A method of fluorescently labeling N 6 - isopentenyladenosine

Abstract

The application discloses a method for fluorescent labeling of N 6 A method for fluorescent labeling of isopentenyladenosine. The method comprises the following steps: 1) i 6 A nucleotide monomer or shorter i 6 A-RNA fragment as the substrate, blue light with a wavelength of 440-470 nm is irradiated, and through the action of manganese bromide tetrahydrate (MnBr2.4H2O) and azidotrimethylsilane, an azido group is introduced at the i 6 A modified N-isopentenyl double bond, to obtain an azidated nucleoside or an azidated nucleoside-RNA fragment; 2) the azidated nucleoside or the azidated nucleoside-RNA fragment is combined with an azide reaction probe by using a click reaction, and fluorescent labeling of i 6 A modification is realized.

Description

Technical Field

[0001] This invention belongs to the field of chemical biology technology, specifically relating to a method for treating N 6 - A method for fluorescent labeling of isopentenyl adenosine. Background Technology

[0002] N 6 -Isopentenyl adenosine (i 6 A) usually refers to the conversion of adenosine (A) into isopentenyl adenosine (i) 6 Chemical modification of tRNA (A). This modification mainly occurs on the anticodon loop of tRNA and is an important form of chemical modification in the tRNA molecule. It is widely involved in various biological processes, such as enhancing the codon recognition efficiency of tRNA, improving translation accuracy, and promoting protein synthesis (RNA. 2018; 24:1277–1284; J Biol Chem. 1981; 256:11580–11584.). Furthermore, i 6 A modification is also closely related to processes such as cell growth and stress response, and may be associated with the pathogenesis of certain diseases (RNA. 2011; 17:1846–1857.). By developing [a specific treatment targeting i...] 6 A's fluorescent labeling technology can monitor i 6 The modification level and dynamic changes of A on tRNA will provide a deeper understanding of its biological functions under different physiological and pathological conditions. This technology will not only help explore i 6 A modifications interact with specific enzymes or receptors, and also provide insights into the interaction between i and 6 This provides new insights into biological processes related to A-modification. Its application will provide important theoretical basis and practical value for discovering new drug targets and advancing the research and development of antibacterial and antiviral drugs.

[0003] Currently, researchers have developed a variety of methods for labeling i 6 A modifications mainly include the following: 1) Antigen-antibody method, through i 6 Modification A causes changes in tRNA hydrogen bond formation and steric hindrance, leading to weakened binding to the anticodon loop (ACL) probe, which is reflected in Northern blotting as a weaker signal compared to unmodified tRNA (RNA. 2022; 28:418–432.). The disadvantage of this method is its complexity and time-consuming operation. 2) Chemical precipitation method: This method utilizes specific chemical reactions (such as PTAD, selectfluor, and nitroso derivatives) to interact with isoprene-modified biomolecules under physiological conditions, and then labels the i-molecule with a red fluorescent group using a fluorescent probe (such as PTAD-DBCO-Cy5). 6On A-RNA (Tetrahedron Lett. 2022; 97, 153793.). However, this method has a limitation: under the same conditions, it may also label other modifications containing double bonds, leading to erroneous signals. 3) Iodine labeling method, which uses elemental iodine to label i 6 The olefinic functional group of A undergoes a cyclization reaction, leading to the cyclization of i during RNA reverse transcription. 6 A base mismatch occurred (Nucleic Acids Res. 2024: gkae150.). Although this method is suitable for single-base resolved sequencing, the weak fluorescence signal of the cyclized product makes it difficult to apply to visualized fluorescent labeling.

[0004] In recent years, photocatalysis has been widely used in the field of chemical biology. Its advantages lie in its strong reaction selectivity, good atom economy, few side reactions, and fast reaction rate, thus it has high practical value. Summary of the Invention

[0005] In order to address the shortcomings of existing technologies and improve the understanding of i 6 This invention provides a simple photocatalytic labeling method that leverages the reaction specificity of A, reduces complex operations, and aligns with the principles of green chemistry. 6 The method of modification by A, that is, for N 6 - A method for fluorescent labeling of isopentenyl adenosine.

[0006] The present invention provides for N 6 A method for fluorescent labeling of isopentenyl adenosine includes the following steps:

[0007] 1) With N 6 -Isopentenyl adenosine or shorter N-containing compounds 6 Using isopentenyl adenine-modified RNA as a substrate, irradiation with blue light at wavelengths of 440–470 nm, and the reaction of manganese bromide tetrahydrate (MnBr2·4H2O) and azidotrimethylsilane (TMSA), [the following text appears to be incomplete and requires further context: "i"] 6 An azide group is introduced into the N-isopentenyl double bond of the A-modified compound to obtain an azide nucleoside or an azide nucleoside-RNA fragment.

[0008] 2) By utilizing the click reaction, the azide-substituted nucleoside or azide-substituted nucleoside-RNA fragment binds to the azide reaction probe, achieving the targeting of i 6 A-modified fluorescent label.

[0009] In step 1) of the above method, the RNA is a single-stranded small RNA fragment with a length of less than 15 bases.

[0010] In step 1) of the above method, the molar ratio of the substrate, manganese bromide tetrahydrate and trimethyl azidosilane is 1:(10-200):(100-2000).

[0011] In step 1) of the above method, replacing the manganese bromide tetrahydrate with manganese fluoride, manganese chloride, manganese iodide or manganese acetate will reduce the yield; trimethylsilane azide can be replaced with sodium azide, but the experimental operation is quite dangerous.

[0012] In step 1) of the above method, the reaction of introducing the azide group is carried out in an oxygen atmosphere.

[0013] In step 1) of the above method, the reaction of introducing the azide group is carried out in a solvent, which may be methanol or a mixture of methanol and water (such as a mixture of methanol and water in a volume ratio of 1:1).

[0014] In step 1) of the above method, the reaction time for introducing the azide group is 2.5-5 hours.

[0015] In step 2) of the above method, the azide reaction probe may be selected from at least one of the following: DBCO-Cy3, DBCO-Cy5, DBCO-Cy5.5, and DBCO-Cy7.

[0016] In step 2) of the above method, the molar ratio of the azidonucleotide or azidonucleotide-RNA fragment to the azido compound reaction probe is 1:(2-20).

[0017] In step 2) of the above method, the click reaction is carried out in a solvent, which is a mixed solvent of methanol and water, specifically a mixed solvent of methanol and water in a volume ratio of 1:1.

[0018] In step 1) of the above method, a shorter N-containing... 6 The reaction system is subjected to enzymatic hydrolysis to obtain azide nucleosides after step 1) and before step 2) using isopentenyl adenine modified RNA as the substrate.

[0019] In step 2) of the above method, the azide-nucleotide-RNA fragment is bound to the azide reaction probe. After the click reaction is completed, the following step is further included: enzymatic digestion of the reaction system to obtain fluorescently labeled i 6 A.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention enables photocatalytic control of RNA fragment labeling, is simple to operate, and conforms to the principles of green chemistry. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0024] Example 1, ai 6 Synthesis of A

[0025] The reaction procedure is as follows:

[0026]

[0027] Add N to a 10 ml Shrek tube 6 -Isopentenyladenine nucleoside (i 6 A) (34 mg, 0.1 mmol), manganese bromide tetrahydrate (14 mg, 0.05 mmol), and trimethylsilane azide (39 μL, 0.3 mmol) were added, followed by three gas purgings. An oxygen bulb was inserted, and 3 mL of methanol was injected. The mixture was stirred at room temperature for 12–16 h under a 3W LED lamp at 440 nm. After the reaction was complete, the reaction solution was rotary evaporated to dryness, and thin-layer chromatography (dichloromethane:methanol = 10:1, v / v) was used to separate the pale yellow solid ai. 6 A (azidonucleotide), yield 11 mg, yield 28%. Structural characterization data of the product are as follows:

[0028] 1 H NMR (300MHz, DMSO-d6) δ8.82(s,1H),8.75(d,J=4.5Hz,1H),6.43(d,J=6.0Hz,1H),6.05(s,1H),5.32(s,1H),5.14(s,1H),4 .84(s,1H),4.62(s,1H),4.28(d,J=12.4Hz,1H),4.16(s,1H),4.01(d,J=7.2Hz,2H),3.75(s,3H),2.99(s,1H),1.31(s,6H).

[0029] Example 2, i 6 A's mark

[0030] (1) Synthesize ai according to the following reaction procedure 6 A:

[0031]

[0032] In a 1 mL sample vial (solvent: water:methanol = 1:1), inject 2 μL of AR, UR, CR, GR and i sequentially. 6 Solution A (50 mM) was prepared to bring the concentration of each nucleoside to 100 μM. 20 μL of 50 mM MnBr₂·4H₂O solution and 10 μL of 1 M TMSA solution were added. The reaction system was irradiated with 100 mW 470 nm blue light for 2.5 hours at room temperature, ultimately yielding i 6 A's azidation product ai 6 A (Experiments show that the original ai in the presence of excessive TMSA) 6 The hydroxyl group -OH in A is changed to peroxy group -OOH), with a yield of 72% (HPLC). This reaction has no significant effect on the remaining nucleosides.

[0033] The characterization data of the product are as follows: HRMS(ESI) calcd for C 15 H 23 N8O6 + (M+H) + :411.1735,Found411.1735.

[0034] Table 1 Nucleoside recovery data

[0035] Nucleosides Recovery rate (%) AR 100 UR 98 CR 98 GR 85

[0036] (2) Follow the reaction procedure below to react with ai 6 A is fluorescently labeled.

[0037]

[0038] In a solution containing 1 ml of 100 μM ai 6 In a small sample vial of solution A (solvent: water:methanol = 1:1, v / v), add 20 μL of DBCO-Cy3 solution (concentration 10 mM). React at room temperature for 1 h. Monitor the reaction by HPLC to ensure complete reaction, yielding the DBCO-labeled nucleoside product ai. 6 A-DBCO-Cy3.

[0039] Product characterization data: HRMS(ESI) calcd for C 65 H 78 N 12 O 17 S3 2+ (M+2H) 2+ :1394.4759,Found 1394.4713.

[0040] Example 3, i 6 A-RNA labeling

[0041]

[0042] Take 6 μL i 6 A-RNA solution (15 bases or less in length, 500 μM, prepared with DEPC water) was added to a microreaction tube, along with 8 μL of 75 mM MnBr2·4H2O solution, 4 μL of 1.5 M TMSA solution, and 12 μL of methanol. An oxygen balloon was inserted and sealed, and the tube was placed in a photoreactor. The reaction system was irradiated with 1 W 470 nm blue light at room temperature for 2.5 hours. After the reaction was completed, a portion of the reaction solution was digested enzymatically (procedure: 7.5 μL of the reaction solution was transferred to a PEEK tube, 2 μL of LCP enzyme solution and 1 μL of LPI enzyme solution were added, and DEPC water was added to a total volume of 50 μL. The tube was vortexed, centrifuged, and incubated at 37 °C for 7 hours). The yield was determined to be 53% by HPLC. The target product ai in the enzymatic digest was... 6 A is characterized.

[0043] Take another micro-reaction tube and add ai to it. 6 A-RNA solution and DBCO-Cy3 solution (concentration 10mM), add DEPC water to ai 6 The A-RNA concentration was 100 μM, the DBCO-Cy3 concentration was 2 mM, and the reaction was carried out at room temperature for 1 h. The reaction was monitored for completeness by HPLC. A portion of the reaction solution was digested enzymatically, and the target product ai in the digest was analyzed. 6 A-DBCO-Cy3 was characterized.

[0044] Characterization data:

[0045] ai 6 A: HRMS(ESI)calcd for C 15 H 21 N8O6 - (MH) - :409.1590,Found 409.1600.

[0046] ai 6 A-DBCO-Cy3:HRMS(ESI)calcd for C 65 H 78 N 12 O 17 S3 2+ (M+2H) 2+ :1394.4759,Found1394.4713.

[0047] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method of fluorescently labeling N 6 - isopentenyladenosine, comprising the steps of: 1) With N 6 -Isopentenyl adenosine or shorter N-containing compounds 6 Using isopentenyl adenine-modified RNA as a substrate, irradiation with blue light at wavelengths of 440–470 nm, and the reaction of manganese bromide tetrahydrate and azidotrimethylsilane, in N... 6 - An azido group is introduced into the N-isopentenyl double bond of isopentenyl adenine to obtain an azidonucleotide or an azidonucleotide-RNA fragment; 2) By utilizing the click reaction, the azide-substituted nucleoside or azide-substituted nucleoside-RNA fragment is bound to the azide reaction probe, thus achieving the targeting of N... 6 - Fluorescent labeling modified with isopentenyl adenosine.

2. The method of claim 1, wherein: In step 1), the RNA is a single-stranded small RNA fragment with a length of less than 15 bases.

3. The method according to claim 1 or 2, characterized in that: In step 1), the molar ratio of the substrate, manganese bromide tetrahydrate and trimethyl azidosilane is 1:(10-200):(100-2000).

4. The method according to any one of claims 1-3, characterized in that: In step 1), the reaction of introducing the azide group is carried out in an oxygen atmosphere; And / or, in step 1), the reaction of introducing the azide group is carried out in a solvent, which may be methanol or a mixture of methanol and water.

5. The method according to any one of claims 1-4, characterized by: In step 1), the reaction time for introducing the azide group is 2.5-5 hours.

6. The method of any one of claims 1-5, wherein: In step 2), the azide reaction probe is selected from at least one of the following: DBCO-Cy3, DBCO-Cy5, DBCO-Cy5.5, and DBCO-Cy7.

7. The method of any one of claims 1-6, wherein: In step 2), the molar ratio of the azidonucleotide or azidonucleotide-RNA fragment to the azido compound reaction probe is 1:(2-20).

8. The method of any one of claims 1-7, wherein: In step 2), the click reaction is carried out in a solvent, which is a mixture of methanol and water.

9. The method of any one of claims 1-8, wherein: In step 1), N-containing 6 The reaction system is subjected to enzymatic hydrolysis to obtain azide nucleosides after step 1) and before step 2) using isopentenyl adenine modified RNA as the substrate.

10. The method of any one of claims 1-8, wherein: In step 2), after the click reaction, the following step is included: enzymatic digestion of the reaction system to obtain the fluorescently labeled N 6 - isopentenyladenosine.

Images