A method for synthesizing photo-responsive circular RNAs based on click chemistry and its applications

Abstract

The present invention discloses a novel method for synthesizing photo-responsive circular RNAs based on click chemistry and its application in CRISPR technology. During the synthesis of the RNA strand, a photo-responsive alkynyl-modified monomer is introduced at any site in the RNA strand to obtain a modified RNA. Through click chemistry, the modified RNA undergoes an intermolecular cyclization reaction with an organic compound containing bis / multi-azide groups to obtain a photo-responsive circular RNA. Existing RNA cyclization methods are only applicable to short RNA strands (20–25 nt), while the method disclosed in the present invention can achieve high-efficiency cyclization of long-chain functional RNAs; moreover, existing methods can only be used for cyclization between the head and tail of the RNA strand, while this method can achieve cyclization between any two sites in the middle / end of the strand according to different experimental requirements. Therefore, this method provides new ideas for RNA cyclization, especially responsive cyclization.

Description

Technical Field

[0001] The present invention relates to the fields of chemical biology and organic chemistry, and particularly relates to a new method for synthesizing photo-responsive circular RNA based on click chemistry and its application in CRISPR technology. Background Art

[0002] Natural circular RNA refers to a kind of RNA with a covalently closed circular structure at both ends. Since it does not have the 3' or 5'-ends of a conventional nucleic acid strand, it can effectively resist the degradation of nucleases, and thus has higher stability than linear RNA. In addition, recent studies have shown that circular RNA has various physiological functions such as a "molecular sponge" for proteins, a scaffold for protein complexes, and regulation of gene transcription and translation. Therefore, circular RNA has become a research hotspot in the current RNA field.

[0003] At present, the main methods for synthesizing circular RNA are enzymatic synthesis and chemical synthesis. The enzymatic method is a commonly used method for synthesizing circular RNA at present, but this method is only limited to natural RNA, and the cyclization efficiency for short chains is relatively low; while the chemical synthesis method mainly covalently connects the two ends of nucleic acids through simple and efficient chemical reactions, and the commonly used methods include the formation of amide bonds, cyanogen bromide-catalyzed phosphodiester bond connection, and click reaction. These methods make up for the deficiencies of the enzymatic synthesis method and can achieve the cyclization of modified RNA, especially the cyclization of stimulus-responsive RNA. For example, a previous literature (Tang, X. et al. Chem. Sci., 2018, 9, 44) reported the cyclization of siRNA by forming an amide bond with an RNA carrying a photo-responsive linker, and achieved photo-responsive regulation of the function of siRNA. However, these similar cyclization methods are all for cyclization between the head and tail of the nucleic acid strand, the reaction conditions are relatively harsh, and these head-to-tail connection methods are only applicable to relatively short nucleic acid strands (20–25 nt in length). Therefore, it is necessary to develop a new responsive RNA cyclization method for cyclization at any site in a longer functional RNA, while improving its stability against nucleases and achieving high spatiotemporal resolution regulation of its function.

[0004] The CRISPR / Cas system is a very powerful gene editing tool. In recent years, CRISPR technology has targeted genes through Cas proteins and manipulated them at specific sites, thereby mutating or correcting target genes. At the same time, CRISPR technology has also shown good application prospects in gene therapy, nucleic acid localization, and nucleic acid detection. Among them, guide RNA plays an important bridging role in the CRISPR / Cas system. On the one hand, guide RNA with a specific secondary structure needs to bind to Cas protein to form a stable RNP complex. On the other hand, the Spacer region in the guide RNA needs to be complementary to the target DNA sequence to induce the Cas protein with cutting activity to cut the target sequence. However, in the case where the guide RNA does not completely match the target DNA sequence, such as a single base mismatch between the DNA sequence and the guide RNA or a multi-base mismatch at the far end of the PAM, the CRISPR / Cas system can still recognize and cut the target sequence, resulting in an off-target effect. Therefore, applying the responsive RNA cyclization strategy to the modification of guide RNA can precisely regulate the function of the CRISPR / Cas system with high spatiotemporal resolution, which is conducive to reducing off-target effects, improving its specificity and its application potential in research / treatment. Summary of the invention

[0005] One of the purposes of the present invention is to provide a ribonucleoside phosphoramidite monomer with a photoresponsive alkynyl modification group and the synthesis of an RNA chain.

[0006] The ribonucleoside phosphoramidite monomer with a photoresponsive alkynyl modification group provided by the present invention has a structural formula as shown in Formula 1:

[0007] In the above formula 1, R 1 represents a hydroxyl protecting group, which may specifically be a DMTr group;

[0008] R 2 express (R 3 represents H, amino protecting group); (R 3 represents H, amino protecting group), (R 3 , R 4 represents any one of H, an amino protecting group).

[0009] When R 2 for When , the compound represented by Formula 1 is a responsive adenosine monomer;

[0010] When R 2 for When , the compound represented by Formula 1 is a responsive cytidine monomer;

[0011] When R 2 is the compound shown in Formula 1 is a responsive uridine monomer;

[0012] When R 2 is the compound shown in Formula 1 is a responsive guanosine monomer.

[0013] The present invention also provides a method for preparing the compound shown in Formula 1.

[0014] The method for preparing the compound shown in Formula 1 provided by the present invention comprises the following steps:

[0015] 1) Reacting the compound shown in Formula I with the compound shown in Formula II to obtain the compound shown in Formula III;

[0016]

[0017] In Formula I and Formula III, R 1 , R 2 are the same as R 1 , R 2 in Formula 1;

[0018] 2) Reacting the compound shown in Formula III with the compound shown in Formula IV to obtain the compound shown in Formula 1;

[0019]

[0020] In step 1) of the above method, the reaction is carried out in the presence of an organic amine and Bu 2 SnCl 2 ;

[0021] The organic amine may specifically be diisopropylethylamine;

[0022] The molar ratio of the compound shown in Formula I to the organic amine, Bu 2 SnCl 2 and the compound shown in Formula II may be 1:5:1.2:1.5 in sequence;

[0023] The temperature of the reaction may be 70 - 90 °C, specifically 80 °C; the time of the reaction may be 20 - 25 min, specifically 20 min;

[0024] The reaction is carried out in an organic solvent, and the organic solvent may specifically be dichloromethane.

[0025] The operation of the reaction may specifically be: dissolving the compound shown in Formula I in dry dichloromethane, adding DIPEA at room temperature at one time, and then adding Bu 2 SnCl 2, after stirring at room temperature for 2 hours, the compound shown in Formula II was slowly added. The reaction mixture was heated to 80 °C, stirred for 20 minutes, and then the reaction was stopped. TLC showed that the reaction was complete. The organic phase was washed with saturated aqueous sodium bicarbonate solution, separated, dried, and a little solid was removed by filtration. After evaporating the solvent, column chromatography separation (methylene chloride:ethyl acetate = 3:1 - 3:2) was carried out to obtain the product.

[0026] In step 2) of the above method, the reaction is carried out in the presence of an organic amine.

[0027] The organic amine can specifically be diisopropylethylamine (DIPEA);

[0028] The molar ratio of the compound shown in Formula III, the organic amine, and the compound shown in Formula IV can be 1:5:2;

[0029] The reaction is carried out at room temperature; the reaction time can be 3 - 4 h, specifically 3 h;

[0030] The reaction is carried out in an organic solvent, and the organic solvent can specifically be methylene chloride;

[0031] The reaction is carried out under the protection of an inert gas, and the inert gas can specifically be nitrogen.

[0032] The specific operation of the reaction is as follows: Under nitrogen protection, the compound shown in Formula III was dissolved in dry methylene chloride, DIPEA was added in one portion, and after stirring for 5 minutes, the compound shown in Formula IV was added. Stirring was carried out at room temperature for 3 hours. TLC showed that the reaction was complete (methylene chloride:methanol = 10:1, R f = 0.6). The mixture was diluted with ethyl acetate and washed three times with saturated aqueous sodium bicarbonate solution. The organic phase was separated, dried, and the solvent was removed. Column chromatography separation (methylene chloride:methanol = 150:1, adding 0.5% triethylamine) was carried out to obtain the product.

[0033] In step 1) of the above method, the compound shown in Formula II was prepared by a method including the following steps:

[0034] 1) Under the catalysis of Zn powder, o-nitrobenzaldehyde was reacted with propargyl bromide to obtain the compound shown in Formula S1-2 (Reference: Heckel, A. Angew. Chem. Int. Ed. 2017, 56, 359–363.).

[0035]

[0036] 2) In the presence of NaH, the compound shown in Formula S1-2 was reacted with a reagent containing a MOM group to obtain the compound shown in Formula S1-3.

[0037] 3) React the compound shown in Formula S1-3 with BCl 3 to obtain the compound shown in Formula II.

[0038] In step 2) of the above method, the reagent containing the MOM group can be MOMBr, MOMCl, etc.

[0039] The application of the above method for preparing the compound shown in Formula 1 to introduce a photo-responsive alkynyl-modified monomer at any site in the RNA strand also belongs to the protection scope of the present invention.

[0040] Another object of the present invention is to provide a method for synthesizing a photo-responsive circular RNA based on click chemistry.

[0041] The method for synthesizing a photo-responsive circular RNA based on click chemistry provided by the present invention includes the following steps:

[0042] 1) During the process of synthesizing the RNA strand by solid-phase synthesis, use the above method for preparing the compound shown in Formula 1 to introduce a photo-responsive alkynyl-modified monomer at any site in the RNA strand to obtain a modified RNA, wherein the structure of the site of the RNA strand after introducing the photo-responsive alkynyl-modified monomer is as shown in Formula 1;

[0043] 2) Through click chemistry, cause the modified RNA to undergo an intermolecular cyclization reaction with an organic compound containing a bis / multi-azide group to obtain a photo-responsive circular RNA;

[0044] In step 2), the organic compound containing a bis / multi-azide group can specifically be 1,4-bis(azidomethyl)benzene;

[0045] The operation of step 2) is: sequentially add the modified RNA, triethylammonium acetate buffer solution, 1,4-bis(azidomethyl)benzene, DMSO, MgCl 2 and ascorbic acid into the reaction tube. After degassing the reaction system, add CuSO 4 -tris(3-hydroxypropyltriazolylmethyl)amine solution and react to obtain a photo-responsive circular RNA.

[0046] The concentration of the triethylammonium acetate buffer solution is 2M and pH = 7;

[0047] 1,4-bis(azidomethyl)benzene is added in the form of a solution, and the solvent composition is H 2 O / DMSO / tBuOH with a volume ratio of 4 / 3 / 1;

[0048] The molar ratio of the modified RNA to 1,4-bis(azidomethyl)benzene can be 1:1;

[0049] Ascorbic acid and CuSO 4The molar ratio of tris(3 - hydroxypropyltriazolylmethyl)amine can be 1:1;

[0050] The reaction is carried out at room temperature, and the reaction time can be 4 - 6 h, specifically 4 h.

[0051] The photo - responsive circular RNA prepared by the above method also belongs to the protection scope of the present invention.

[0052] The synthesis method of the above photo - responsive circular RNA and the application of the obtained photo - responsive circular RNA in the cyclic modification and functional switch regulation of CRISPR / gRNA, siRNA, ribozyme and riboswitch also belong to the protection scope of the present invention.

[0053] The present invention has the following advantages compared with the existing RNA cyclization methods: the existing RNA cyclization methods can only be applied to short RNA chains (20 - 25 nt), while the method disclosed in the present invention can achieve high - efficiency cyclization of long - chain functional RNAs; and the existing methods can only be used for cyclization between the head and tail of RNA chains, while this method can achieve cyclization between any two sites in the middle / at the end of the chain according to different experimental requirements. Therefore, this method provides a new idea for RNA cyclization, especially responsive cyclization. Description of the Drawings

[0054] Figure 1 It is the cyclization result of gfp - crRNA.

[0055] Figure 2 It is the photolysis result after the cyclization of gfp - crRNA.

[0056] Figure 3 It is the stability result after the cyclization of gfp - crRNA.

[0057] Figure 4 It is the in vitro digestion result after the cyclization of gfp - crRNA.

[0058] Figure 5 It is the cyclization result of gfp - cpf1 - crRNA.

[0059] Figure 6 It is the in vitro digestion result of cpf1 - crRN - CA1.

[0060] Figure 7 It is the in vitro digestion result of cpf1 - crRN - CA2 / 3.

[0061] Figure 8 It is the MALDI - TOF of crRNA - CA1.

[0062] Figure 9 It is the MALDI - TOF of crRNA - CA2.

[0063] Figure 10 MALDI-TOF of Cpf1-crRNA-CA1.

[0064] Figure 11 MALDI-TOF of Cpf1-crRNA-CA2.

[0065] Figure 12 MALDI-TOF of Cpf1-crRNA-CA3. Detailed implementation manners

[0066] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0067] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0068] The present invention protects a new method for photo-responsive RNA cyclization based on click chemistry, which mainly includes the synthesis of RNA phosphoramidite monomers with a novel photo-responsive alkynyl modification group, the synthesis of an RNA strand containing two modification sites by solid-phase synthesis, and finally an intermolecular cyclization reaction with a small molecule bis-azide compound by click chemistry. In addition, the present invention discloses the application of this method in the cyclization and functional regulation of crRNA in the CRISPR system.

[0069] Example 1

[0070] First, we synthesized the RNA phosphoramidite monomers with this modification (including adenosine monomer, cytidine monomer, uridine monomer, and guanosine monomer) through the following design route.

[0071] The following is the synthesis of the photo-responsive alkyne derivative:

[0072]

[0073] S1-2: Activate Zn powder with dilute hydrochloric acid, stir for a moment, filter by suction, and wash with water, ethanol, and ether respectively, then dry for later use. At 0 °C, add the activated Zn powder (130 mmol, 8.3 g) to ultradry DMF (100 mL) at one time, slowly add propargyl bromide (1.9 eq, 94 mmol, 8.1 mL), after stirring for 1 hour, slowly add o-nitrobenzaldehyde S1-1 (1 eq, 50 mmol, 7.6 g), stir for 15 minutes, and TLC shows that the reaction is complete (petroleum ether: ethyl acetate = 3:1, R f = 0.3). Quench the reaction with 150 mL of saturated ammonium chloride aqueous solution, extract three times with ether, combine the organic phases, wash three times with water, separate the organic phase, dry and concentrate, and then separate by column chromatography (petroleum ether: ethyl acetate = 5:1 - 3:1), with a yield of 91%. 1 1H-NMR (300 MHz, CDCl 3 ): δ [ppm] = 7.97 - 7.94 (d, J = 9.0 Hz, 1H), 7.90 - 7.87 (d, J = 8.1 Hz, 1H), 7.70 - 7.65 (t, J = 7.4 Hz, 1H), 7.49 - 7.43 (d, J = 7.7 Hz, 1H), 5.47 - 5.45 (m, 1H), 2.95 - 2.87 (m, 2H), 2.71 - 2.63 (m, 1H), 2.12 - 2.10 (t, J = 2.6 Hz, 1H). 13 13C-NMR (75 MHz, DMSO-d 6 ): δ [ppm] = 147.7, 138.7, 133.1, 128.6, 128.5, 123.7, 81.0, 72.9, 66.3, 28.1.

[0074] S1-3: At 0 °C, dissolve NaH (60%, 1.5 eq, 15 mmol, 600 mg) in dry THF (20 mL), slowly add S1-2 (10 mmol, 1.91 g), and raise the temperature to room temperature after 15 minutes. After reacting at room temperature for 2 hours, slowly add MOMBr (1.2 eq, 12 mmol, 0.91 mL), and react at room temperature for 5 min. TLC shows that the reaction is complete (petroleum ether: ethyl acetate = 3:1, R f = 0.5). Spread about 1 cm of diatomaceous earth on a Buchner funnel with filter paper, filter, rotary evaporate the filtrate, and then separate by column chromatography (petroleum ether: ethyl acetate = 15:1), with a yield of 94%. 1 1H-NMR (500 MHz, CDCl 3): δ [ppm] = 7.97 - 7.96 (d, J = 8.2 Hz, 1H), 7.86 - 7.84 (d, J = 7.9 Hz, 1H), 7.68 - 7.65 (t, J = 7.6 Hz, 1H), 7.48 - 7.45 (t, J = 7.8 Hz, 1H), 5.44 - 5.42 (m, 1H), 4.68 - 4.67 (dd, J = 2.9 Hz, 6.9 Hz, 1H), 4.55 - 4.53 (dd, J = 3.2 Hz, 6.9 Hz, 1H), 3.37 (s, 1H), 2.89 - 2.85 (m, 1H), 2.78 - 2.73 (m, 1H), 2.034 - 2.028 (d, J = 2.9 Hz, 1H). 13 C-NMR (125 MHz, CDCl 3 ): 148.3, 136.4, 133.3, 128.95, 128.67, 124.5, 95.3, 80.0, 71.4, 70.7, 56.0, 27.3. HRMS (ESI): m / z [M + H] + calcd for C 12 H 14 NO 4 : 236.0917; found: 236.0917.

[0075] S1-4: S1-3 (2.3 mmol, 540 mg) was added to dry n-pentane (15 mL). S1-3 was only slightly soluble in the system, so it was made as evenly distributed as possible. BCl 3 (0.5 eq, 1.1 mmol, 1 M hexane solution) was slowly added dropwise at 0 °C. The system changed from slightly soluble to clear, and there was a brown oily liquid adsorbed on the container wall. After stirring for 15 minutes, the temperature was raised to room temperature, and stirring was continued for 2 - 3 hours. A small amount of the reaction solution was taken, the solvent was removed by rotary evaporation, and the reaction progress was determined by NMR of the crude product. After the reaction was complete, the solvent was removed by rotary evaporation and directly used for the next reaction. 1 H-NMR (500 MHz, CDCl 3 ): δ [ppm] = 8.02 - 8.01 (d, J = 8.2 Hz, 1H), 7.79 - 7.77 (d, J = 7.9 Hz, 1H), 7.70 - 7.67 (t, J = 7.4 Hz, 1H), 7.52 - 7.49 (t, J = 7.4 Hz, 1H), 5.61 - 5.59 (m, 2H), 5.33 - 5.32 (d, J = 6.1 Hz, 1H), 2.93 - 2.78 (m, 2H), 2.05 - 2.04 (t, J = 2.5 Hz, 1H). 13 C-NMR (125 MHz, CDCl 3): 148.2, 134.7, 133.5, 129.2, 129.0, 124.7, 80.5, 78.9, 74.2, 71.4, 26.9。

[0076] The following is the synthesis of the responsive adenosine monomer:

[0077]

[0078] S2-2: Dissolve S2-1 (1 eq, 1.5 mmol, 1.03 g) in dry dichloromethane (25 mL). At room temperature, add DIPEA (5 eq, 7.5 mmol, 1.24 mL) all at once, and then add Bu 2 SnCl 2 (1.2 eq, 1.84 mmol, 559 mg). Stir at room temperature for 2 hours and then slowly add S1-4 (1.5 eq, 2.3 mmol). Heat the reaction mixture to 80 °C, stir for 20 minutes and then stop the reaction. TLC shows that the reaction is complete (dichloromethane:ethyl acetate = 1:2, R f = 0.6). Wash the organic phase with saturated aqueous sodium bicarbonate, separate the organic phase, dry it, filter off a little solid, evaporate the solvent and then separate by column chromatography (dichloromethane:ethyl acetate = 3:1 - 3:2), with a yield of 37%. 1 1H-NMR (300 MHz, CDCl 3 ): δ [ppm] = 9.05 - 9.03 (m, 1H), 8.74 - 8.67 (m, 1H), 8.23 - 8.11 (m, 1H), 8.04 - 7.90 (m, 3H), 7.72 - 7.19 (m, 14H), 6.82 - 6.79 (m, 4H), 6.28 - 6.11 (m, 1H), 5.46 - 5.29 (m, 1H), 5.06 - 4.91 (m, 2H), 4.79 - 4.62 (m, 1H), 4.40 - 4.35 (m, 1H), 4.26 - 4.19 (m, 1H), 3.78 (s, 6H), 3.51 - 3.36 (m, 2H), 2.83 - 2.55 (m, 3H). 13 13C-NMR (125 MHz, DMSO-d 6): 158.5, 152.3, 152.2, 151.0, 148.5, 145.3, 143.9, 136.0, 135.9, 135.3, 133.8, 132.9, 130.1, 129.6, 129.3, 129.0, 128.9, 128.2, 128.1, 127.1, 124.6, 113.6, 85.9, 80.1, 73.4, 71.8, 29.5, 27.0, 26.7, 22.6, 14.4. HRMS(ESI): m / z [M+H] + calcd for C 49 H 45 N 6 O 10 : 877.3192; found: 877.3200.

[0079] S2-3: Under nitrogen protection, dissolve S2-2 (1 eq, 0.51 mmol, 450 mg) in dry dichloromethane (5 mL), add DIPEA (5 eq, 2.60 mmol, 424 μL) in one portion, and after stirring for 5 minutes, add 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (2 eq, 1.02 mmol, 228 μL). Stir at room temperature for 3 hours. TLC shows that the reaction is complete (dichloromethane:methanol = 10:1, R f = 0.6). Dilute the mixture with ethyl acetate, wash it three times with saturated aqueous sodium bicarbonate, separate the organic phase, dry it, remove the solvent, and perform column chromatography separation (dichloromethane:methanol = 150:1, adding 0.5% triethylamine), with a yield of 56%. 1 H-NMR(400 MHz, CDCl 3 ): δ [ppm] = 9.04 - 8.98 (m, 1H), 8.77 - 8.62 (m, 1H), 8.30 - 7.83 (m, 5H), 7.61 - 7.26 (m, 12H), 6.82 - 6.79 (m, 5H), 6.28 - 5.89 (m, 1H), 5.36 - 5.20 (m, 2H), 4.94 - 4.56 (m, 3H), 4.38 - 4.31 (m, 1H), 3.78 (s, 6H), 3.67 - 3.32 (m, 6H), 2.70 - 2.54 (m, 5H), 1.32 - 1.01 (m, 12H). 13 C-NMR(125 MHz, CDCl 3): 158.9, 153.2, 151.8, 136.5, 135.8, 134.0, 133.1, 130.2, 130.1, 129.0, 128.9, 128.28, 128.25, 128.2, 127.9, 127.8, 113.2, 92.2, 91.5, 71.7, 71.2, 43.4, 42.0, 29.8, 29.7, 29.6, 29.5, 29.3, 29.2, 27.2, 27.1, 27.0, 24.62, 24.58, 24.5。 31 P NMR: 150.66, 150.51. HRMS(ESI): m / z [M+H] + calcd for C 58 H 62 N 8 O 11 P: 1077.4270; found: 1077.4270。

[0080] The following is the synthesis of responsive cytidine monomers:

[0081]

[0082] S3-2: Dissolve S3-1 (1 eq, 1.5 mmol, 881 mg) in dry dichloromethane (25 mL). After adding DIPEA (5 eq, 7.5 mmol, 1.24 mL) all at once at room temperature, then add Bu 2 SnCl 2 (1.2 eq, 1.84 mmol, 559 mg). Stir for 2 hours at room temperature and then slowly add S1-4 (1.5 eq, 2.3 mmol). Heat the reaction mixture to 80 °C, stir for 20 minutes and then stop the reaction. TLC shows that the reaction is complete (dichloromethane:methanol = 20:1, R f = 0.5). Wash the organic phase with saturated aqueous sodium bicarbonate, separate the organic phase and dry it, filter off a little solid, evaporate the solvent and then separate by column chromatography (dichloromethane:methanol = 60:1), with a yield of 58%. 1 H-NMR (400 MHz, DMSO-d 6): δ [ppm] = 10.93 (s, 1H), 8.27 - 8.21 (m, 1H), 7.99 - 7.95 (m, 1H), 7.81 - 7.70 (m, 2H), 7.58 - 7.53 (m, 1H), 7.41 - 7.25 (m, 9H), 6.91 - 6.89 (m, 4H), 5.76 - 5.73 (m, 1H), 5.52 - 5.36 (m, 2H), 5.12 - 4.97 (m, 1H), 4.79 - 4.68 (m, 1H), 4.33 - 4.16 (m, 2H), 4.02 - 4.00 (m, 1H), 3.76 (s, 6H), 3.33 - 3.31 (m, 2H), 2.86 - 2.73 (m, 3H), 2.11 (s, 3H). 13 C-NMR (100 MHz, DMSO-d 6 ): 171.4, 162.9, 158.6, 154.7, 154.7, 148.6, 148.5, 144.9, 135.9, 135.62, 135.57, 133.8, 133.7, 130.24, 130.17, 129.7, 129.5, 128.4, 128.2, 127.3, 124.6, 113.7, 95.9, 92.6, 89.7, 86.4, 82.3, 80.5, 80.4, 79.1, 78.9, 74.0, 73.9, 71.4, 71.3, 68.3, 67.9, 62.1, 61.9, 26.8, 26.4, 24.8. HRMS (ESI): m / z [M + H] + calcd for C 43 H 43 N 4 O 11 : 791.2923; found: 791.2909.

[0083] S3-3: Under nitrogen protection, dissolve S3-2 (1 eq, 406 mg, 0.51 mmol) in dry dichloromethane (5 mL), add DIPEA (5 eq, 2.60 mmol, 424 μL) in one portion, stir for 5 minutes, and then add 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (2 eq, 1.02 mmol, 228 μL). Stir at room temperature for 3 hours. TLC shows that the reaction is complete (DCM:EA = 1:2, R f = 0.4). Dilute the mixture with ethyl acetate, wash it three times with saturated aqueous sodium bicarbonate solution, separate the organic phase, dry it, remove the solvent, and perform column chromatography separation (dichloromethane:ethyl acetate = 1:1, adding 0.5% triethylamine), with a yield of 63%. 1 H-NMR (500 MHz, CDCl 3): δ [ppm] = 9.16 - 9.12 (m, 1H), 8.45 - 8.23 (m, 1H), 7.88 - 7.80 (m, 2H), 7.57 - 7.53 (m, 1H), 7.36 - 7.19 (m, 9H), 6.82 - 6.77 (m, 4H), 6.03 - 5.87 (m, 1H), 5.59 - 5.50 (m, 1H), 5.14 - 5.04 (m, 1H), 4.79 - 4.62 (m, 1H), 4.42 - 4.07 (m, 3H), 3.75 - 3.74 (d, 6H), 3.50 - 3.36 (m, 6H), 2.83 - 2.53 (m, 5H), 2.17 - 2.16 (m, 3H), 1.22 - 1.03 (m, 12H). 13 C-NMR (125 MHz, CDCl 3 ): 170.2, 162.5, 158.8, 155.3, 148.8, 148.6, 144.9, 144.2, 135.9, 135.6, 135.4, 135.3, 135.2, 133.4, 133.1, 130.4, 130.2, 129.6, 129.5, 128.6, 128.4, 128.0, 127.3, 124.3, 117.7, 113.4, 113.3, 96.5, 93.3, 92.8, 90.1, 89.6, 87.1, 87.0, 79.9, 79.7, 79.5, 78.9, 72.2, 71.6, 71.3, 71.23, 71.16, 60.9, 60.5, 58.4, 58.2, 47.2, 45.4, 45.3, 43.3, 27.1, 26.9, 25.0, 24.6, 24.5, 24.4, 23.0, 22.9, 20.2, 20.1. 31 P NMR: 150.81. HRMS (ESI): m / z [M + H] + calcd for C 52 H 59 N 6 O 12 PNa: 1013.3821; found: 1013.3824.

[0084] The following is the synthesis of the responsive uridine monomer:

[0085]

[0086] S4-2: Dissolve S4-1 (1 eq, 1.5 mmol, 819 mg) in dry dichloromethane (25 mL). At room temperature, add DIPEA (5 eq, 7.5 mmol, 1.24 mL) all at once, and then add Bu 2SnCl 2 (1.2 eq, 1.84 mmol, 559 mg), and S1-4 (1.5 eq, 2.3 mmol) was slowly added thereto after stirring at room temperature for 2 h. The reaction mixture was heated to 80 °C, stirred for 20 min and then the reaction was stopped. TLC showed that the reaction was complete (dichloromethane:ethyl acetate = 2:1, R f = 0.7). The organic phase was washed with saturated aqueous sodium bicarbonate, separated, dried, filtered to remove a little solid, the solvent was evaporated, and then column chromatography was carried out (dichloromethane:ethyl acetate = 3:1), with a yield of 38%. 1 1H-NMR (400 MHz, DMSO-d 6 ): δ [ppm] = 11.4 - 11.3 (m, 1H), 7.99 - 7.93 (m, 1H), 7.78 - 7.52 (m, 4H), 7.39 - 7.30 (m, 4H), 7.26 - 7.24 (m, 5H), 6.91 - 6.89 (m, 4H), 5.84 - 5.68 (m, 1H), 5.42 - 5.34 (m, 2H), 5.25 - 5.20 (m, 1H), 4.94 - 4.87 (m, 1H), 4.73 - 4.60 (m, 1H), 4.28 - 4.13 (m, 2H), 3.93 - 3.92 (m, 1H), 3.74 (s, 1H), 3.25 - 3.22 (m, 2H), 2.77 - 2.73 (m, 2H), 2.71 - 2.69 (m, 1H). 13 13C-NMR (100 MHz, DMSO-d 6 ): 163.4, 158.6, 150.7, 148.6, 145.2, 135.8, 135.6, 133.8, 130.3, 129.6, 128.4, 128.2, 124.7, 113.7, 102.3, 87.9, 86.4, 83.2, 80.3, 78.0, 74.0, 71.3, 69.0, 65.5, 63.3, 60.4, 27.0, 26.5, 14.6. HRMS (ESI): m / z [M-H] - calcd for C 41 H 38 N 3 O 11 : 748.2512; found: 748.2501.

[0087] S4-3: Under nitrogen protection, dissolve S4-2 (1 eq, 450 mg, 0.6 mmol) in dry dichloromethane (10 mL), add DIPEA (5 eq, 3.0 mmol, 496 μL) in one portion, and after stirring for 5 minutes, add 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (2 eq, 1.2 mmol, 268 μL). Stir at room temperature for 3 hours. TLC shows that the reaction is complete (dichloromethane:ethyl acetate = 1:1, R f = 0.8). Dilute the mixture with ethyl acetate, wash it three times with saturated aqueous sodium bicarbonate solution, separate the organic phase, dry it, remove the solvent, and perform column chromatography separation (dichloromethane:methanol = 150:1, adding 0.5% triethylamine), with a yield of 47%. 1 1H-NMR (500 MHz, CDCl 3 3): δ [ppm] = 7.90 - 7.84 (m, 2H), 7.78 - 7.75 (m, 1H), 7.57 - 7.51 (m, 1H), 7.40 - 7.30 (m, 3H), 7.24 - 7.19 (m, 7H), 6.79 - 6.77 (m, 4H), 6.02 - 5.85 (m, 1H), 5.47 - 5.45 (m, 1H), 5.27 - 5.09 (m, 1H), 4.97 - 4.87 (m, 1H), 4.72 - 4.55 (m, 1H), 4.48 - 4.31 (m, 2H), 4.17 - 4.10 (m, 1H), 3.73 (s, 6H), 3.55 - 3.30 (m, 6H), 2.80 - 2.30 (m, 5H), 1.11 - 1.03 (m, 12H). 13 13C-NMR (125 MHz, CDCl 3): 163.0, 158.8, 150.09, 150.06, 150.0, 148.7, 148.5, 148.2, 148.0, 144.30, 144.27, 144.2, 140.1, 140.0, 136.0, 133.2, 130.30, 130.27, 129.3, 129.2, 129.1, 128.81, 128.77, 128.7, 128.3, 128.03, 128.01, 127.2, 124.5, 124.4, 117.8, 117.5, 117.4, 113.3, 102.33, 102.28, 93.4, 92.9, 92.6, 88.1, 88.0, 87.7, 87.2, 87.1, 82.9, 82.7, 82.6, 79.7, 79.6, 78.3, 71.5, 71.4, 71.3, 71.2, 71.1, 61.7, 61.5, 60.4, 58.6, 58.5, 58.0, 57.9, 49.4, 43.4, 43.34, 43.28, 43.2, 43.1, 30.7, 29.7, 29.6, 27.1, 26.9, 24.71, 24.65, 24.6, 24.51, 24.46, 21.1, 20.44, 20.38, 20.23, 20.17, 17.7, 14.2。 31 P NMR: 150.76, 150.19. HRMS(ESI): m / z [M+Na] + calcd for C 50 H 56 N 5 O 12 PNa: 972.3555; found: 972.3552。

[0088] The following is the synthesis of responsive guanosine monomers:

[0089]

[0090] S5-2: Dissolve S5-1 (1 eq, 1.5 mmol, 984 mg) in dry dichloromethane (25 mL). After adding DIPEA (5 eq, 7.5 mmol, 1.24 mL) all at once at room temperature, then add Bu 2 SnCl 2 (1.2 eq, 1.84 mmol, 559 mg). Stir at room temperature for 2 hours and then slowly add S1-4 (1.5 eq, 2.3 mmol). Heat the reaction mixture to 80 °C, stir for 20 minutes and then stop the reaction. TLC shows that the reaction is complete (dichloromethane:methanol = 20:1, R f= 0.4). The organic phase was washed with saturated aqueous sodium bicarbonate, separated, dried, filtered to remove a little solid, and the solvent was evaporated and then separated by column chromatography (methylene chloride: methanol = 100:1), with a yield of 54%. 1 H-NMR (400 MHz, DMSO-d 6 ): δ [ppm] = 12.14 - 12.05 (m, 1H), 11.63 - 11.58 (m, 1H), 8.11 - 7.80 (m, 2H), 7.70 - 7.52 (m, 2H), 7.42 - 7.16 (m, 10H), 6.86 - 6.77 (m, 4H), 5.99 - 5.57 (m, 1H), 5.38 - 5.03 (m, 2H), 4.93 - 4.49 (m, 3H), 4.39 - 4.29 (m, 1H), 4.06 - 3.98 (m, 2H), 3.73 - 3.71 (d, J = 6.68, 6H), 3.30 - 3.15 (m, 2H), 2.78 - 2.58 (m, 3H), 2.46 (s, 1H), 1.23 - 1.13 (m, 6H). 13 C-NMR (100 MHz, DMSO-d 6 ): 180.9, 169.3, 158.6, 158,5, 155.5, 149.2, 147.7, 137.8, 135.9, 133.8, 133.5, 130.2, 129.7, 128.3, 128.2, 124.8, 120.9, 113.6, 86.1, 80.5, 80.0, 79.1, 73.8, 71.0, 69.6, 64.1, 60.3, 35.2, 26.9, 21.2, 19.4, 19.3. HRMS (ESI): m / z [M+Na] + calcd for C 46 H 46 N 6 O 11 Na: 881.3117; found: 881.3112.

[0091] S5-3: Under nitrogen protection, S5-2 (1 eq, 438 mg, 0.51 mmol) was dissolved in dry methylene chloride (10 mL), and DIPEA (5 eq, 3.0 mmol, 424 μL) was added in one portion. After stirring for 5 minutes, 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (2 eq, 1.0 mmol, 228 μL) was added. Stir at room temperature for 3 hours. TLC showed that the reaction was complete (methylene chloride: ethyl acetate = 1:2, R f= 0.5). The mixture was diluted with ethyl acetate and washed three times with saturated aqueous sodium bicarbonate. The organic layer was separated, dried, and the solvent was removed. Column chromatography separation (methylene chloride:ethyl acetate = 1:1, plus 0.5% triethylamine) gave a yield of 31.0%. 1 H-NMR(500MHz,CDCl 3 ): δ [ppm] = 11.79(s, 1H), 7.85 - 7.19(m, 13H), 7.16 - 7.15(m, 1H), 6.76 - 6.71(m, 4H), 5.97 - 3.88(m, 8H), 3.71(s, 6H), 3.52 - 3.13(m, 5H), 2.69 - 2.22(m, 4H), 2.01 - 1.95(m, 1H), 1.73 - 1.70(m, 2H), 1.21 - 0.81(m, 18H). 13 C-NMR(125MHz,DMSO-d 6 ): 178.8, 178.6, 158.72, 158.68, 155.6, 155.5, 155.4, 148.8, 148.4, 148.3, 148.1, 148.0, 147.5, 147.1, 138.6, 138.4, 137.8, 137.2, 136.0, 135.8, 135.5, 135.3, 135.0, 133.4, 133.2, 133.0, 130.12, 130.06, 130.0, 129.1, 129.0, 128.5, 128.4, 128.3, 128.11, 128.08, 128.0, 127.2, 127.1, 124.5, 124.3, 124.2, 122.4, 121.82, 121.75, 118.0, 117.4, 113.3, 113.2, 93.9, 92.3, 87.9, 87.6, 86.8, 86.6, 86.4, 86.3, 86.1, 84.1, 83.5, 79.7, 79.6, 79.3, 79.0, 78.6, 78.4, 72.9, 72.2, 71.6, 71.4, 71.3, 71.2, 71.1, 70.9, 70.8, 63.6, 63.3, 63.1, 60.4, 58.9, 43.4, 43.3, 43.1, 43.0, 36.2, 36.1, 36.0, 27.3, 27.13, 27.06, 27.0, 24.61, 24.56, 24.51, 24.46, 21.1, 20.2, 20.1, 19.0, 18.83, 18.79, 18.6, 14.2. 31 P NMR: 150.69, 149.83. HRMS(ESI): m / z [M+Na] +Calculated for C 55 H 63 N 8 O 12 NaP: 1081.4195; found: 1081.4193.

[0092] Example 2

[0093] The bis - azide compound for click reaction with alkynyl was synthesized according to the method reported in the previous literature (Mayor, M. Small 2014, 10, 349 - 359.). The steps are as follows:

[0094]

[0095] Sodium azide (163 mg, 2.5 mmol) was dissolved in DMSO (4 mL), and 1,4 - bis(bromomethyl)benzene (264 mg, 1 mmol) was added. The reaction mixture was stirred at room temperature for 18 hours until the starting material spots disappeared. After the reaction was quenched with H 2 O, it was extracted with diethyl ether. The organic phase was washed with water five times to remove DMSO, dried with Na 2 SO 4 filtered, and the solvent was removed under reduced pressure to obtain pure 1,4 - bis(azidomethyl)benzene.

[0096] Secondly, we selected the crRNA (36 nt) targeting the GFP gene in the CRISPR / Cas9 system as the cyclization object, and arbitrarily selected two sites from it for the incorporation of photo - responsive alkynyl - modified monomers. We completed the synthesis of the modified crRNA (crRNA - LA1, crRNA - LA2) by nucleic acid solid - phase synthesis and characterized it by mass spectrometry (Table 1). Finally, we screened out the most suitable click reaction conditions for the synthesis of photo - responsive cyclic crRNA (crRNA - CA1, crRNA - CA2). The cyclization conditions were as follows: sequentially add crRNA - LA1 / crRNA - LA2 (1.5 nmol), triethylammonium acetate buffer (2 M, pH = 7, 2 μL), 1,4 - bis(azidomethyl)benzene solution (0.75 mM H 2 O / DMSO / tBuOH 4 / 3 / 1 solution, 2 μL), DMSO (55 V% of the final solution), MgCl 2 (100 mM, 1 μL) and fresh ascorbic acid (125 mM, 2 μL). After degassing the reaction system with nitrogen, add CuSO 4 - tris(3 - hydroxyphenyltriazolylmethyl)amine solution (250 mM, 55% DMSO / H 2(O(v / v), 1 μL) to make the final volume 20 μL. After reacting for 4 hours at room temperature, the sample was diluted with RNase-free water and desalted using an Amicon Ultra Centrifugal Filter (Millipore), usually requiring 6 washes. Finally, analysis was performed by 15% denaturing polyacrylamide gel electrophoresis( Figure 1 ), and the cyclized oligonucleotide was purified by HPLC (PLRP-S column, 250 mm × 4.6 mm, 8 μm; mobile phase A = 0.1 M triethylammonium acetate pH 7, B = methanol. The methanol ratio was 15% to 85% within 25 minutes, and the flow rate was 1 mL / min), and the results were characterized by MALDI-TOF (Table 1; Figure 8 for MALDI-TOF of crRNA-CA1; Figure 9 for MALDI-TOF of crRNA-CA2).

[0097] After the cyclization of crRNA was completed, we first verified its photosensitivity under ultraviolet light( Figure 2 ). We detected the deprotection of crRNA-CA1 after irradiation with ultraviolet light (λ = 365 nm, 20 mW / cm 2 ) for 30 s, 1 min, 2 min, 3 min, and 5 min respectively, and characterized this experiment by polyacrylamide gel electrophoresis. The results showed that compared with the standard guide RNA (gfp-crRNA) of the control group, the cyclized guide RNA (crRNA-CA1) could achieve complete deprotection within 30 s. Secondly, the stability of the cyclized crRNA was verified( Figure 3 ). The standard guide RNA (gfp-crRNA) of the control group and the cyclized guide RNA (crRNA-CA1) (10 μM, 10 μL) were placed in 1X NEB 3.1 buffer, and 1 μL of 0.01 mg / mL RNase A was added to make the total volume of the reaction system 100 μL. The reaction system was placed at 37 °C, and 15 μL of the reaction solution was taken at 1 min, 2.5 min, 5 min, 10 min, 30 min, and 60 min respectively, 5 μL of EDTA was added for quenching, and it was quickly frozen in dry ice. After all the samplings were completed, the stability results were analyzed and characterized by 15% non-denaturing polyacrylamide gel electrophoresis. The experimental results showed that the stability of crRNA was significantly improved after cyclization.

[0098] Finally, we verified the ability of the photo-responsive cyclized crRNA to regulate CRISPR activity( Figure 4)。Add the standard guide RNA / circularized guide RNA / photoactivated circularized crRNA (10 pmol), tracrRNA (10 pmol), Cas9 protein (10 pmol), and 100 ng of target DNA to 1x NEB 3.1 Buffer to make the total volume 10 μL. After incubating the reaction system at 37 °C for 90 min, add proteinase K (20 mg / mL, 1 μL), and react at 50 °C for 10 minutes to inactivate the Cas9 protein. Finally, heat at 95 °C for 5 minutes and characterize the results by 2% agarose gel. From the results of in vitro digestion experiments, for both crRNA-CA1 and crRNA-CA2, the circularized group almost lost the cleavage ability in the CRISPR system, while after light activation, the cleavage function was restored. In addition, we also applied this method to the longer gfp-cpf1-crRNA (43 nt). First, the modifications at the head and tail in Cpf1-crRNA (cpf1-crRNA-LA1), the modification in the spacer region (cpf1-crRNA-LA3), and the modification in the scaffold region (cpf1-crRNA-LA2) were completed according to the method described previously. Then, the corresponding crRNAs were circularized (gfp-cpf1-crRNA-CA1 / CA2 / CA3) through click reaction conditions ( Figure 5 )(Table 1; Figure 10 is the MALDI-TOF of Cpf1-crRNA-CA1; Figure 11 is the MALDI-TOF of Cpf1-crRNA-CA2; Figure 12 is the MALDI-TOF of Cpf1-crRNA-CA3). Finally, the in vitro digestion experiment was carried out according to the following conditions: Add the standard guide RNA / circularized guide RNA / photoactivated circularized guide RNA (5 pmol), Cpf1 protein (5 pmol), and 100 ng of target DNA to 1x NEB3.1 Buffer to make the total volume 10 μL. After incubating the reaction system at 37 °C for 30 min, add proteinase K (20 mg / mL, 1 μL), and react at 50 °C for 10 minutes to inactivate the Cpf1 protein. Finally, heat at 95 °C for 5 minutes and characterize the results by 2% agarose gel ( Figure 6 、 Figure 7 ). The digestion experiment also proved that the responsive circularization of the longer Cpf1-crRNA (43 nt) could also effectively regulate the CRISPR activity.

[0099] Table 1: RNA sequences used in the present invention

[0100]

[0101] The red nucleoside moiety is a nucleoside site containing a modified alkynyl group.

[0102] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any variations, uses, or improvements of the present invention, including those that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Applications of some basic features can be made within the scope of the following appended claims.

Claims

1. A ribonucleoside phosphoramidite monomer with a photo-responsive alkynyl modification group, and its structural formula is shown in Formula 1: In Formula 1, R 1 represents a hydroxyl protecting group; R 2 represents , , , any one of; Wherein, R 3 represents H, an amino protecting group; R 4 represents H, an amino protecting group.

2. A method for preparing the compound shown in Formula 1 of Claim 1, comprising the following steps: 1) Reacting the compound shown in Formula I with the compound shown in Formula II to obtain the compound shown in Formula III; In Formula I and Formula III, R 1 , R 2 is the same as R 1 , R 2 ; 2) Reacting the compound shown in Formula III with the compound shown in Formula IV to obtain the compound shown in Formula 1; 3. According to the method described in Claim 2, It is characterized in that: In step 1), the compound shown in Formula II is prepared by a method comprising the following steps: 1) Under the catalysis of Zn powder, reacting o-nitrobenzaldehyde with propargyl bromide to obtain the compound shown in Formula S1-2, 2) In the presence of NaH, reacting the compound shown in Formula S1-2 with a reagent containing MOM group to obtain the compound shown in Formula S1-3 3) Reacting the compound shown in Formula S1-3 with BCl3 to obtain the compound shown in Formula II.

4. The application of the method for preparing the compound shown in Formula 1 in Claim 2 or 3 in introducing a photo-responsive alkynyl modification monomer at any site in an RNA chain.

5. A method for synthesizing a photo-responsive circular RNA based on click chemistry, comprising the following steps: 1) During the synthesis of an RNA chain by solid-phase synthesis, using the method for preparing the compound shown in Formula 1 in Claim 2 or 3 to introduce a photo-responsive alkynyl modification monomer at any site in the RNA chain to obtain a modified RNA, wherein the site of the RNA chain after introducing the photo-responsive alkynyl modification monomer has a structural formula as shown in Formula 1 of Claim 1; 2) Making the modified RNA undergo an intermolecular cyclization reaction with an organic compound containing bis / multi-azide groups by click chemistry to obtain a photo-responsive circular RNA.

6. According to the method described in Claim 5, It is characterized in that: In step 2), the organic compound containing bis / multi-azide groups is 1,4-bis(azidomethyl)benzene; The operation in step 2) is as follows: Add modified RNA, triethylammonium acetate buffer, 1,4-bis(azidomethyl)benzene, DMSO, MgCl 2 and ascorbic acid into the reaction tube in sequence. After degassing the reaction system, add CuSO 4 - tris(3-hydroxypropyltriazolylmethyl)amine solution, react to obtain the photo-responsive circular RNA.

7. According to the method described in Claim 5 or 6, It is characterized in that: The molar ratio of the modified RNA to 1,4-bis(azidomethyl)benzene is 1:1; The molar ratio of ascorbic acid to CuSO4-tris(3-hydroxypropyltriazolylmethyl)amine is 1:1; The reaction is carried out at room temperature, and the reaction time is 4-6 h.

8. A photo-responsive circular RNA prepared by the method described in any one of Claims 5-7.

9. The application of the synthesis method of the photo-responsive circular RNA described in any one of Claims 5-7 and the photo-responsive circular RNA described in Claim 8 in the cyclic modification and functional switch regulation of CRISPR / gRNA, siRNA, ribozyme, and riboswitch.

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