Method for fixed-point oxidation modification of RNA (Ribonucleic Acid)

Abstract

The invention discloses a method for fixed-point oxidation modification of RNA (Ribonucleic Acid). According to the method, an oxidation reaction is directionally guided to a preset guanine site in a post-transcriptional stage by utilizing guide DNA (Deoxyribose Nucleic Acid). According to the LOCAL technology, by enhancing the reaction activity of unpaired nucleotides in a DNA induction loop structure, site selective oxidation close to stoichiometric efficiency is achieved, and the precision close to single nucleotide is achieved. According to the invention, the guide DNA and the reaction conditions are systematically optimized, so that the reaction rule depending on the sequence and the structure in the oxidation reaction is preliminarily disclosed. The malachite green aptamer is used as a model, site selective oxidation is carried out near a ligand binding site of the malachite green aptamer, the fluorescence generation function of the malachite green aptamer is found to be completely lost, and it is powerfully proved that specific oxidative damage can cause RNA function damage. The modularization method can also be applied to intracellular RNA visualization and affinity enrichment research through click chemistry to adapt to a biological coupling strategy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for site-specific oxidative modification of RNA. Background Art

[0002] Post-transcriptional RNA modifications, from methylation to oxidative damage, are increasingly recognized as important regulators of RNA stability, localization, and function. Although these modifications have been extensively studied, establishing causal relationships between specific chemical changes and biological outcomes remains an ongoing challenge in the field. This challenge is particularly prominent in the study of RNA oxidative damage (such as 8-oxoguanine, 8-oxoG). 8-oxoG is not only a biomarker of cellular stress but is also associated with neurodegenerative diseases, cancer, and aging. For example, in Alzheimer's disease, elevated levels of oxidative damage to G have been observed prior to the formation of amyloid plaques, and certain oxidized microRNAs have been shown to play a role in cancer metastasis signaling. Despite their potential pathophysiological significance, the molecular consequences of site-specific oxidative events remain largely speculative, in part due to persistent technical gaps. Namely, the lack of modular and adjustable technologies to introduce oxidative damage into RNA site-selectively, and the lack of such tools is crucial for clarifying cause-and-effect relationships versus correlations in complex biological systems.

[0003] Site-specific base editing tools would allow for the precise study of individual oxidative lesions to validate hypotheses regarding their functional outcomes. Current chemical methods (e.g., treating RNA with H2O2, ionizing radiation, or Fe 2+ / ascorbic acid) result in widespread, non-specific damage to RNA. This non-selective oxidation can simultaneously have confounding effects on RNA folding, interactions, and function, making it difficult to attribute biological effects to individual RNA oxidative lesions. Of course, solid-phase RNA synthesis allows for the introduction of pre-oxidized nucleotides at specific sites, but this method is mainly limited to specialized laboratories and is only applicable to relatively short RNAs (less than about 100 nucleotides). Recently, it has been reported that 8-oxoGTP can be introduced into RNA site-specifically by in vitro transcription with engineered RNA polymerases. Although this technique is very sophisticated, it may not be applicable to post-synthetic modification of native or already transcribed RNAs.

[0004] In addition, chemical tools such as functional transfer oligonucleotides and enzymatic tools such as transferases and base editors, although transformative in RNA editing and functionalization, have not been developed for introducing oxidative damage. Therefore, many fundamental questions regarding RNA oxidation remain unanswered: the biological consequences of individual RNA oxidative lesions; how oxidation affects the enzymatic stability of RNA, etc. Without site-specific editing tools, these molecular-level questions will remain confined to correlative analysis, hindering the achievement of mechanistic breakthroughs.

[0005] Recent studies have shown that unpaired nucleotides generally exhibit higher chemical reactivity than paired nucleotides. This unique reactivity has facilitated the development of various chemical applications, including base editing and bioconjugation; for example, designed complementary DNA (cDNA) can induce the formation of circular structures, thereby promoting the chemical modification of internal nucleotides, while the remaining sequence is protected by the RNA-cDNA duplex. This strategy has been successfully used to confine RNA 2´-OH acylation within the DNA-induced loop, enabling site-selective RNA labeling and caging of RNA by acyl imidazole reagents. In addition, circular structures are reactive towards acyl carbenes and have been reported to selectively deposit acetyl adducts at the guanosine O6 site of the RNA loop. However, to date, the key questions of whether circular structures exhibit high oxidative reactivity and how to utilize potential selective oxidative reactions for local oxidative base editing remain unanswered. Despite the increasing interest in the study of RNA oxidative lesions, there is currently no method capable of directing oxidation to specific RNA sites. SUMMARY OF THE INVENTION

[0006] Based on the deficiencies in the prior art, in order to develop a post-synthetic method capable of achieving site-selective oxidative editing, we aimed to overcome three key challenges: namely, achieving regioselectivity and chemoselectivity to target specific nucleotides, being able to perform modifications between different nucleotides in an RNA sequence, and ensuring the broad practicality of the method for chemists and biologists. First, we believed that regioselectivity could be achieved by exploiting the reactivity differences between paired and unpaired guanines. At the same time, it was found that an oxidant selective for G was also crucial for achieving chemoselectivity among different nucleobase types. Second, we envisioned achieving programmability for modular adaptation to multiple RNA sequences by designing guide DNAs to induce circular structures at user-defined sites. Third, we anticipated that practicality would require the use of widely available reagents and instruments to meet the needs of non-specialist laboratories. The successful development of this method would allow for the programmable modification of RNA with the desired oxidative modifications by simply replacing the corresponding cDNA strand.

[0007] Here, we present a post-synthetic modification method called "Localized Oxidation Constrained at Loops" (LOCAL) for introducing site-selective oxidative damage at predetermined G residues. LOCAL exploits the resistance of RNA-DNA duplexes to oxidation, directing the oxidative reactivity to unpaired nucleotides within loops induced by inexpensive DNA oligonucleotides. We demonstrate that appropriately designed guide DNAs can induce loops in RNA, enabling high-yield oxidative reactions at selected sites. This sequence-selective reaction occurs in a programmable manner with near single-nucleotide resolution. Additionally, this modular strategy also supports site-selective functional labeling (e.g., affinity tags, fluorophores) in a plug-and-play manner, enabling applications such as RNA visualization and potential interactome analysis. We anticipate that the LOCAL method will become a practical strategy for probing site-specific oxidative damage in diverse RNA contexts and may be applicable to many chemical and biological laboratories.

[0008] The specific embodiments of the present invention are as follows: The present invention provides a method for site-directed oxidation modification of RNA, comprising the following steps: (a) Hybridizing a guide DNA with a target RNA to form an RNA-DNA duplex with a predetermined loop structure, wherein the guide DNA comprises a sequence complementary to the target RNA and introduces at least one nucleotide mismatch at a preset site; (b) Performing an oxidation reaction on the duplex formed in step (a) in the presence of an oxidant; (c) Removing the guide DNA to obtain an RNA product with specific oxidation modification at the preset site.

[0009] Preferably, in step (a), the guide DNA induces a 1-3 nt bulge loop or internal loop structure on the target RNA through 1-3 nucleotide mismatches.

[0010] In particular, in the embodiments of the present invention, the guide DNA designed to induce a 1 nt RNA bulge reacts with the photocatalyst Ru(bpy)3 under blue light irradiation (10 - 40 minutes at room temperature) and can achieve near-stoichiometric modification (>95%) at the selected G site. 3+

[0011] Preferably, the preset site is within the complementary region corresponding to at least the 6th nucleotide to at least the penultimate 6th nucleotide in the target RNA sequence. There will be a problem of decreased oxidation selectivity when the preset site is set at the first 5 bases before the 5' or 3' end of the target RNA.

[0012] ​Preferably, in step (a), the guide DNA hybridizes with the target RNA by annealing. The annealing conditions are: pH value is 7.0 - 7.5 and salt concentration (final concentration of NaCl is 50 mM or 100 mM).

[0013] The data of the embodiments of the present invention prove that pre - annealed DNA protects RNA from oxidation. The inventors fine - tuned the pH value and salt concentration, significantly reducing the background oxidation in RNA, so that a higher concentration of oxidant can be used at the selected site to achieve near - complete modification. The data show that the easily accessible and unmodified guide DNA can protect RNA under highly oxidative conditions, inhibiting the reactivity by at least more than 3 times.

[0014] Preferably, the target RNA ≤ 200 nt. In the current form, LOCAL may not be applicable to the modification of long RNAs (>200 nt) because the length of the synthetic guide DNA is limited by solid - phase synthesis, approximately 200 nt.

[0015] Preferably, the oxidant is a photocatalyst or a singlet oxygen generator; the photocatalyst is Ru(bpy)3 3+ ; the singlet oxygen generator is eosin B.

[0016] Specifically, when using the photocatalyst, the conditions for photo - excited oxidation reaction include: irradiating with a light source of 456 nm wavelength for 10 - 40 minutes, and the reaction system contains Co(NH3)5Cl 2+ as an electron acceptor.

[0017] Furthermore, the method for site - specific oxidation modification of RNA of the present invention further includes functionalizing the oxidized RNA by click chemistry. The specific steps are: (1) In the presence of propargylamine, covalently link the oxidation product 8 - oxoG with an alkyne group; (2) Use copper - catalyzed azide - alkyne cycloaddition reaction to directionally couple the functional group to the oxidation site. Achieve RNA functionalization through local oxidation labeling.

[0018] Furthermore, the functional group is selected from biotin, fluorescein or azide group.

[0019] The beneficial effects of the present invention: The method for site - specific oxidation modification of RNA provided by the present invention uses guide DNA to direct the oxidation reaction to the preset guanine site at the post - transcriptional stage. The LOCAL technology realizes site - selective oxidation with near - stoichiometric efficiency and achieves precision close to that of a single nucleotide by enhancing the reactivity of unpaired nucleotides in the DNA - induced loop structure.

[0020] Through systematic optimization of the guide DNA and reaction conditions, the present invention initially reveals the sequence- and structure-dependent reaction rules in the oxidation reaction.

[0021] In addition, using the malachite green aptamer as a model, site-selective oxidation was performed near its ligand-binding site, and it was found that its fluorescence generation function was completely lost, which strongly proves that specific oxidative damage can lead to impaired RNA function. This modular approach can also be adapted to bioconjugation strategies through click chemistry for intracellular RNA visualization and affinity enrichment studies. The present invention fills the key technical gap in the field of research on oxidative damage of transcribed RNA and can be extended to RNA bioconjugation applications. Brief Description of the Drawings

[0022] Figure 1 Rational design of site-localized RNA oxidation guided by DNA; wherein, a is the mechanism of RNA oxidation reaction mediated by Ru(bpy)3 2+ and the quencher Co(NH3)5Cl 2+ ; b is the experimental procedure of primer extension analysis for locating RNA oxidation editing sites.

[0023] Figure 2 Results of the evaluation of the oxidation resistance of single-stranded RNA and RNA-DNA double-stranded; wherein, a is the experimental procedure for measuring the oxidation resistance of RNA based on flash quenching; b and c are the chemical conversion analysis of ribonucleotides based on high performance liquid chromatography (HPLC).

[0024] Figure 3 Results of the evaluation of the oxidation resistance of single-stranded RNA and RNA-DNA double-stranded; wherein, a is the experimental procedure for measuring the oxidation resistance of RNA based on singlet oxygen; b and c are the chemical conversion analysis of ribonucleotides based on high performance liquid chromatography (HPLC).

[0025] Figure 4Localized oxidation restricted by a circular structure; a shows the formation of unpaired regions through DNA-RNA hybridization, exposing bases originally protected by double strands to the oxidation reaction system. b shows the introduction of 1 to 3 nucleotide potential bulges at the target site of the model RNA (tRF3005): When the guide DNA is complementary to the target site, at least 1 ribonucleotide is deliberately deleted at the labeled site. c shows the premature termination of reverse transcription of oxidized RNA (characterized using PAGE gels). After pre-annealing RNA with the guide DNA that can induce bulges and internal loops, damage is formed by flash quenching oxidation. d shows the guide DNA inducing 1×1, 1×2, 1×3, and 1×4 internal loops at the rG9 site of the model RNA: Different-sized circular structures are designed by regulating the number of DNA deletion sites. e shows the PAGE gel characterization of premature termination of reverse transcription in the singlet oxygen (¹O2)-mediated oxidation reaction. The C and G lanes label the template strand bases, and the target guanosine site (rG9) is highlighted in red.

[0026] Figure 5 Positions for guide DNA-programmed nucleobase oxidation; a shows the guide DNA determining the localization of oxidative editing. Site-specific oxidation of rG6, rG9, and rG15 sites in the model RNA (tRF3005) is achieved by inducing a 1nt bulge with the guide DNA. b shows the workflow for localizing RNA oxidative modifications using primer extension analysis. c shows that primer extension analysis shows that the sites of oxidative modification change with the migration of the bulge loop. After pre-annealing RNA with a fully complementary guide DNA or a guide DNA that induces different bulge loops, it is oxidized under flash quenching conditions. The gel lanes labeled C and G represent the template strand bases, and the target guanosines rG6, rG9, and rG15 are highlighted in blue, magenta, and cyan, respectively.

[0027] Figure 6 Simultaneous oxidative editing of multi-target guide DNA; a shows that LOCAL enables the simultaneous introduction of multiple oxidative damages at multiple preset RNA sites (rG6 and rG15). Reverse transcription primers labeled with FAM and Cy5 are used respectively, and the oxidative damages at rG6 and rG15 are detected by primer extension analysis. b shows that through rationally designed guide DNA, LOCAL can synchronously edit multiple sites: Demonstrating the co-localization analysis of multiple oxidative damages, and the target color-coding scheme is the same as Figure 5 in c.

[0028] Figure 7 Oxidation-regulated fluorescent RNA aptamer; a shows the evaluation of the impact of the oxidation modification of the malachite green aptamer. b shows that primer extension analysis shows that G 23 G 24 of the wild-type malachite green aptamer undergoes oxidation modification. c shows the determination of the linear range by titrating the concentration of the wild-type malachite green aptamer. d shows the evaluation of the binding luminescence effects of the wild-type / oxidized malachite green aptamer and the malachite green dye.

[0029] Figure 8 To achieve RNA site-selective bioconjugation by site-directed oxidation. a. Post-synthesis RNA bioconjugation methods are of great significance for enriching and visualizing RNA analysis. b. Experimental procedure for achieving RNA site-selective bioconjugation through LOCAL oxidation. Specific guanosine (G) in the DNA-induced loop can be oxidized by singlet oxygen (¹O2), and then "captured" by propargylamine to form an alkynylated RNA complex, which is then derivatized by copper-catalyzed click chemistry.

[0030] Figure 9 Possibility of local labeling at pre-determined sites; among them, top: Schematic diagram of the design of model RNA miR-124 and its guiding DNA (targeting 5 G-rich sites of miR-124); bottom: Primer extension analysis shows that the oxidized modification sites change with the migration of the loop structure. The duplex is oxidized by singlet oxygen in the presence of propargylamine.

[0031] Figure 10 Practicality of the LOCAL technique in target enrichment; a. The LOCAL technique can achieve site-selective biotinylation of model RNA. b. Procedure for enriching biotinylated RNA from a nucleic acid mixture. c. Top: Quantitative map of the enrichment level of biotinylated RNA; bottom: Dot blot shows the enrichment effect of biotinylated RNA.

[0032] Figure 11 Fluorescently labeled RNA imaging under cellular stress conditions; a. Experimental procedure for fluorescently labeled RNA imaging under cellular stress conditions. After liposome transfection with FAM-labeled miR-124, heat shock treatment is performed at 42 °C for 40 minutes. b. Fluorescence emission scans of FAM-labeled miR-124 (green) and unmodified miR-124 (gray). The emission peak of FAM-miR-124 is around 520 nm. c. Comparison of fluorescence imaging of FAM-labeled miR-124 in cells before and after heat shock. d. Left: Number of fluorescent spots per cell before and after heat shock; right: Statistical analysis of the fluorescent spot area (data are presented as mean ± standard error, n = 3 independent experiments), scale bar is 10 μm. Detailed implementation method

[0033] Example 1: Rational design of DNA-guided site-local RNA oxidation

[0034] To test the feasibility of site - specific RNA oxidation, we constructed duplexes containing a circular structure at the pre - defined site, which was achieved through the designed guide DNA. The guide DNA is the reverse complementary sequence of the target RNA, introducing one or more mismatches at the selected RNA sites. Previous studies have shown that this design can induce unpaired RNA circular structures at these sites. In the prototype design, we employed an easily accessible photocatalyst, Ru(bpy)3 3+ , which has previously been shown to efficiently oxidize guanosine by outer - sphere electron transfer under blue - light irradiation ( Figure 1 ). We hypothesized that complementary DNA might inhibit the oxidation of nucleobases within the protected duplex because these nucleobases are less solvent - exposed and have lower intermolecular accessibility in the duplex. In contrast, the circular structures induced by the designed guide DNA would expose otherwise non - reactive nucleobases, thus promoting the oxidation reaction. Subsequently, the oxidized - modified RNA can be obtained by enzymatic removal of DNA and applied to further studies.

[0035] Example 2: Pre - annealed DNA Protects RNA from Oxidation There is currently no relevant data on whether RNA - DNA duplexes can resist oxidation, especially under the reaction conditions required for stoichiometric oxidative modification. Therefore, we explored the degree of antioxidant protection of complementary DNA (cDNA) on paired RNA. To verify this, we used a 35 - nt model RNA containing three guanines (G) located in different sequence environments, which can be used to study the influence of neighboring base - pair redox reactions. We adopted the reported "flash - quench" oxidation method, in which Ru(bpy)3 2+ and Co(NH3)5Cl 2+ were used to in - situ generate Ru(bpy)3 3+ to selectively oxidize unpaired guanines in the single - stranded region of RNA. The experimental procedure is as Figure 2 shown: First, a strong oxidant was generated by photo - excitation, and then after removing DNA and digesting RNA, the chemical transformation of ribonucleotides was analyzed by high - performance liquid chromatography (HPLC). The results showed that almost all Gs in the unprotected single - stranded RNA (ssRNA) were oxidized and consumed, while adenine (A), cytosine (C), and uracil (U) showed antioxidant properties. High - resolution mass spectrometry (HRMS) confirmed that the main oxidation product of G was the physiologically stable Oz.

[0036] To further explore whether DNA has a protective effect on other physiologically relevant oxidants, we tested the oxidative effect of singlet oxygen (¹O2). Although previous studies have shown that singlet oxygen oxidizes single-stranded RNA (ssRNA) more strongly than double-stranded RNA (dsRNA), the antioxidant property of RNA-DNA duplexes remains unclear. In the experiment, we co-incubated the model ssRNA or its corresponding RNA-DNA duplex with 25 μM Eosin B, and singlet oxygen was generated by blue light excitation. After 20 minutes, DNA was removed and RNA was recovered. HPLC analysis showed that 87% of G in ssRNA was oxidized, while the RNA-DNA duplex exhibited significant antioxidant property ( Figure 3 ). Further identification by HRMS showed that the main product of singlet oxygen oxidation of G was 8-oxoG. Collectively, pre-annealed complementary DNA can effectively protect RNA from damage by various oxidants.

[0037] Specific experimental procedures: tRF-3005-35nt: 5’-AUCCUGCCGACUACGCCAagacuguuaaaugacug-3’; tRF-3005-35mer-cDNA: 5’-cagtcatttaacagtctTGGCGTAGTCGGCAGGAT-3’; All of the above were chemically synthesized.

[0038] 1. Construction of photocatalyst oxidation reaction system a. Formation of hybridization complex: Take 200 pmol tRF-3005-35nt and 400 pmol tRF-3005-35mer-cDNA, add NaCl to a final concentration of 100 mM, heat at 70 °C for 4 minutes, and cool to 25 °C at a gradient of 0.1 °C / second. Subsequently, add 1 μL of 100 mM sodium phosphate buffer (pH 7.0) and 1 μL of 60 mM MgCl2, and incubate at 25 °C for 20 minutes.

[0039] b. Construction of oxidation reaction system: 1 µL of 22.5 mM [Co(NH3)5Cl]Cl2 (Merck, catalog number 298301) and 0.5 µL of 4.5 mM [Ru(bpy)3]Cl2 (Merck, catalog number 224758, dissolved in 20 mM sodium phosphate buffer, pH 7.0) were successively added to the above-mentioned mixture, and the total volume was adjusted to 10 µL. The reaction tube was placed in a temperature mixer and incubated in the dark (25 °C). After 15 minutes, the tube cap was removed, and the mixture was shaken at 800 rpm and irradiated with a blue LED (wavelength 456 nm, 40 W, 6 cm away from the sample) for 10 minutes to trigger the oxidation reaction.

[0040] c. RNA purification: The RNA was purified using an RNA purification and concentration kit (Jianshi, catalog number TR113-200) according to the instructions, and the final elution volume was approximately 14 µL. Subsequently, 4 µL of DNase I (NEB, catalog number M0303S) and 2 µL of 10× DNase I reaction buffer (NEB) were added, and the mixture was incubated at 37 °C for 2 hours to degrade the cDNA. After purification, the RNA purification and concentration kit (Jianshi, catalog number TR113-200) was used again, and the RNA was eluted with RNase-free water (elution volume approximately 20 µL).

[0041] The eluted RNA was mixed with 10 µL of 3 M sodium acetate (pH 5.2, Beyotime, catalog number ST342), 1 µL of glycogen (20 mg / mL, Beyotime, catalog number D0812), 80 µL of RNase-free water, and 500 µL of absolute ethanol. It was precipitated at -20 °C for 2 hours, and the RNA precipitate was collected by centrifugation at 14,500×g for 20 minutes. The precipitate was washed twice with 700 µL of pre-cooled 75% ethanol and finally resuspended in 14 µL of RNase-free water for subsequent HPLC-HRMS analysis.

[0042] The summary of the configured system is shown in Table 1.

[0043] Table 1

[0044] 2. Construction of singlet oxygen reaction system Take 200 pmol of tRF-3005-35nt, 400 pmol of tRF-3005-35mer-cDNA, and 4 µL of 0.5 M NaCl (all three dissolved in D2O), and make up the volume to a total of 10 µL. Heat this mixture at 70 °C for 4 minutes and cool it to 25 °C at a gradient of 0.1 °C / second. Add 6 µL of 3.33× premixed PBS buffer (containing 33.3 mM Na2HPO4, 5.8 mM KH2PO4, 0.46 M NaCl, 8.8 mM KCl, pH 7.2 - 7.6, 20 mM MgCl2, with D2O as the solvent), gently mix, and equilibrate at 25 °C for 20 minutes. Subsequently, add 1 µL of 0.5 mM Eosin B (dissolved in D2O) and 3 µL of D2O, and mix well. Place the reaction tube in a thermostatic mixer (25 °C, 800 rpm), and irradiate it with a blue light LED source (wavelength 456 nm, power 40 W, distance from the sample 6 cm) for 20 minutes. After the reaction, add 10 µL of 3 M sodium acetate (NaOAc), 1 µL of glycogen (20 mg / mL, Beyotime, product number D0812), 80 µL of RNase-free water, and 500 µL of absolute ethanol for ethanol precipitation and purification. Centrifuge at 4 °C to collect the RNA precipitate, wash it with 75% ethanol, and resuspend the RNA precipitate in 14 µL of RNase-free water. Subsequently, add 4 µL of DNase I (NEB, product number M0303S) and 2 µL of 10× DNase I reaction buffer (NEB), incubate at 37 °C for 2 hours to degrade the cDNA. After purification, use the RNA purification and concentration kit (Jianshi, product number TR113-200) again, and elute with RNase-free water (elution volume is approximately 20 µL). Mix the eluted RNA with 10 µL of 3 M sodium acetate (pH 5.2, Beyotime, product number ST342), 1 µL of glycogen (20 mg / mL, Beyotime, product number D0812), 80 µL of RNase-free water, and 500 µL of absolute ethanol. Precipitate at -20 °C for 2 hours, centrifuge at 14,500×g for 20 minutes to collect the RNA precipitate. Wash the precipitate twice with 700 µL of pre-cooled 75% ethanol, and finally resuspend it in 14 µL of RNase-free water for subsequent HPLC-HRMS analysis.) The summary of the preparation system is shown in Table 2 below.

[0045] Table 2

[0046] Example 3: Locally Oxidized by Ring Structure Restriction (LOCAL) Since oxidation is inhibited in the paired helical structure, we predicted that designed guide DNAs, if inducing unpaired sequences, would re-expose these protected residues to oxidation. To this end, we initially designed and tested guide DNAs that deleted 1 to 3 nucleotides at the target position, which should induce potential bulges of 1 nt to 3 nt in size.

[0047] The guide DNA designed to induce a 1-nt RNA bulge reacted with the photocatalyst Ru(bpy)3 under blue light irradiation 3+ or singlet oxygen generator and was able to achieve near-stoichiometric modification at the selected G site (>95% in flash quenching), as shown by PAGE analysis of RT primer extension, with a concentrated dark band appearing 3´ downstream of the modified G site. Bulge loops containing more than one nucleotide also led to local oxidation at the selected G site, but the regioselectivity seemed to decrease. This may be due to the larger bulge exacerbating the helical twist, thereby weakening the thermodynamic stability of the duplex. In addition, the effect of the bulge on duplex stability is known to be sequence-dependent in DNA. Similarly, the relative position of the selected G in the bulge seems to affect the regioselectivity. In a dinucleotide bulge, the G residue showed stronger oxidizability at the 3´ end of the asymmetric bulge than at the 5´ end. These data indicate that the yield and selectivity of oxidation can be regulated by modulating the size and symmetry of the induced bulge loop. The chemoselectivity for G was confirmed by LC-MS analysis of the modified X as a model.

[0048] The physicochemical properties of the microenvironment around the pre-target site may affect the reactivity. Therefore, we tested how another type of internal loop in a circular structure affects local oxidation. We designed guide DNAs containing one or more unpaired nucleotide insertions, which were expected to induce 1×1, 1×2, or 1×3 internal loops. Preliminary experiments inducing internal loops at the pre-target site also led to local oxidation, but the oxidation yield was approximately 3-fold lower than that of the bulge loop. In addition, the internal loop seemed to exacerbate the regioselectivity of photocatalyst oxidation modification, as PAGE analysis showed diffused off-target modification on both sides of the selected residue. This may be due to the internal loop greatly disrupting the stability of the helix, thereby re-exposing the originally protected residues in the model RNA to oxidation. Taken together, our data show that guide DNAs inducing loops are very effective in protecting RNA from oxidation and that the oxidation efficiency and selectivity at the pre-target site can be enhanced by fine-tuning the RNA circular structure.

[0049] A set of guide DNAs induces the formation of potential bulge loop structures of 1 to 3 nucleotides at selected positions in the model RNA tRF3005. These guide DNAs omit ≥1 ribonucleotide at the target site. In addition, the guide DNAs induce internal loop structures with sizes of 1×1, 1×2, 1×3, and 1×4 at the G9 site of the model RNA tRF3005 through base complementary pairing.

[0050] PAGE analysis of representative primer extension demonstrated premature termination of oxidized RNA reverse transcription. Figure 4 The shown RNAs were pre-annealed with guide DNAs to form different bulge loop and internal loop double-stranded complex structures, and were subjected to oxidative modification under flash quenching and singlet oxygen conditions.

[0051] Corresponding guide DNA sequences: tRF-3005-35mer-G9-B1: AAAcagtcatttaacagtctTGGCGTAGTGGCAGGATAAA, tRF-3005-35mer-G9-B2a: AAAcagtcatttaacagtctTGGCGTAGGGCAGGATAAA, tRF-3005-35mer-G9-B2b: AAAcagtcatttaacagtctTGGCGTAGTGCAGGATAAA, tRF-3005-35mer-G9-B3: AAAcagtcatttaacagtctTGGCGTAGGCAGGATAAA, tRF-3005-35mer-G9-L1: AAAcagtcatttaacagtctTGGCGTAGTGGGCAGGATAAA, tRF-3005-35mer-G9-L2: AAAcagtcatttaacagtctTGGCGTAGTGGGGCAGGATAAA, tRF-3005-35mer-G9-L3: AAAcagtcatttaacagtctTGGCGTAGTGGGGGCAGGATAAA, tRF-3005-35mer-G9-L4: AAAcagtcatttaacagtctTGGCGTAGTAGGTGGCAGGATAAA.

[0052] The flash quenching and singlet oxygen oxidation system is as described in Example 1 and Example 2.

[0053] Primer Extension Analysis: Mix 4 pmol of RNA (the same as in Example 2) with 4 pmol of a 5'-end fluorescently labeled reverse transcription primer (Cy5-RT-primer), add 0.25 μL of dNTP mixture (10 mM each, Yeasen, #10122ES74), and make up to a total volume of 6.5 μL with RNase-free water. For the sequencing lane, replace dGTP or dCTP with a ddGTP (APE×BIO, #B8137) / dGTP mixture (molar ratio 9:1) or a ddCTP (APE×BIO, #B8140) / dCTP mixture (molar ratio 9:1), respectively. Incubate the reaction solution at 65 °C for 5 minutes and immediately cool it on ice for 2 minutes. Subsequently, add 2 μL of 5× First Strand Buffer (Invitrogen), 1 μL of 0.1 M DTT, 0.25 μL of RiboLock RNase Inhibitor (40 U / μL, ThermoScientific, #EO0382), and 0.25 μL of SuperScript III Reverse Transcriptase (200 U / μL, ThermoScientific, #18080044) (final volume 10 μL), and perform the reaction according to the following program: 25 °C for 10 minutes, 42 °C for 50 minutes, 52 °C for 50 minutes. After the reaction, add 11 μL of loading dye (containing 8 M urea, 0.05% bromophenol blue), and load it onto a denaturing 18% polyacrylamide gel (UREA TBE PAGE Kit, APE×BIO, product number K4138). Electrophorese at a constant current of 19 mA in 1× TBE buffer (pH 8.3) for approximately 2.5 hours. Finally, visualize the cDNA bands by fluorescence imaging.

[0054] The summary of the prepared systems is shown in Table 3.

[0055] Table 3

[0056] Example 4: Positioning the Oxidation of Programmable Nucleobases by Guide DNA To gain insights into the programmability of loop structure-restricted oxidation, we deployed new guide DNAs to characterize the oxidation at positions other than G9. If programmable, these guide DNAs should relocate the oxidation reactivity by transferring potential bulges or internal loops to other sites. Subsequently, we measured the reverse transcription termination points after photocatalyst oxidation, enzymatic DNA removal, and column purification. Primer extension analysis showed ( Figure 5 and Figure 6), the modification sites do change as the circular structure moves. Similar experiments using another oxidant, singlet oxygen, also confirmed this view. Therefore, the data support the programming of local oxidation through easily accessible guide DNA. In addition, multiple oxidation sites can be written simultaneously.

[0057] Corresponding guide DNA sequences: tRF-3005-35mer-G6-B1: 5’-AAAcagtcatttaacagtctTGGCGTAGTCGGAGGATAAA-3’, tRF-3005-35mer-G9-B1: 5’-AAAcagtcatttaacagtctTGGCGTAGTGGCAGGATAAA-3’, tRF-3005-35mer-G 15 -B1: 5’-AAAcagtcatttaacagtctTGGGTAGTCGGCAGGATAAA-3’, tRF-3005-35mer-G6G 15 -B1: 5’-AAAcagtcatttaacagtctTGGGTAGTCGGAGGATAAA-3’.

[0058] The singlet oxygen oxidation system is as in Example 2.

[0059] The photocatalyst configuration system was slightly modified (the concentration of the photocatalyst was reduced and the illumination time was extended to 40 min), as shown in Table 4 specifically.

[0060] Table 4

[0061] Example 5: Oxidation-regulated fluorescent RNA aptamer Next, we investigated the utility of the LOCAL method in introducing local oxidation into fluorescent RNA aptamers. Previous studies have mainly focused on how modifications such as m5C, pseudouridine, and phosphorothioate modulate the stability and intermolecular binding affinity of RNA aptamers. However, how nucleobase oxidation affects fluorescent RNA remains unclear, possibly in part due to the lack of a post-synthetic method capable of synthesizing fluorescent RNA with oxidized modifications at any pre-defined site. For this purpose, we selected the malachite green (MG)-malachite green aptamer (MGA) system to evaluate how local nucleotide oxidation affects RNA function. Triphenylmethane dyes such as MG usually exhibit extremely low quantum yields due to rapid vibrational de-excitation. However, when MG binds to its aptamer, MG is stabilized in a planar conformation, resulting in a fluorescence enhancement of up to 2360-fold. To investigate whether site-specific guanine (G) oxidation would disrupt the MGA-MG interaction, we employed a flash quenching method to oxidize G residues at different positions within the aptamer. RT-termination and PAGE analysis confirmed the high oxidation efficiency of G23 and G24, with a conversion rate approaching 100%. Therefore, the oxidized variant "G23G24-B4" was selected for further functional testing. Before measuring MG binding, we first performed titrations to determine the linear detection range and ultimately selected 0.5 µM MGA (or oxoMGA). Fluorescence assays showed ( Figure 7 ), compared to the native aptamer, oxidation of G23G24 significantly weakened the MG binding ability, presumably because local structural perturbations near the dye-binding pocket hindered the necessary interaction between the aptamer and MG. The loss of function of oxidized MGA highlights that even single or double G modifications can significantly alter RNA structure and ligand affinity. Overall, these findings emphasize the crucial role of intact guanine in maintaining aptamer function. By demonstrating that local oxidation near the MG-binding region disrupts complex formation, this study further validates our "LOCAL" method as a powerful tool for probing the impact of specific oxidative damage on RNA-ligand interactions.

[0062] Specific experimental procedures: Mix 10 pmol of MGA or oxoMGA with 2 μL of 1 M KCl, and adjust the volume to 10 μL with RNase-free water. Heat the mixture at 70 °C for 4 minutes, then cool it down to 25 °C at a gradient of 0.1 °C / second. Add 2 μL of 100 mM HEPES buffer (pH 7.5, modified with 50 mM MgCl2) to the solution and incubate at 25 °C for 20 minutes. Then add 2 μL of different concentrations of MG (600, 200, 66.7, 22.2, 7.4, 2.5, 0 μM), and adjust the final volume to 20 μL with RNase-free water. Measure the fluorescence signal of the mixture (excitation wavelength / emission wavelength = 620 nm / 656 nm) using an imaging multimode microplate reader (Agilent).

[0063] Example 6: RNA Functionalization by Site-Specific Oxidation Labeling Post-synthetic methods capable of achieving site-specific functionalization labeling on RNA are highly demanded for the enrichment and visualization of RNA and its intermolecular binding partners. Multiple enzymatic strategies have been described for site-selective labeling of RNA. However, the sequence range of the enzymes used in these methods is limited, restricting the degree of RNA modification. Recently, two classes of chemical strategies have been developed to overcome these problems: label-transfer oligonucleotide probes and pre-positioned directed 2'-acylation alleviated sequence bias. Although these methods are ingenious, they require the synthesis of chemically modified oligonucleotides or acyl imidazole reagents. Therefore, we sought to expand our RNA functionalization method due to its simplicity, high yield, and the absence of synthetic and enzymatic engineering. Recent studies have shown that singlet oxygen can efficiently oxidize G, and its product can be "captured" by covalent conjugation with propargylamine. Subsequently, the conjugated alkyne can be further derivatized by copper-catalyzed "click" chemistry ( Figure 8 ). Given that oxidation can be restricted to selected sites and inexpensive reagents are readily available, these characteristics prompted us to consider that site-specific oxidation labeling might provide a simple solution for RNA functionalization.

[0064] As a preliminary test, we explored the possibility of site-specific labeling. We designed a set of guide DNAs to gradually move the potential RNA secondary bulge structure towards the 3'-end of the model RNA to a G residue, and initiated the labeling cascade reaction by irradiation with the photosensitizer eosin B in the presence of propargylamine. Subsequently, we characterized the RT termination points after oxidation labeling, enzymatic removal of DNA, and RNA purification. PAGE analysis showed ( Figure 9 ) that the labeling sites changed as the loop structure moved, confirming the feasibility and programmability of this labeling method. Interestingly, the guide DNA that induced a relatively small bulge (e.g., 1 nt) significantly improved the labeling efficiency compared to the guide DNA that induced an internal loop, which is consistent with the reaction pattern observed in site-specific oxidation.

[0065] Encouraged by the labeling efficiency and selectivity, we further explored the utility of this method in target enrichment. To this end, we derivatized propargylamine-labeled miR-124 with biotin azide via copper-catalyzed alkyne-azide cycloaddition “click” reaction to achieve biotinylation of miR-124 at specific sites. Subsequently, we measured the quality of RNA by streptavidin bead enrichment, stringent bead washing, and formamide elution of RNA. Notably, biotinylated RNA bound to streptavidin beads almost stoichiometrically, and approximately 80% of the RNA input was eluted after formamide denaturation ( Figure 10 a in Figure 10 ). Dot blot analysis of biotinylated RNA before and after enrichment ( Figure 10 b and c in

[0066] Figure 11 further confirmed this finding, demonstrating the utility of this method in RNA enrichment and potentially in the study of sequence-specific biomolecular interactions. Figure 9 and Figure 11 We applied this labeling strategy to the visualization of RNA in living cells by binding fluorophores to target RNA ( Figure 11 a in Figure 11 ). As a preliminary test, propargylamine-labeled miR-124 was derivatized with FAM-containing azide via CuAAC “click” chemistry. Fluorescence measurements verified the presence of the FAM fluorophore on miR-124, and denaturing PAGE analysis further confirmed this view (

[0067] Specific experimental steps for oxidation labeling: 200 pmol of RNA (miR-124-RNA-37mer: 5’P-UAAGGCACGCGGUGAAUGCCagacuguuaaaugacug-3’) was heated with 400 pmol of complementary DNA in folding buffer (containing 50 mM NaCl) at 70 °C for 4 minutes and then cooled to 25 °C. 6 µL of 3.33× modified PBS buffer (containing 33.3 mM Na2HPO4, 5.8 mM KH2PO4, 0.46 M NaCl, 8.8 mM KCl, pH 7.2 - 7.6, 20 mM MgCl2) was added, gently mixed and equilibrated at 25 °C for 20 minutes. Then 2 µL of 1 mM Eosin B and 2 µL of 200 mM propargylamine were added to make the final volume 20 µL. The reaction solution was placed in a thermostatic shaker (shaking at 25 °C, 800 rpm) and irradiated with a blue LED lamp (λ = 456 nm, 40 W, 6 cm away from the sample) for 15 minutes. After irradiation, 10 µL of 3 M NaOAc (pH 5.2, Beyotime, catalog number ST342), 1 µL of glycogen (20 mg / mL, Beyotime, catalog number D0812), 80 µL of RNase-free water and 500 µL of absolute ethanol were added in sequence, and precipitated at -20 °C for 2 hours. The RNA precipitate was collected by centrifugation (14,500 ×g, 20 minutes), washed twice with 700 µL of ice-cold 75% ethanol, and finally resuspended in 14 µL of RNase-free water. Then 4 µL of DNase I (NEB, catalog number M0303S) and 2 µL of 10× DNase I reaction buffer (NEB, catalog number M0303S) were added and incubated at 37 °C for 2 hours to remove DNA. The RNA was purified using an RNA purification and concentration kit (Jianshi, catalog number TR113-200) and used for primer extension experiments.

[0068] Copper-catalyzed click chemical reaction: For the RNA sample (11.4 µL, approximately 1 µg) treated with local oxidation labeling, successively add 3.6 µL of freshly prepared click chemical reaction mixture, the components of which are: 0.5 mM CuSO4 (Sigma, catalog number 451657), 2 mM THPTA (MCE, catalog number HY-W021042), 5 mM sodium ascorbate (Sigma-Aldrich, catalog number 11140), 1 mM biotin-conjugated azide (Sigma-Aldrich, catalog number 762024) or 6-FAM-PEG3-azide (Shanghai Yuanye, catalog number S55093). Place the mixture in a thermostatic oscillator and carry out copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) at 22 °C and 500 rpm for 30 minutes. After the reaction, purify the RNA using the purification column of the RNA purification and concentration kit (Jianshi, catalog number TR113-200).

[0069] Enrichment of biotinylated RNA: For each sample, use 40 µL of Dynabeads MyOne streptavidin C1 magnetic beads (Thermo Scientific, catalog number 65001) and place them in a 1.5 mL low-binding tube. Place the magnetic beads on a magnetic rack (stand still at 20 °C for 1 minute), carefully aspirate and discard the supernatant. Add 1 mL of RNA binding buffer and wash the magnetic beads three times through the magnetic rack. Add 20 µL of RNA binding buffer (final volume is about 40 µL) and incubate at room temperature for 1 hour. The RNA mixture for enrichment analysis consists of 4.5 µg of biotinylated miR-124 and 4.5 µg of yeast 5S rRNA. Add RNA binding buffer to each sample to a final volume of 200 µL. Transfer 200 µL of the RNA mixture to 40 µL of pre-equilibrated magnetic beads, place them on a sample rotary mixer (22 rpm) and incubate at 20 °C for 1 hour. Then transfer the sample to a magnetic rack (stand still at 20 °C for 1 minute), carefully aspirate and discard the supernatant (reserve the supernatant for subsequent analysis). Wash the magnetic beads 5 times by inverting and mixing with 1 mL of RNA wash buffer, transfer to the magnetic rack and then aspirate and discard the supernatant. Repeat the washing step 4 times. Then add 1 mL of PBS buffer to wash and aspirate and discard the supernatant. To elute the biotinylated RNA, add 100 µL of RNA elution buffer, incubate at 65 °C for 5 minutes, and then at 90 °C for 5 minutes. Place the sample on a magnetic rack (Promega, catalog number Z5332) (stand still at 20 °C for 1 minute) and collect the eluate (about 90 µL). Repeat the elution once, collect a total of 180 µL of eluate, and purify it using the RNA purification and concentration kit (Jianshi, catalog number TR113-200). After washing with the purification column, elute the sample with 6 µL of RNase-free water. Use dot blot analysis to evaluate the enrichment degree of biotinylated miR-124.

[0070] Table 5 Composition of RNA Binding Buffer

[0071] Table 6 Composition of RNA Washing Buffer

[0072] Table 7 Composition of RNA Elution Buffer

[0073] Dot Blot Analysis: Spot 1 µL of the eluted RNA samples (including input, supernatant, and eluate, with 3 replicates per group) onto an Ambion® BrightStar®-Plus nylon membrane (Thermo Scientific, catalog number AM10100). Fix the RNA on the membrane using a UV crosslinker (energy 180 mJ / cm²). After fixation, wash the membrane with 10 mL of TBST buffer (1× TBS containing 0.1% Tween-20, Beyotime, catalog number ST673-500ml) on a horizontal shaker at room temperature for 5 minutes to remove unbound RNA. Subsequently, block the membrane with TBST buffer containing 5% BSA (Yeasen, catalog number 36105ES25) for 1 hour at room temperature. Incubate the membrane with 1 µg / mL streptavidin-HRP (Pierce, catalog number 21124, dissolved in 5% BSA) at room temperature for 1 hour. Wash the membrane three times with TBST buffer, 10 minutes each time. Finally, evenly add Clarity Western ECL substrate (Bio-Rad, catalog number 1705061) to the surface of the membrane. After incubating for 2 minutes, collect the chemiluminescence signal using a Bio-Rad ChemiDocTM MP imaging system.

[0074] Fluorescence Scanning of FAM-Labeled miR-124 Mix 200 ng of FAM-labeled miR-124 or unlabeled miR-124 with RNase-free water to a final volume of 20 µL. Load the mixture into a 384-well plate (LABSELECT, catalog number 31431). Perform emission wavelength scanning using an imaging multimode microplate reader (Agilent, model MS-SP104). Fix the excitation wavelength (Ex) at 460 ± 9 nm, and scan the emission wavelength (Em) in the range of 480 - 700 nm (interval 2 nm).

[0075] Cell Culture and RNA Transfection 100,000 HeLa cells were seeded into a 24-well plate containing cover slips (CITOTEST, catalog number 80346-1210, 12 mm in diameter). After 24 hours of seeding (when the cell confluence was approximately 40%), 120 ng of FAM-labeled miR-124 and 3 μL of Lipofectamine RNAiMAX (Invitrogen, catalog number 13778100) were transfected in Opti-MEM medium (Thermo Scientific, catalog number 31985070) according to the instructions. Six hours after transfection, the cover slips were washed three times with PBS, and 0.5 mL of fresh medium was added to each well. Subsequently, the cells were incubated at 42 °C for 40 minutes and fixed rapidly with 4% paraformaldehyde (Macklin, catalog number P804536). After fixation, the cells were washed with PBS for 10 minutes, stained with DAPI (Sigma-Aldrich, catalog number D9542) for 10 minutes, and rinsed with PBS for 10 minutes. Finally, the cover slips were mounted with Fluoromount-G mounting medium (Southern Biotech, catalog number 0100-01) for microscopy imaging.

[0076] In summary, oxidative damage to RNA is increasingly recognized as being associated with cancer, neurodegenerative diseases, and cardiovascular diseases, but deciphering its site-specific functional consequences remains challenging. Existing methods, including random bulk oxidation and solid-phase synthesis, have limitations in terms of precision, scalability, and compatibility with transcribed RNA. Here, we introduce LOCAL, a post-synthetic strategy that enables the introduction of oxidative damage at pre-defined sites with near-nucleotide resolution. By exploiting the differential reactivity of unpaired and paired nucleotides, LOCAL combines DNA-guided reactivity with photochemical oxidation to systematically investigate how individual lesions impair RNA function. This may represent a crucial step towards deciphering the causal relationship between RNA oxidation and dysfunction.

[0077] LOCAL achieves regioselectivity by using RNA-DNA hybridization to protect double-stranded regions from oxidation while directing the oxidation reaction towards the nucleotides exposed in DNA-induced loops. This control enables near-complete oxidation at pre-defined sites even under highly oxidative reaction conditions, while minimizing off-target effects. Additionally, the physicochemical properties of the induced RNA loops appear to influence the efficiency and selectivity of oxidative editing. For example, 1nt bulged loops provide the highest chemical yields to date with minimal off-target oxidation, likely due to their maintenance of duplex stability. In contrast, internal loops exhibit lower selectivity, highlighting the loop geometry as an adjustable parameter for regioselectivity in optimizing editing accuracy. Notably, the chemoselectivity of this method towards guanine (confirmed by mass spectrometry) is consistent with the redox sensitivity of guanine. Future work exploring alternative oxidants may expand the scope to oxidative damage of other native or modified nucleobases.

[0078] We demonstrate the utility of LOCAL by inducing oxidative damage near the ligand-binding site of the malachite green aptamer, which abolishes its fluorescence activity. This initial experiment exemplifies how oxidation of key functional residues can impair RNA activity, with implications for RNA dysfunction in oxidative stress-driven pathologies. Moreover, the utility of LOCAL is not limited to oxidation studies - the combination of propargylamine-based bioconjugation with "click" chemistry enables downstream applications such as RNA visualization and affinity-based enrichment. Importantly, its photochemical properties allow for fine-tuning of the oxidation level through control of the reaction time, enabling the simulation of dose-dependent effects of oxidative damage. Overall, these features highlight the potential of LOCAL as a versatile method in mechanistic studies and as a potentially broadly applicable biotechnological tool.

Claims

1. A method for site-directed oxidation modification of RNA, characterized in that, Comprising the following steps: (a) Hybridize the guide DNA with the target RNA to form an RNA-DNA duplex with a predetermined circular structure, wherein the guide DNA contains a sequence complementary to the target RNA and introduces at least one nucleotide mismatch at a preset site; (b) Perform an oxidation reaction on the duplex formed in step (a) in the presence of an oxidant; (c) Remove the guide DNA to obtain an RNA product with specific oxidation modification at the preset site.

2. The method for site-specific oxidation modification of RNA according to claim 1, wherein In step (a), the guide DNA induces the formation of a 1nt to 3nt bulge loop or internal loop structure on the target RNA through 1-3 nucleotide mismatches.

3. The method for site-directed oxidation modification of RNA according to claim 1, wherein, In step (a), the preset site is within the complementary region corresponding to at least the 6th nucleotide to at least the 6th nucleotide from the end in the target RNA sequence.

4. The method for site-directed oxidation modification of RNA according to claim 1, wherein, In step (a), the guide DNA is annealed and hybridized with the target RNA; The annealing conditions are: pH value is 7.0 - 7.5, and the final concentration of NaCl is 50 mM or 100 mM.

5. The method for site-directed oxidation modification of RNA according to claim 1, wherein, The target RNA ≤ 200 nt.

6. The method for site-directed oxidative modification of RNA according to claim 1, wherein The oxidant is a photocatalyst or a singlet oxygen generator; The photocatalyst is Ru(bpy)3 3+ ; The singlet oxygen generator is eosin B.

7. The method for site-directed oxidation modification of RNA according to claim 1, wherein When using a photocatalyst, the conditions for the photoexcitation oxidation reaction include: irradiating with a light source of 456 nm wavelength for 10 - 40 minutes, and the reaction system contains Co(NH3)5Cl 2+ as an electron acceptor.

8. The method for site-directed oxidation modification of RNA according to claim 1, wherein It further includes functionalizing and labeling the oxidized RNA by click chemistry, and the specific steps are: (1) In the presence of propargylamine, covalently link the oxidized product 8-oxoguanosine with an alkyne group; (2) Use copper-catalyzed azide-alkyne cycloaddition reaction to directionally couple a functional group to the oxidation site.

9. The method for site-directed oxidation modification of RNA according to claim 8, wherein, The functional group is selected from biotin, fluorescein or azide group.

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