riboSAM;

[0040] Figure 2 The yield detection (A) and quantitative analysis (B) of U43Cy3-riboSAM generated by the reaction of equal amounts of U43azide-riboSAM under different concentrations of DBCO-Cy3;

[0041] Figure 3 The yield detection (A) and quantitative analysis (B) of U43Cy3-riboSAM generated by the reaction of U43azide-riboSAM and excess DBCO-Cy3 after different reaction times;

[0042] Figure 4 It is the urea-PAGE detection picture of the synthesized U55azide-riboSAM;

[0043] Figure 5 The yield detection (A) and quantitative analysis (B) of U55Cy3-riboSAM generated by the reaction of equal amounts of U55azide-riboSAM under different concentrations of DBCO-Cy3;

[0044] Figure 6 The yield detection (A) and quantitative analysis (B) of U55Cy3-riboSAM generated by the reaction of U55azide-riboSAM and excess DBCO-Cy3 after different reaction times;

[0045] Figure 7 The yield detection (A) and quantitative analysis (B) of U55Cy3-riboSAM generated after U55azide-riboSAM reacted with excess DBCO-Cy3 for 5 and 10 minutes at different reaction temperatures;

[0046] Figure 8 It is the urea-PAGE detection picture of the synthesized U74azide-riboSAM;

[0047] Fig. 9 The yield detection (A) and quantitative analysis (B) of U74Cy3-riboSAM generated by the reaction of equal amounts of U74azide-riboSAM under different concentrations of DBCO-Cy3;

[0048] Fig.10 The yield detection (A) and quantitative analysis (B) of U74Cy3-riboSAM generated by the reaction of U74azide-riboSAM and excess DBCO-Cy3 after different reaction times;

[0049] Fig.11 It is the urea-PAGE detection picture of the synthesized U84azide-riboSAM;

[0050] Fig.12 The yield detection (A) and quantitative analysis (B) of U84Cy3-riboSAM generated by the reaction of equal amounts of U84azide-riboSAM under different concentrations of DBCO-Cy3;

[0051] Fig.13 The yield detection (A) and quantitative analysis (B) of U84Cy3-riboSAM generated by the reaction of U84azide-riboSAM and excess DBCO-Cy3 after different reaction times. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0053] Example 1

[0054] This example uses the above invention to efficiently synthesize a SAM-VI riboswitch RNA (U43Cy3-riboSAM) with a Cy3 fluorescent label at the U43 site on the convex ring. The secondary structure of U43Cy3-riboSAM is as follows:

[0055]

[0056] The synthesis process is divided into two stages: the synthesis of U43azide-riboSAM and the post-transcriptional SPAAC reaction.

[0057] 1. Synthesis of U43azide-riboSAM

[0058] 1) DNA template preparation: According to the sequence of riboSAM, the DNA template and primer sequences were designed as shown in Table 1.

[0059] Table 1 Sequence of riboSAM and DNA template and primer sequences used for riboSAM synthesis

[0060]

[0061]

[0062] Note: The underlined sequence indicates the promoter sequence recognized and bound by T7 RNA polymerase. The italic sequence is the linker, which is used to relieve the steric hindrance between T7 RNA polymerase and beads after DNA is connected to beads. mC refers to the 2'-methylation modification of the terminal C.

[0063] The target DNA sequence was sent to the gene synthesis department of Sangon Biotech Co., Ltd., and the department was asked to insert the sequence into the multiple cloning site of the pUC57 plasmid to obtain a recombinant plasmid (pUC57-riboSAM) containing the target DNA sequence. At the same time, the primer sequence was sent to Sangon Biotech Co., Ltd. for synthesis to obtain primers for PCR amplification.

[0064] 2) PCR amplification to obtain biotin-DNA. The plasmid obtained in 1) was used as a PCR template, and the target DNA fragment was amplified in large quantities by the PCR amplification system shown in Table 2 (the amplification program is shown in Table 3), and the PCR product was purified by urea-polyacrylamide gel, and finally about 11 nmole of biotin-DNA template was obtained.

[0065] Table 2 PCR amplification system

[0066]

[0067] Table 3 PCR amplification program

[0068]

[0069] 3) Preparation of bead-DNA. The biotin-DNA obtained in 2) was incubated with 110uL of streptavidin-coated agarose beads (produced by Changzhou Tiandirenhe Biotechnology Co., Ltd.) at 4°C overnight to obtain about 10nmole of bead-DNA.

[0070] 4) Synthesis of U43azide-riboSAM. The following 7-step PLOR reaction was designed to carry out azide labeling on the U43 site of the riboSAM convex ring to obtain U43azide-riboSAM. The reagent ratio, reaction temperature and time of each step of the reaction are shown in Table 4.

[0071]

[0072] Table 4 Reagent ratios and reaction conditions for the 7-step PLOR reaction to synthesize U43azide-riboSAM

[0073]

[0074] Experimental results: Figure 1 As shown, the synthesized U43azide-riboSAM was subjected to urea-polyacrylamide gel (urea-PAGE) detection, and the unmodified riboSAM obtained by in vitro transcription was used as a reference, and a single gel band of U43azide-riboSAM was successfully detected, indicating the successful synthesis of high-purity U43azide-riboSAM.

[0075] 2. Explore the optimal reaction conditions for generating U43Cy3-riboSAM through post-transcription SPAAC click chemistry reaction between U43azide-riboSAM and DBCO-Cy3.

[0076] 1) The reaction yield is close to 100% when DBCO-Cy3 reaches 500 times that of U43azide-riboSAM.

[0077] Since the SPAAC reaction between azide and DBCO requires the presence of an excess of DBCO, under the condition that an equal amount of U43azide-riboSAM exists, the present invention sets the DBCO-Cy3 concentration to be 1, 10, 50, 100, 250, 500, 750, and 1000 times (1×-1000×) of azide-riboSAM, respectively, reacts at 37° C. for 30 minutes, and uses urea-PAGE to detect the amount of U43Cy3-riboSAM generated at different DBCO-Cy3 concentrations, in order to obtain the DBCO-Cy3 dosage with the highest yield.

[0078] Experimental results: Figure 2 As shown in A, fluorescent gel imaging shows that when there is 1 and 10 times of DBCO-Cy3, almost no U43Cy3-riboSAM is generated until the DBCO-Cy3 concentration is increased to 50 times ( Figure 2 A, the upper figure), U43Cy3-riboSAM began to be generated. As the concentration of DBCO-Cy3 further increased, the amount of U43Cy3-riboSAM generated gradually increased ( Figure 2 A), gel quantitative analysis showed that DBCO-Cy3 tended to produce the maximum reaction yield after reaching 500 times ( Figure 2 B). UV gel imaging showed the same results ( Figure 2A). In the UV gel imaging, compared with U43azide-riboSAM, the addition of the fluorescent group Cy3 makes U43Cy3-riboSAM appear as a slightly delayed band. At the same time, it can be seen that when the concentration of DBCO-Cy3 reaches 500 times or higher, almost all U43azide-riboSAM is converted into U43Cy3-riboSAM ( Figure 2 The lower figure in A also shows that the SPAAC reaction occurring on RNA also exhibits a reaction efficiency of nearly 100%.

[0079] 2) The SPAAC reaction between U43azide-riboSAM and DBCO-Cy3 is extremely rapid.

[0080] The reaction rate of the two was further tested in the presence of an equal amount of U43azide-riboSAM and a DBCO-Cy3 concentration 500 times that of U43azide-riboSAM. At a reaction temperature of 37°C, the reaction time was set at 2, 4, 6, 8, 10, 20, and 30 min, and the amount of U43Cy3-riboSAM generated was detected by urea-PAGE.

[0081] Experimental results: Figure 3 As shown in the figure, gel imaging detection shows that the reaction between the two is extremely rapid, and the reaction is basically complete after 2 minutes, which is manifested by the generation of equal amounts of U43Cy3-riboSAM under fluorescence imaging and the conversion of all U43azide-riboSAM to U43y3-riboSAM under UV imaging after different reaction times (2, 4, 6, 8, 10, 20, 30 minutes). Figure 3 A). It can be seen that the SPAAC reaction occurring on the riboSAM convex ring is fast and efficient, and Cy3 can be efficiently introduced into the U43 site to finally obtain U43Cy3-riboSAM.

[0082] Example 2

[0083] This example explores whether the azide labeling and SPAAC reaction of the U55 site on the inner ring of riboSAM are equally fast and efficient, in order to clarify whether this technology can efficiently fluorescently label designated sites in different structural regions of the target RNA. The specific research process also includes the synthesis of U55azide-riboSAM and the establishment and optimization of the post-transcription SPAAC reaction system. Among them, the secondary structure of U55Cy3-riboSAM is as follows:

[0084]

[0085] 1. Synthesis of U55azide-riboSAM

[0086] The bead-DNA obtained in Example 1 was used as a transcription template, and the U55 site of the inner loop of riboSAM was azide-labeled by the following 9-step PLOR reaction to obtain U55azide-riboSAM. The reagent ratio, reaction temperature and time of each step of the reaction are shown in Table 5.

[0087]

[0088] Table 5 Reagent ratios and reaction conditions for the 9-step PLOR reaction to synthesize U55azide-riboSAM

[0089]

[0090]

[0091] Experimental results: Figure 4 As shown, the synthesized U55azide-riboSAM was subjected to urea-PAGE detection, and the unmodified riboSAM obtained by in vitro transcription was used as a reference. A single gel band of U55azide-riboSAM was successfully detected, indicating that high-purity U55azide-riboSAM was successfully synthesized.

[0092] 2. Explore the optimal reaction conditions for generating U55Cy3-riboSAM through post-transcription SPAAC click chemistry reaction between U55azide-riboSAM and DBCO-Cy3.

[0093] 1) DBCO-Cy3 tends to produce the maximum reaction yield when it reaches 500 times that of U55azide-riboSAM.

[0094] In the presence of an equal amount of U55azide-riboSAM, the DBCO-Cy3 concentration was set to 1, 10, 50, 100, 250, 500, 750, and 1000 times that of U55azide-riboSAM, respectively. The reaction was carried out at 37°C for 30 min, and urea-PAGE was used to detect the amount of U55Cy3-riboSAM generated at different DBCO-Cy3 concentrations.

[0095] Experimental results: Figure 5 As shown in A, fluorescent gel imaging showed that, similar to the U43 site, with the increase of DBCO-Cy3 concentration, the amount of U55Cy3-riboSAM generated gradually increased. Gel quantitative analysis further showed that when DBCO-Cy3 reached 500 times (500×), the maximum reaction yield was generated ( Figure 5B).

[0096] 2) The SPAAC reaction between U55azide-riboSAM and DBCO-Cy3 is slightly slower than that between U43azide-riboSAM.

[0097] In the presence of equal amounts of U55azide-riboSAM and a DBCO-Cy3 concentration 500 times that of U55azide-riboSAM, the reaction rates of the two were further tested at 37°C. The reaction times were set to 2, 4, 6, 8, and 10 min, respectively, and the amount of U43Cy3-riboSAM generated was detected by urea-PAGE.

[0098] Experimental results: Figure 6 As shown, fluorescent gel imaging detection and quantitative analysis show that, unlike the U43 site, the reaction between U55azide-riboSAM and DBCO-Cy3 needs 6 minutes to complete. The crystal structure shows that in the ligand-bound state, U35 (corresponding to the U55 site in the present invention) of the truncated 55nt riboSAM of the same family is wrapped inside the RNA, so this also indicates that the contact between the U55 site and DBCO-Cy3 may not be as direct and sufficient as the U43 site, so the reaction speed of SPAAC is also slightly slower.

[0099] 3) The yield of post-transcription SPAAC reaction at 37°C was higher than that at 25°C and 30°C.

[0100] In the presence of an equal amount of U55azide-riboSAM and a DBCO-Cy3 concentration 500 times that of U55azide-riboSAM, three different reaction temperatures were set at 25°C, 30°C, and 37°C, and the amount of product U55Cy3-riboSAM generated was detected after 5 and 10 minutes of reaction, respectively.

[0101] Experimental results: Figure 7 As shown, fluorescent gel imaging detection and quantitative analysis show that the reaction yield at 30°C and 37°C is significantly higher than that at 25°C, especially after 10 minutes of reaction at 37°C. In view of this, all SPAAC reactions in the present invention are carried out at 37°C.

[0102] Example 3

[0103] This example further explores the azide labeling of the U74 site on the riboSAM double helix structure and the system and yield of the SPAAC reaction. The specific steps also include the synthesis of U74azide-riboSAM and the establishment and optimization of the post-transcription SPAAC reaction system. Among them, the secondary structure of U74Cy3-riboSAM is as follows:

[0104]

[0105] 1. Synthesis of U74azide-riboSAM

[0106] The bead-DNA obtained in Example 1 was used as a transcription template, and the U74 site of the riboSAM double helix region was azide-labeled by the following 12-step PLOR reaction to obtain U74azide-riboSAM. The reagent ratio, reaction temperature and time of each step of the reaction are shown in Table 6.

[0107]

[0108] Table 6 Reagent ratios and reaction conditions for the 12-step PLOR reaction to synthesize U74azide-riboSAM

[0109]

[0110] Experimental results: Figure 8 As shown, the synthesized U74azide-riboSAM was subjected to urea-PAGE detection, and the unmodified riboSAM obtained by in vitro transcription was used as a reference. A single gel band of U74azide-riboSAM was successfully detected, indicating that high-purity U74azide-riboSAM was successfully synthesized.

[0111] 2. Explore the optimal reaction conditions for generating U74Cy3-riboSAM through post-transcription SPAAC click chemistry reaction between U74azide-riboSAM and DBCO-Cy3.

[0112] 1) DBCO-Cy3 tends to produce the maximum reaction yield when it reaches 500 times that of U74azide-riboSAM.

[0113] In the presence of an equal amount of U74azide-riboSAM, the DBCO-Cy3 concentration was set to 1, 10, 50, 100, 250, 500, 750, and 1000 times that of U74azide-riboSAM, respectively. The reaction was carried out at 37°C for 90 min, and urea-PAGE was used to detect the amount of U74Cy3-riboSAM generated at different DBCO-Cy3 concentrations.

[0114] Experimental results: Fig. 9 As shown in Figure 2, fluorescent gel imaging showed that, similar to the U43 and U55 sites, with the increase of DBCO-Cy3 concentration, the amount of U74Cy3-riboSAM generated gradually increased ( Fig. 9A). Further gel quantitative analysis showed that the maximum reaction yield tended to be produced after DBCO-Cy3 reached 500 times (500×).

[0115] 2) The SPAAC reaction between U74azide-riboSAM and DBCO-Cy3 is slow.

[0116] Under the condition that the concentration of DBCO-Cy3 was 500 times that of U74azide-riboSAM, the reaction rate of the two was further tested at 37°C. The reaction time was set to 10, 20, 30, 40, 50, 60, and 90 min, and urea-PAGE was used to detect the amount of U74Cy3-riboSAM generated after different reaction times.

[0117] Experimental results: Fig.10 As shown, fluorescent gel imaging showed that, unlike the fast SPAAC reaction at U43 and U55 sites, the reaction between U74azide-riboSAM and DBCO-Cy3 was slower ( Fig.10 A). Further gel quantitative analysis showed that only half of the substrate had completed the reaction after 10 minutes, and the reaction was not complete until 90 minutes ( Fig.10 A, 10B). This may be because the U74 site is located on the double helix and is buried inside the RNA molecule when riboSAM folds to form a spatial structure, so the contact with DBCO-Cy3 is not as direct as other sites (U43 on the convex loop and U55 on the inner loop).

[0118] Example 4

[0119] This example further explores the azide labeling of the U84 site on the riboSAM single-stranded structure and the system and yield of the SPAAC reaction. The specific steps also include the synthesis of U84azide-riboSAM and the establishment and optimization of the post-transcription SPAAC reaction system. Among them, the secondary structure of U84Cy3-riboSAM is as follows:

[0120]

[0121] 1. Synthesis of U84azide-riboSAM

[0122] The bead-DNA obtained in Example 1 was used as a transcription template, and the U84 site of the riboSAM single-stranded region was azide-labeled by the following 12-step PLOR reaction to obtain U84azide-riboSAM. The reagent ratio, reaction temperature and time of each step of the reaction are shown in Table 7.

[0123]

[0124] Table 7 Reagent ratios and reaction conditions for the 12-step PLOR reaction to synthesize U84azide-riboSAM

[0125]

[0126]

[0127] Experimental results: Fig.11 As shown, the synthesized U84azide-riboSAM was subjected to urea-PAGE detection, and the unmodified riboSAM obtained by in vitro transcription was used as a reference, and a single gel band of U84azide-riboSAM was successfully detected, indicating that high-purity U84azide-riboSAM was successfully synthesized.

[0128] 2. Explore the optimal reaction conditions for generating U84Cy3-riboSAM through post-transcription SPAAC click chemistry reaction between U84azide-riboSAM and DBCO-Cy3.

[0129] 1) DBCO-Cy3 tends to produce the maximum reaction yield when it reaches 750 times that of U84azide-riboSAM.

[0130] In the presence of an equal amount of U74azide-riboSAM, the DBCO-Cy3 concentration was set to 1, 10, 50, 100, 250, 500, 750, and 1000 times that of U84azide-riboSAM, respectively. The reaction was carried out at 37°C for 30 min, and urea-PAGE was used to detect the amount of U84Cy3-riboSAM generated at different DBCO-Cy3 concentrations.

[0131] Experimental results: Fig.12 As shown, fluorescent gel imaging showed that, similar to other sites, with the increase of DBCO-Cy3 concentration, the amount of U55Cy3-riboSAM generated gradually increased and tended to produce the maximum reaction yield after reaching 500-750 times.

[0132] 2) The SPAAC reaction between U84azide-riboSAM and DBCO-Cy3 is extremely fast.

[0133] In the presence of equal amounts of U74azide-riboSAM and a DBCO-Cy3 concentration 750 times that of U84azide-riboSAM, the reaction rates of the two were further tested at 37°C. The reaction times were set to 2, 4, 6, 8, and 10 min, and urea-PAGE was used to detect the amount of U84Cy3-riboSAM generated after different reaction times.

[0134] Experimental results: Fig.13 As shown in the figure, fluorescent gel imaging detection and quantitative analysis show that the reaction between U84azide-riboSAM and DBCO-Cy3 can also be completed after 2 minutes. U84 is located on the single chain, and after riboSAM folds to form a three-dimensional structure, it is still exposed on the surface of the molecule and can directly contact DBCO-Cy3 and react quickly.

[0135] In summary, combining site-selective azide labeling with post-transcriptional SPAAC click chemistry reaction can achieve efficient fluorescent labeling of designated sites of long-chain RNA, making large-scale synthesis of fluorescently labeled long-chain RNA possible, and providing an important methodological reference for the fluorescent labeling and synthesis of more target RNAs with important biological functions.

Claims

1. A method for synthesizing site-selective azide-modified RNA, characterized in that: The following steps are involved: (1) Incubate the target DNA with biotin modification at the 5' end of the coding strand with agarose beads coated with streptavidin to obtain solid-phase bead-DNA; (2) Using the solid phase bead-DNA of step (1) as a template for transcription of the PLOR system, and starting transcription by adding liquid phase T7 RNA polymerase and NTPs; the NTPs are of only three or less types, and the T7 RNA polymerase is forced to pause transcription at a specific site due to the lack of the corresponding type of NTP; through solid-liquid phase separation and solid phase cleaning, a new NTP combination is re-added to the transcription system to restart transcription; through multiple rounds of transcription pause-restart, until the T7 RNA polymerase transcription reaches the upstream of the labeling site, azide-NTP is added to the transcription system to replace the corresponding unmodified NTP, so as to introduce azide into the corresponding site of the target RNA; after the labeling is completed, all four types of NTPs are added to the transcription system, so that the transcription enters the termination stage, the synthesis of the full-length RNA is completed, and azide-RNA is obtained.

2. The synthesis method according to claim 1, characterized in that: In step (1), the target DNA with biotin modification at the 5' end of the coding chain is obtained by PCR amplification using a plasmid containing the target DNA sequence as a template; the 5' end of the forward primer of the PCR amplification is modified with biotin.

3. The synthesis method according to claim 2, characterized in that: The plasmid containing the target DNA sequence contains a linker sequence and a promoter sequence recognized and bound by T7 RNA polymerase.

4. An azide-RNA synthesized by the method according to any one of claims 1 to 3.

5. Use of the azide-RNA according to claim 4 in synthesizing a long-chain RNA with a fluorescent marker at a designated site.

6. A method for synthesizing a long-chain RNA with a fluorescent label at a designated site, characterized in that: The azide-RNA described in claim 4 and the fluorescent group modified with an orthogonal reagent are subjected to a copper-free catalyzed SPAAC click chemistry reaction to generate the fluorescently labeled long-chain RNA.

7. The synthesis method according to claim 6, characterized in that: The fluorescent groups include Cy3 and Cy5.

8. The synthesis method according to claim 6, characterized in that: The molar ratio of the fluorescent group modified by the orthogonal reagent to the azide-RNA is 500-1000:

1.

9. The synthesis method according to claim 6, characterized in that: The click chemistry reaction time is 2-90 min; the temperature is 25-37°C.

10. The synthesis method according to claim 6, characterized in that: The designated sites include designated sites of four different structural regions of RNA, namely, convex loop, inner loop, double helix and single strand.

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