A method for site-directed oxidation modification of RNA

Abstract

The present invention discloses a method for site-directed oxidative modification of RNA, which utilizes guide DNA to directionally guide the oxidation reaction to a preset guanine site in the post-transcriptional stage. LOCAL technology achieves site-selective oxidation with near-stoichiometric efficiency and achieves accuracy close to that of a single nucleotide by enhancing the reactivity of unpaired nucleotides in DNA-induced ring structures. The present invention systematically optimizes the guide DNA and reaction conditions, and preliminarily reveals the reaction rules that depend on sequence and structure in the oxidation reaction. The present invention uses the malachite green aptamer as a model, implements site-selective oxidation near its ligand binding site, and finds that its fluorescence generation function is completely lost, which strongly proves that specific oxidative damage can lead to impaired RNA function. This modular approach can also be adapted to biocoupling strategies through click chemistry for intracellular RNA visualization and affinity enrichment studies.

Description

Technical Field

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

[0002] Posttranscriptional RNA modifications, ranging 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 acute in the study of RNA oxidative lesions, such as 8-oxoguanosine (8-oxoG). 8-oxoG is not only a biomarker of cellular stress but has also been implicated in neurodegenerative diseases, cancer, and aging. For example, elevated levels of oxidative damage to G have been observed prior to amyloid plaque formation in Alzheimer's disease, while 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. Specifically, there is a lack of modular and tunable technologies for site-selectively introducing oxidative lesions into RNA, a critical tool for clarifying causality and correlation in complex biological systems.

[0003] Site-specific base editing tools will allow precise study of individual oxidative lesions to test hypotheses about their functional consequences. Current chemical methods (e.g., with H2O2, ionizing radiation, or Fe 2+ Treatment of RNA with oxidative stress (e.g., oxidative stress / ascorbic acid) can lead to widespread, nonspecific damage to RNA. This nonselective oxidation can have mixed effects on RNA folding, interactions, and function, making it difficult to attribute biological effects to a single RNA oxidative lesion. Of course, solid-phase RNA synthesis allows for the introduction of preoxidized nucleotides at specific sites, but this approach is largely limited to specialized laboratories and is only applicable to relatively short RNA lengths (less than approximately 100 nucleotides). Recently, it has been reported that 8-oxoGTP can be site-specifically incorporated into RNA by in vitro transcription using an engineered RNA polymerase. While this technique is elegant, it may not be suitable for postsynthetic modification of native or transcribed RNA.

[0004] Furthermore, while chemical tools such as function-transferring oligonucleotides and enzymatic tools such as transferases and base editors have been transformative for RNA editing and functionalization, they have yet to be developed for the introduction of oxidative lesions. Consequently, many fundamental questions regarding RNA oxidation remain unanswered, including the biological consequences of oxidative lesions on individual RNAs and how oxidation affects the enzymatic stability of RNA. Without site-specific editing tools, these molecular-level questions will remain limited to correlative analyses, hindering 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 be induced to form a ring structure, facilitating chemical modification of nucleotides within the ring, while the remaining sequence is protected by the RNA-cDNA duplex. This strategy has been successfully used to restrict RNA 2´-OH acylation to the DNA-induced ring, enabling site-selective RNA labeling and RNA caging with acyl imidazole reagents. Furthermore, the ring structure is reactive toward acetyl carbenes, and it has been reported that acetyl adducts can be selectively deposited at the guanosine O6 position of the RNA ring. However, key questions remain: whether the ring structure possesses high oxidative reactivity and how this potential for selective oxidation can be exploited for localized oxidative base editing. Despite the growing interest in studying RNA oxidative damage, methods to direct oxidation to specific RNA sites remain unanswered. Summary of the Invention

[0006] To develop a post-synthetic method capable of site-selective oxidative editing, we aimed to overcome three key challenges: achieving regioselectivity and chemoselectivity to target specific nucleotides, enabling modification across different nucleotides within an RNA sequence, and ensuring the broad applicability of this method to chemists and biologists. First, we proposed that regioselectivity could be achieved by exploiting the differences in reactivity between paired and unpaired guanines. Furthermore, identifying an oxidant selective for G was crucial for achieving chemoselectivity across different nucleobase types. Second, we envisioned modular programmability to adapt to diverse RNA sequences by designing guide DNA to induce looping at user-defined sites. Third, we foresaw that practicality would require the use of widely available reagents and instrumentation, making it suitable for non-specialized laboratories. Successful development of this method would allow programmable modification of RNA with desired oxidative modifications by simply replacing the corresponding cDNA strand.

[0007] Here, we present a post-synthetic modification method, termed "Localized Oxidation Constrained at Loops" (LOCAL), for the site-selective introduction of oxidative lesions at predetermined G residues. LOCAL exploits the resistance of RNA-DNA duplexes to oxidation, directing oxidative reactivity to unpaired nucleotides within loops induced by inexpensive DNA oligonucleotides. We demonstrate that appropriately designed guide DNA can induce loops in RNA, enabling high-yield oxidation reactions at selected sites. This sequence-selective reaction occurs programmably with near-single-nucleotide resolution. Furthermore, this modular strategy enables plug-and-play implementation of site-selective functional labeling (e.g., affinity tags, fluorophores), supporting 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 lesions in a variety of RNA contexts and will likely be applicable to many chemistry and biology laboratories.

[0008] The specific embodiments of the present invention are as follows:

[0009] The present invention provides a method for site-directed oxidative modification of RNA, comprising the following steps:

[0010] (a) hybridizing a guide DNA with a target RNA to form an RNA-DNA duplex having a predetermined circular structure, wherein the guide DNA comprises a sequence complementary to the target RNA and introduces at least one nucleotide mismatch at a predetermined position;

[0011] (b) subjecting the duplex formed in step (a) to an oxidation reaction in the presence of an oxidizing agent;

[0012] (c) The guide DNA is removed to obtain an RNA product that undergoes specific oxidative modification at the predetermined site.

[0013] Preferably, in step (a), the guide DNA induces the formation of a 1 nt to 3 nt protruding loop or internal loop structure on the target RNA through 1-3 nucleotide mismatches.

[0014] In particular, in an embodiment of the present invention, the guide DNA designed to induce 1nt RNA bulge is irradiated with blue light (10-40 minutes at room temperature) and the photocatalyst Ru(bpy)3 3+ The reaction was able to achieve near-stoichiometric modification (>95%) at the selected G site.

[0015] Preferably, the predetermined site is located within the complementary region corresponding to at least the sixth nucleotide to at least the sixth-to-last nucleotide in the target RNA sequence. Setting the predetermined site within the first five bases of the 5' or 3' end of the target RNA may result in decreased oxidation selectivity.

[0016] Preferably, in step (a), the guide DNA and the target RNA are annealed and hybridized;

[0017] The annealing conditions are: pH 7.0-7.5 and salt concentration (final NaCl concentration 50 mM or 100 mM).

[0018] Data from the present invention demonstrate that pre-annealed DNA protects RNA from oxidation. By fine-tuning the pH and salt concentration, the inventors significantly reduced background oxidation in RNA, enabling the use of higher concentrations of oxidant at selected sites to achieve near-complete modification. The data demonstrate that readily available, unmodified guide DNA can protect RNA under highly oxidizing conditions, suppressing reactivity by at least threefold.

[0019] Preferably, the target RNA is ≤200 nt. In its current form, LOCAL may not be suitable for the modification of long RNA (>200 nt) because the length of the synthetic guide DNA is limited to approximately 200 nt by solid-phase synthesis.

[0020] 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.

[0021] Specifically, when using a photocatalyst, the conditions for the light-induced oxidation reaction include: irradiating with a 456 nm wavelength light source for 10-40 minutes, and the reaction system contains Co(NH3)5Cl 2+ As an electron acceptor.

[0022] Furthermore, the method for site-directed oxidative modification of RNA of the present invention further comprises functionalizing the oxidatively modified RNA by click chemistry, and the specific steps are as follows:

[0023] (1) In the presence of propargylamine, the oxidation product 8-oxoG is covalently linked to the alkyne group;

[0024] (2) Using copper-catalyzed azide-alkyne cycloaddition reaction, functional groups are directed to the oxidation sites. RNA functionalization is achieved through localized oxidation labeling.

[0025] Furthermore, the functional group is selected from biotin, fluorescein or azide group.

[0026] Beneficial effects of the present invention:

[0027] The site-directed oxidative modification method for RNA provided by this invention utilizes guide DNA to direct the oxidation reaction to a predetermined guanine site during the post-transcriptional phase. By enhancing the reactivity of unpaired nucleotides within DNA-induced loop structures, the LOCAL technique achieves near-stoichiometric site-selective oxidation with near-single-nucleotide precision.

[0028] The present invention systematically optimizes the guide DNA and reaction conditions, preliminarily revealing the sequence- and structure-dependent reaction rules in oxidation reactions.

[0029] Furthermore, using the malachite green aptamer as a model, we performed site-selective oxidation near its ligand-binding site and found that its fluorescence-generating function was completely lost, strongly demonstrating that specific oxidative damage can lead to impaired RNA function. This modular approach can also be adapted to bioconjugation strategies via click chemistry for intracellular RNA visualization and affinity enrichment studies. This invention fills a key technological gap in the research of oxidative damage to transcribed RNA and can be expanded to RNA bioconjugation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A rational design for DNA-guided site-localized RNA oxidation; wherein a is based on the Flash-quench method, and Ru(bpy)3 2+ and quencher Co(NH3)5Cl 2+ a) The mechanism of mediating RNA oxidation reaction; b) The experimental process of primer extension analysis used to locate RNA oxidation editing sites.

[0031] Figure 2 The figures are the results of the oxidation resistance evaluation of single-stranded RNA and double-stranded RNA-DNA. Among them, a is the experimental process for RNA oxidation resistance determination based on flash quenching; b and c are ribonucleotide chemical conversion analysis based on high-performance liquid chromatography (HPLC).

[0032] Figure 3 The figures are the results of the oxidation resistance evaluation of single-stranded RNA and double-stranded RNA-DNA. Among them, a is the experimental process for RNA oxidation resistance determination based on singlet oxygen; b and c are ribonucleotide chemical conversion analysis based on high-performance liquid chromatography (HPLC).

[0033] Figure 4Figure 1 shows localized oxidation restricted by loop structure. a) Unpaired regions are formed through DNA-RNA hybridization, exposing previously double-stranded protected bases to the oxidation reaction system. b) Introduction of a potential bulge of 1 to 3 nucleotides into the target site of a model RNA (tRF3005): The guide DNA, when complementary to the target site, intentionally lacks at least one ribonucleotide at the labeled site. c) Premature termination of reverse transcription of oxidized RNA (demonstrated using PAGE gel). RNA was preannealed with a guide DNA capable of inducing bulges and internal loops, followed by flash-quenching oxidation to form damage. d) Guide DNA was used to induce 1×1, 1×2, 1×3, and 1×4 internal loops at the rG9 site of the model RNA: loop structures of varying sizes were designed by manipulating the number of DNA deletion sites. e) PAGE gel depicting premature termination of reverse transcription during singlet oxygen (¹O₂)-mediated oxidation. Lanes C and G mark template strand bases, with the target guanine nucleotide site (rG9) highlighted in red.

[0034] Figure 5 Figure 1 shows the location of nucleobase oxidation programmed by guide DNA; a shows the location of oxidative editing determined by guide DNA. Site-specific oxidation of rG6, rG9, and rG15 in a model RNA (tRF3005) was achieved by inducing a 1-nt bulge with guide DNA. b shows the workflow for targeting RNA oxidative modifications using primer extension assays. c shows that the site of oxidative modification shifts with the migration of the bulge loop as shown in primer extension assays. RNA was preannealed with either a fully complementary guide DNA or a guide DNA that induced a different bulge loop and then oxidized under flash quenching conditions. Gel lanes labeled C and G indicate template strand bases, and the target guanosine nucleotides rG6, rG9, and rG15 are highlighted in blue, magenta, and cyan, respectively.

[0035] Figure 6 Guide DNA for simultaneous oxidative editing of multiple targets; a shows LOCAL's simultaneous introduction of multiple oxidative lesions at multiple preset RNA sites (rG6 and rG15). FAM and Cy5 fluorescently labeled reverse transcription primers were used, respectively, to detect oxidative lesions at rG6 and rG15 by primer extension analysis. b shows LOCAL's ability to simultaneously edit multiple sites using rationally designed guide DNA: demonstrating co-localization analysis of multiple oxidative lesions, with the target site color scheme being the same. Figure 5 c in.

[0036] Figure 7 The oxidative modification of fluorescent RNA aptamers is shown in Figure 1. a) Evaluation of the effect of oxidative modification of malachite green aptamers. b) Primer extension analysis shows that malachite green aptamers G 23 G 24 Oxidative modification occurs. c shows the linear range determined by titration of wild-type malachite green aptamer concentration. d shows the luminescence effect evaluation of the wild-type / oxidatively modified malachite green aptamer binding to malachite green dye.

[0037] Figure 8 Site-directed oxidation enables site-selective bioconjugation of RNA. (a) Post-synthesis RNA bioconjugation methods are important for RNA enrichment and visualization. (b) The experimental workflow for site-selective bioconjugation of RNA via local oxidation. Specific guanosine nucleotides (G) in the DNA-induced loop can be oxidized by singlet oxygen (¹O2) and subsequently "captured" by propargylamine to form an alkynylated RNA complex, which is then derivatized via copper-catalyzed click chemistry.

[0038] Figure 9 The possibility of localized labeling at predetermined sites. Figure 1: Top: Schematic diagram of the model RNA miR-124 and its guide DNA design (targeting five G-rich sites of miR-124); Bottom: Primer extension analysis reveals that the site of oxidative modification shifts with loop structure migration. The duplex was oxidized with singlet oxygen in the presence of propargylamine.

[0039] Figure 10 Figure 1 demonstrates the utility of LOCAL technology for target enrichment. (a) LOCAL technology enables site-selective biotinylation of model RNA. (b) The process for enriching biotinylated RNA from a nucleic acid mixture. (c) Top: Quantitative graph of biotinylated RNA enrichment levels. (bottom) Dot blot demonstrating the enrichment of biotinylated RNA.

[0040] Figure 11 Figure 2 shows imaging of fluorescently labeled RNA under cellular stress conditions. a) Experimental workflow for imaging fluorescently labeled RNA under cellular stress conditions. After lipofectamine transfection with FAM-labeled miR-124, cells were heat-shocked 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 located around 520 nm. c) Comparison of fluorescence imaging of FAM-labeled miR-124 before and after heat shock. d) Center (left): Number of fluorescent spots per cell before and after heat shock; (right): Statistical analysis of fluorescent spot area (data are presented as mean ± standard error, n = 3 independent experiments). Scale bar, 10 μm. DETAILED DESCRIPTION

[0041] Example 1: Rational Design of DNA-Guided Site-Localized RNA Oxidation

[0042] To test the feasibility of site-localized RNA oxidation, we constructed duplexes containing loops at predetermined sites and implemented them with designed guide DNA. The guide DNA is the reverse complement of the target RNA and introduces one or more mismatches at the selected RNA sites. Previous studies have shown that this design can induce unpaired RNA loops at these sites. In the prototype design, we used a readily available photocatalyst, Ru(bpy)3 3+ , which has previously been shown to efficiently oxidize guanosine ( Figure 1 We hypothesized that complementary DNA might inhibit the oxidation of nucleobases within the protected duplex due to their reduced solvent exposure and intermolecular accessibility. Conversely, the designed guide DNA-induced looping would expose otherwise unreactive nucleobases, thereby promoting oxidation. Subsequently, enzymatic removal of the DNA could yield oxidatively modified RNA for further investigation.

[0043] Example 2: Pre-annealed DNA protects RNA from oxidation

[0044] There is currently no data on whether RNA-DNA duplexes are resistant to oxidation, particularly under the reaction conditions required for stoichiometric oxidative modification. Therefore, we investigated the extent to which complementary DNA (cDNA) provides protection against oxidation of paired RNA. To address this issue, we used a 35-nt model RNA containing three guanines (G) in different sequence contexts, which can subsequently be used to study the effects of neighboring bases on redox reactions. We employed a previously reported “flash-quench” oxidation method to photocatalyze Ru(bpy)3 2+ With Co(NH3)5Cl 2+ In situ generation of Ru(bpy)3 3+ , selectively oxidize unpaired guanines in single-stranded regions of RNA. Figure 2 As shown: A strong oxidant was generated by photoexcitation, followed by DNA removal and RNA digestion, and the chemical conversion of ribonucleotides was analyzed by high-performance liquid chromatography (HPLC). The results showed that nearly all G in unprotected single-stranded RNA (ssRNA) was oxidized, while adenine (A), cytosine (C), and uracil (U) exhibited resistance to oxidation. High-resolution mass spectrometry (HRMS) confirmed that the primary oxidation product of G was physiologically stable Oz.

[0045] To further explore whether DNA has a protective effect against other physiologically relevant oxidants, we tested the oxidative effect of singlet oxygen (¹O2). Although studies have shown that singlet oxygen oxidizes single-stranded RNA (ssRNA) more strongly than double-stranded RNA (dsRNA), the antioxidant properties of RNA-DNA duplexes are still unclear. In the experiment, we co-incubated the model ssRNA or its corresponding RNA-DNA duplex with 25 μM Eosin B, and generated singlet oxygen by blue light excitation. After 20 minutes, the DNA was removed and the RNA was recovered. HPLC analysis showed that 87% of the G in the ssRNA was oxidized, while the RNA-DNA duplex showed significant antioxidant properties ( Figure 3 HRMS further identified 8-oxoG as the primary product of singlet oxygen oxidation of G. In summary, pre-annealed complementary DNA can effectively protect RNA from damage by various oxidants.

[0046] Specific experimental steps:

[0047] tRF-3005-35nt: 5'-AUCCUGCCGACUACGCCAagacuguuaaaugacug-3';

[0048] tRF-3005-35mer-cDNA: 5'-cagtcatttaacagtctTGGCGTAGTCGGCAGGAT-3';

[0049] All of the above are chemically synthesized.

[0050] 1. Construction of photocatalytic oxidation reaction system

[0051] a. Hybridization complex formation:

[0052] 200 pmol of tRF-3005-35nt and 400 pmol of tRF-3005-35mer-cDNA were added with NaCl to a final concentration of 100 mM, heated at 70°C for 4 minutes, and cooled to 25°C at a gradient of 0.1°C / second. Subsequently, 1 µL of 100 mM sodium phosphate buffer (pH 7.0) and 1 µL of 60 mM MgCl2 were added, and the mixture was incubated at 25°C for 20 minutes.

[0053] b. Construction of oxidation reaction system:

[0054] To the above mixture, 1 µL of 22.5 mM [Co(NH₃)₅Cl]Cl₂ (Merck, Product No. 298301) and 0.5 µL of 4.5 mM [Ru(bpy)₃]Cl₂ (Merck, Product No. 224758, dissolved in 20 mM sodium phosphate buffer, pH 7.0) were added sequentially to adjust the total volume to 10 µL. The reaction tube was incubated in a thermomixer (25°C) in the dark. After 15 minutes, the tube cap was removed, the sample was shaken at 800 rpm, and the oxidation reaction was initiated by irradiation with a blue LED (wavelength 456 nm, 40 W, 6 cm from the sample) for 10 minutes.

[0055] c. RNA purification:

[0056] RNA was purified using an RNA Purification and Concentration Kit (Jianshi, Cat. No. TR113-200) according to the manufacturer's instructions, with a final elution volume of approximately 14 µL. Subsequently, 4 µL of DNase I (NEB, Cat. No. M0303S) and 2 µL of 10× DNase I Reaction Buffer (NEB) were added and incubated at 37°C for 2 hours to degrade cDNA. After purification, the RNA was again purified using an RNA Purification and Concentration Kit (Jianshi, Cat. No. TR113-200) and eluted with RNase-free water (approximately 20 µL).

[0057] The eluted RNA was mixed with 10 µL of 3 M sodium acetate (pH 5.2, Beyotime, Catalog No. ST342), 1 µL of glycogen (20 mg / mL, Beyotime, Catalog No. D0812), 80 µL of RNase-free water, and 500 µL of absolute ethanol. Precipitation was carried out 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 ice-cold 75% ethanol and finally resuspended in 14 µL of RNase-free water for subsequent HPLC-HRMS analysis.

[0058] The configuration system is summarized in Table 1.

[0059] Table 1

[0060]

[0061] 2. Construction of singlet oxygen reaction system

[0062] 200 pmol of tRF-3005-35nt and 400 pmol of tRF-3005-35mer cDNA were mixed with 4 µL of 0.5 M NaCl (all dissolved in D2O) to a total volume of 10 µL. The mixture was heated at 70°C for 4 minutes and then cooled to 25°C at a gradient of 0.1°C / second. Six µL of 3.33× premixed PBS buffer (33.3 mM Na2HPO4, 5.8 mM KH2PO4, 0.46 M NaCl, 8.8 mM KCl, pH 7.2-7.6, 20 mM MgCl2, in D2O) was added, gently mixed, and equilibrated at 25°C for 20 minutes. Subsequently, 1 µL of 0.5 mM Eosin B (dissolved in D2O) and 3 µL of D2O were added and mixed thoroughly. The reaction tube was placed in a thermomixer (25°C, 800 rpm) and irradiated with a blue LED light source (wavelength 456 nm, power 40 W, 6 cm from the sample) for 20 minutes. After the reaction, 10 µL of 3 M sodium acetate (NaOAc), 1 µL of glycogen (20 mg / mL, Beyotime, Cat. No. D0812), 80 µL of RNase-free water, and 500 µL of absolute ethanol were added for ethanol precipitation and purification. The RNA pellet was collected by centrifugation at 4°C, washed with 75% ethanol, and resuspended in 14 µL of RNase-free water. 4 µL of DNase I (NEB, Cat. No. M0303S) and 2 µL of 10× DNase I reaction buffer (NEB) were then added and incubated at 37°C for 2 hours to degrade the cDNA. After purification, the RNA was again eluted with RNase-free water (approximately 20 µL) using an RNA purification and concentration kit (Jianshi, Cat. No. TR113-200). The eluted RNA was mixed with 10 µL of 3 M sodium acetate (pH 5.2, Beyotime, Catalog No. ST342), 1 µL of glycogen (20 mg / mL, Beyotime, Catalog No. D0812), 80 µL of RNase-free water, and 500 µL of absolute ethanol. Precipitation was carried out 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 ice-cold 75% ethanol and finally resuspended in 14 µL of RNase-free water for subsequent HPLC-HRMS analysis.

[0063] The configuration system is summarized in Table 2.

[0064] Table 2

[0065]

[0066] Example 3: Ring-Confined Local Oxidation (LOCAL)

[0067] Because oxidation is inhibited in paired helical structures, we predicted that designed guide DNAs that induce unpaired sequences would re-expose these protected residues for oxidation. To this end, we initially designed and tested guide DNAs with deletions of 1 to 3 nucleotides at the target position, which should induce potential bulged loops of 1 to 3 nt in size.

[0068] Guide DNA designed to induce 1nt RNA bulges under blue light irradiation with photocatalyst Ru(bpy)3 3+ Reactions with singlet oxygen generators, or singlet oxygen generators, can achieve near-stoichiometric modification (>95% in flash quenching) at selected G sites. For example, PAGE analysis of RT primer extension reveals a concentrated dark band 3′ downstream of the modified G site. Bulb loops containing more than one nucleotide also lead to localized oxidation at selected G sites, but appear to have reduced regioselectivity. This is likely because larger bulges increase helical distortion, thereby weakening the thermodynamic stability of the duplex. Furthermore, the effect of bulges on duplex stability is known to be sequence-dependent in DNA. Similarly, the relative position of the selected G within the bulge appears to influence regioselectivity. In dinucleotide bulges, G residues at the 3′ end of the asymmetric bulge exhibit greater oxidative activity than those at the 5′ end. These data suggest that oxidation yield and selectivity can be manipulated by adjusting the size and symmetry of the induced bulge loop. LC-MS analysis of modified X as a model confirmed the chemoselectivity for G.

[0069] The physicochemical properties of the microenvironment surrounding the intended site may influence reactivity. Therefore, we tested how an alternative circular structure, an internal loop, affects local oxidation. We designed guide DNAs containing one or more unpaired nucleotide insertions, predicted to induce 1×1, 1×2, or 1×3 internal loops. Preliminary experiments inducing internal loops at the intended site also led to local oxidation, but the oxidation yield was approximately 3-fold lower than that of the raised loop. Furthermore, the internal loop appeared to exacerbate the regioselectivity of the photocatalytic oxidative modification, as PAGE analysis revealed diffuse non-targeted modifications on both sides of the selected residue. This is likely due to the fact that the internal loop significantly destabilizes the helix, thereby re-exposing previously protected residues in the model RNA for oxidation. Taken together, our data demonstrate that loop-inducing guide DNAs are highly effective in protecting RNA from oxidation and that the efficiency and selectivity of oxidation at the intended site can be enhanced by fine-tuning the RNA loop structure.

[0070] A set of guide DNAs induced the formation of potential bulged loop structures of 1 to 3 nucleotides at selected positions of the model RNA tRF3005. These guide DNAs omitted ≥1 ribonucleotide at the target site. Furthermore, through complementary base pairing, the guide DNAs induced internal loop structures of 1×1, 1×2, 1×3, and 1×4 in size at the G9 site of the model RNA tRF3005.

[0071] PAGE analysis of representative primer extensions demonstrates premature termination of reverse transcription from oxidized RNA. Figure 4 The RNA shown forms different convex loop and internal loop double-stranded complex structures after pre-annealing with guide DNA, and undergoes oxidative modification under flash quenching and singlet oxygen conditions.

[0072] Corresponding guide DNA sequence:

[0073] tRF-3005-35mer-G9-B1:AAAcagtcatttaacagtctTGGCGTAGTGGCAGGATAAA,

[0074] tRF-3005-35mer-G9-B2a:AAAcagtcatttaacagtctTGGCGTAGGGCAGGATAAA,

[0075] tRF-3005-35mer-G9-B2b:

[0076] AAAcagtcatttaacagtctTGGCGTAGTGCAGGATAAA,

[0077] tRF-3005-35mer-G9-B3:AAAcagtcatttaacagtctTGGCGTAGGCAGGATAAA,

[0078] tRF-3005-35mer-G9-L1:AAAcagtcatttaacagtctTGGCGTAGTGGGCAGGATAAA,

[0079] tRF-3005-35mer-G9-L2:AAAcagtcatttaacagtctTGGCGTAGTGGGGCAGGATAAA,

[0080] tRF-3005-35mer-G9-L3:AAAcagtcatttaacagtctTGGCGTAGTGGGGGCAGGATAAA,

[0081] tRF-3005-35mer-G9-L4:AAAcagtcatttaacagtctTGGCGTAGTAGGTGGCAGGATAAA.

[0082] The flash quenching and singlet oxygen oxidation systems are as described in Examples 1 and 2.

[0083] Primer extension analysis:

[0084] Mix 4 pmol of RNA (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 a dNTP mix (10 mM each, Yeasen, #10122ES74), and bring the total volume to 6.5 µL with RNase-free water. For sequencing lanes, replace dGTP or dCTP with a 9:1 molar ratio of ddGTP (APE×BIO, #B8137) / dGTP mixture or a 9:1 molar ratio of ddCTP (APE×BIO, #B8140) / dCTP mixture, respectively. Incubate the reaction at 65°C for 5 minutes and immediately cool on ice for 2 minutes. Subsequently, 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) were added sequentially (final volume 10 µL). The reaction was carried out according to the following schedule: 25°C for 10 minutes, 42°C for 50 minutes, and 52°C for 50 minutes. After the reaction, 11 µL of loading dye (containing 8 M urea and 0.05% bromophenol blue) was added and the sample was loaded onto a denaturing 18% polyacrylamide gel (UREA TBE PAGE Kit, APE×BIO, Cat. No. K4138). Electrophoresis was performed in 1× TBE buffer (pH 8.3) at a constant current of 19 mA for approximately 2.5 hours. cDNA bands were visualized by fluorescence imaging.

[0085] The configuration system is summarized in Table 3.

[0086] Table 3

[0087]

[0088] Example 4: Directing DNA to Program the Location of Nucleobase Oxidation

[0089] To gain insight into the programmability of loop-restricted oxidation, we deployed novel guide DNAs to characterize oxidation at positions other than G9. If programmable, these guide DNAs should redirect oxidative reactivity by shifting potential bulges or internal loops to other sites. We then measured reverse transcription termination sites after photocatalytic oxidation, enzymatic DNA removal, and column purification. Primer extension analysis revealed ( Figure 5 and Figure 6 ), the modification site indeed shifts as the ring structure moves. Similar experiments using another oxidant, singlet oxygen, also confirmed this notion. Therefore, the data support the programming of localized oxidation using readily accessible guide DNA. Furthermore, multiple oxidation sites can be inscribed simultaneously.

[0090] Corresponding guide DNA sequence:

[0091] tRF-3005-35mer-G6-B1: 5'-AAAcagtcatttaacagtctTGGCGTAGTCGGAGGATAAA-3',

[0092] tRF-3005-35mer-G9-B1: 5'-AAAcagtcatttaacagtctTGGCCGTAGTGGCAGGATAAA-3',

[0093] tRF-3005-35mer-G 15 -B1:5'-AAAcagtcatttaacagtctTGGGTAGTCGGCAGGATAAA-3',

[0094] tRF-3005-35mer-G6G 15 -B1: 5'-AAAcagtcatttaacagtctTGGGTAGTCGGAGGATAAA-3'.

[0095] The singlet oxygen oxidation system is as in Example 2.

[0096] 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.

[0097] Table 4

[0098]

[0099] Example 5: Oxidation-regulated fluorescent RNA aptamers

[0100] Next, we investigated the utility of the LOCAL approach for introducing localized oxidation into fluorescent RNA aptamers. Previous studies have primarily focused on how modifications (such as m5C, pseudouridine, and phosphorothioate) modulate RNA aptamer stability and intermolecular binding affinity. However, how nucleobase oxidation affects fluorescent RNA remains unclear, likely due in part to the lack of postsynthetic methods for synthesizing fluorescent RNA with oxidative modifications at any predetermined site. To this end, we employed the malachite green (MG)-malachite green aptamer (MGA) system to assess how localized nucleotide oxidation affects RNA function. Triphenylmethane dyes, such as MG, typically exhibit extremely low quantum yields due to rapid vibrational deexcitation. However, upon binding to its aptamer, MG is stabilized in a planar conformation, resulting in fluorescence enhancement of up to 2360-fold. To investigate whether site-specific guanine (G) oxidation disrupts the MGA-MG interaction, we employed a flash-quenching approach to oxidize G residues at various positions within the aptamer. RT termination and PAGE analysis confirmed the high oxidation efficiency of G23 and G24, with conversion rates 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 ), oxidation of G23G24 significantly impaired MG binding compared to the native aptamer, presumably because local structural perturbations near the dye-binding pocket hindered the necessary interaction between the aptamer and MG. Loss of function of oxidized MGA highlights how even single or double G modifications can significantly alter RNA structure and ligand affinity. Overall, these findings emphasize the critical role of intact guanines in maintaining aptamer function. By demonstrating that local oxidation near the MG-binding region disrupts complex formation, this study further validates our "LOCAL" approach as a powerful tool for probing the effects of specific oxidative lesions on RNA-ligand interactions.

[0101] Specific experimental steps:

[0102] 10 pmol of MGA or oxoMGA was mixed with 2 µL of 1 M KCl, and the volume was adjusted to 10 µL with RNase-free water. The mixture was heated at 70°C for 4 minutes, then cooled to 25°C at a gradient of 0.1°C / s. 2 µL of 100 mM HEPES buffer (pH 7.5, modified with 50 mM MgCl2) was added to the solution, and the mixture was incubated at 25°C for 20 minutes. Subsequently, 2 µL of various concentrations of MG (600, 200, 66.7, 22.2, 7.4, 2.5, and 0 µM) was added, and the final volume was adjusted to 20 µL with RNase-free water. The fluorescence signal of the mixture was measured (excitation / emission = 620 nm / 656 nm) using an Imaging Multi-Mode Microplate Reader (Agilent).

[0103] Example 6: RNA functionalization by local oxidation labeling

[0104] Post-synthetic methods that enable site-localized functional labeling on RNA are in high demand for the enrichment and visualization of RNA and its intermolecular binding partners. A variety of enzymatic strategies have been described for site-selective labeling of RNA. However, the enzymes used in these methods have a limited sequence range, limiting the extent of RNA modification. Recently, two classes of chemical strategies have been developed to overcome these problems: label-transfer oligonucleotide probes and targeted 2´-acylation of predetermined sites mitigate sequence bias. Although elegant, these methods require the synthesis of chemically modified oligonucleotides or acyl imidazole reagents. Therefore, we sought to expand our approach to RNA functionalization due to its simplicity, high yield, and the absence of synthesis and enzyme engineering. Recent studies have demonstrated that singlet oxygen can efficiently oxidize G, the product of which can be “trapped” 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 that inexpensive reagents are readily available, these properties prompted us to consider that localized oxidative labeling might provide a simple solution for RNA functionalization.

[0105] As a preliminary test, we explored the possibility of localized labeling at predetermined sites. We designed a set of guide DNAs that gradually moved the potential RNA secondary bulge structure toward the 3' end of the model RNA to the G residue. The labeling cascade was initiated by irradiation with the photosensitizer Eosin B in the presence of propargylamine. Subsequently, we characterized the RT termination point after oxidative labeling, enzymatic DNA removal, and RNA purification. PAGE analysis showed ( Figure 9), the labeling site changes as the loop moves, confirming the feasibility and programmability of this labeling method. Interestingly, guide DNA that induces relatively small bulges (e.g., 1 nt) significantly improves labeling efficiency compared to guide DNA that induces internal loops, consistent with the reaction pattern observed for localized oxidation.

[0106] Encouraged by the labeling efficiency and selectivity, we further explored the utility of this method for target enrichment. To this end, we biotinylated miR-124 at a specific site by derivatizing propargylamine-tagged miR-124 with biotin azide via a copper-catalyzed alkyne-azide cycloaddition "click" reaction. Subsequently, we measured RNA quality by enrichment with streptavidin beads, stringent bead washes, and RNA elution with formamide. Remarkably, biotinylated RNA bound to the streptavidin beads in an almost stoichiometric ratio, and approximately 80% of the RNA input was eluted after formamide denaturation ( Figure 10 Dot blot analysis of biotinylated RNA before and after enrichment ( Figure 10 Zhongb and Figure 10 This finding is further confirmed in Figure 3c, demonstrating the utility of this method for RNA enrichment and potential sequence-specific biomolecular interaction studies.

[0107] We applied this labeling strategy to the visualization of RNA in living cells by conjugating fluorophores to target RNAs ( Figure 11 As a preliminary test, propargylamine-tagged miR-124 was derivatized with azide containing FAM via CuAAC “click” chemistry. Fluorescence measurements confirmed the presence of the FAM fluorophore on miR-124, which was further confirmed by denaturing PAGE analysis ( Figure 9 and Figure 11 As proof-of-concept for the utility of our method for RNA tracking, we imaged FAM-tagged miR-124 in the presence or absence of cellular stress. Stress granules (SGs) are phase-separated, membrane-less organelles primarily containing RNA and RNA-binding proteins that form under stress conditions. We found that the fluorescent signal of lipofectamine-transfected FAM-tagged miR-124 was primarily localized in condensed, punctate structures after heat shock ( Figure 11 c and Figure 11 This observation is consistent with previous studies showing that some cytoplasmic RNAs localize to SGs, further supporting the idea that LOCAL can be used to fluorescently label RNAs.

[0108] Specific experimental steps for oxidation labeling:

[0109] 200 pmol of RNA (miR-124-RNA-37mer: 5'-P-UAAGGCACGCGGUGAAUGCCagacuguuaaaugacug-3') and 400 pmol of complementary DNA were heated in folding buffer (containing 50 mM NaCl) at 70°C for 4 minutes and then cooled to 25°C. Six 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. Subsequently, 2 µL of 1 mM Eosin B and 2 µL of 200 mM propargylamine were added to a final volume of 20 µL. The reaction mixture was placed in a thermostatic shaker (25°C, 800 rpm) and irradiated with a blue LED lamp (λ = 456 nm, 40 W, 6 cm from the sample) for 15 minutes. After irradiation, 10 µL of 3 M NaOAc (pH 5.2, Beyotime, Catalog No. ST342), 1 µL of glycogen (20 mg / mL, Beyotime, Catalog No. D0812), 80 µL of RNase-free water, and 500 µL of anhydrous ethanol were added to the mixture, followed by precipitation 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. Subsequently, 4 µL of DNase I (NEB, Cat. No. M0303S) and 2 µL of 10× DNase I reaction buffer (NEB, Cat. No. M0303S) were added, and the mixture was incubated at 37°C for 2 hours to remove DNA. RNA was purified using an RNA purification and concentration kit (Jianshi, Cat. No. TR113-200) and used for primer extension experiments.

[0110] Copper-catalyzed click chemistry reactions:

[0111] For the locally oxidatively labeled RNA sample (11.4 µL, approximately 1 µg), 3.6 µL of a freshly prepared click chemistry reaction mixture was added, consisting of: 0.5 mM CuSO₄ (Sigma, Cat. No. 451657), 2 mM THPTA (MCE, Cat. No. HY-W021042), 5 mM sodium ascorbate (Sigma-Aldrich, Cat. No. 11140), and 1 mM biotin-conjugated azide (Sigma-Aldrich, Cat. No. 762024) or 6-FAM-PEG₃-azide (Shanghai Yuanye, Cat. No. S55093). The mixture was incubated in a thermostat at 22°C and 500 rpm for 30 minutes for a copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. Following the reaction, RNA was purified using a column using an RNA purification and concentration kit (Jianshi, Cat. No. TR113-200).

[0112] Enrichment of biotin-modified RNA:

[0113] For each sample, 40 µL of Dynabeads MyOne Streptavidin C1 magnetic beads (Thermo Scientific, Cat. No. 65001) were placed in a 1.5 mL low-binding tube. The beads were placed on a magnetic rack (incubated at 20°C for 1 minute), and the supernatant was carefully aspirated. 1 mL of RNA binding buffer was added, and the beads were washed three times by magnetic racking. 20 µL of RNA binding buffer was added (final volume approximately 40 µL), and the mixture was incubated at room temperature for 1 hour. The RNA mixture used for enrichment analysis consisted of 4.5 µg of biotinylated miR-124 and 4.5 µg of yeast 5S rRNA. RNA binding buffer was added to each sample to a final volume of 200 µL. The 200 µL RNA mixture was transferred to 40 µL of pre-equilibrated magnetic beads and incubated at 20°C for 1 hour on a sample rotator (22 rpm). The samples were then transferred to a magnetic rack (incubated at 20°C for 1 minute), and the supernatant was carefully aspirated (retained for subsequent analysis). Wash the beads five times with 1 mL of RNA wash buffer by inversion, pipetting onto a magnetic rack, and discarding the supernatant. Repeat this wash step four times. Then, wash with 1 mL of PBS buffer, 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, Cat. No. Z5332) (incubate at 20°C for 1 minute), and collect the eluate (approximately 90 µL). Repeat this elution once, collecting a total of 180 µL of eluate, which is then purified using an RNA purification and concentration kit (Jianshi, Cat. No. TR113-200). After washing with the purification column, the sample is eluted with 6 µL of RNase-free water. Dot blot analysis is used to assess the enrichment of biotinylated miR-124.

[0114] Table 5. Composition of RNA Binding Buffer

[0115]

[0116] Table 6 Composition of RNA Wash Buffer

[0117]

[0118] Table 7 Composition of RNA elution buffer

[0119]

[0120] Dot Blot analysis:

[0121] 1 µL of eluted RNA sample (including input, supernatant, and eluate, with three replicates per group) was spotted onto an Ambion® BrightStar®-Plus nylon membrane (Thermo Scientific, Cat. No. AM10100). RNA was fixed to the membrane using a UV crosslinker (energy of 180 mJ / cm²). After fixation, the membrane was washed with 10 mL of TBST buffer (1× TBS containing 0.1% Tween-20, Beyotime, Cat. No. ST673-500ml) on a rocking platform at room temperature for 5 minutes to remove unbound RNA. Subsequently, the membrane was blocked with TBST buffer containing 5% BSA (Yisheng, Cat. No. 36105ES25) for 1 hour at room temperature. The membrane was then incubated with 1 µg / mL streptavidin-HRP (Pierce, Cat. No. 21124, dissolved in 5% BSA) at room temperature for 1 hour. The membrane was then washed three times with TBST buffer for 10 minutes each. Finally, Clarity Western ECL chromogenic substrate (Bio-Rad, Cat. No. 1705061) was evenly added to the membrane surface. After incubation for 2 minutes, the chemiluminescent signal was collected using the Bio-Rad ChemiDoc™ MP imaging system.

[0122] Fluorescence scanning of FAM-labeled miR-124:

[0123] 200 ng of FAM-labeled or unlabeled miR-124 was mixed with RNase-free water to a final volume of 20 µL and loaded into a 384-well plate (LABSELECT, Cat. No. 31431). Emission wavelength scanning was performed using an imaging multi-mode microplate reader (Agilent, Model MS-SP104). The excitation wavelength (Ex) was fixed at 460 ± 9 nm, and the emission wavelength (Em) was scanned from 480 to 700 nm (at 2 nm intervals).

[0124] Cell culture and RNA transfection:

[0125] 100,000 HeLa cells were seeded into 24-well plates containing coverslips (CITOTEST, Cat. No. 80346-1210, 12 mm diameter). Twenty-four hours after seeding (cell confluence approximately 40%), 120 ng of FAM-labeled miR-124 was transfected with 3 μL of Lipofectamine RNAiMAX (Invitrogen, Cat. No. 13778100) in Opti-MEM medium (Thermo Scientific, Cat. No. 31985070) according to the manufacturer's instructions. Six hours after transfection, the coverslips were washed three times with PBS, and 0.5 mL of fresh medium was added to each well. The cells were then incubated at 42°C for 40 minutes and quickly fixed with 4% paraformaldehyde (Macklin, Cat. No. P804536). After fixation, cells were washed with PBS for 10 minutes, stained with DAPI (Sigma-Aldrich, Cat. No. D9542) for 10 minutes, and rinsed with PBS for 10 minutes. Finally, sections were mounted with Fluoromount-G mounting medium (Southern Biotech, Cat. No. 0100-01) for microscopic imaging.

[0126] In summary, RNA oxidative damage is increasingly implicated in cancer, neurodegenerative diseases, and cardiovascular diseases, but dissecting its site-specific functional consequences remains challenging. Existing methods, including random bulk oxidation and solid-phase synthesis, are limited in precision, scalability, and compatibility with transcribed RNA. Here, we introduce LOCAL, a postsynthetic strategy that enables the introduction of oxidative lesions at predetermined 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 could be a key step toward dissecting the causal relationship between RNA oxidation and dysfunction.

[0127] LOCAL achieves regioselectivity by exploiting RNA-DNA hybridization to protect duplex regions from oxidation while directing oxidation to exposed nucleotides within induced loops in the DNA. This control enables near-complete oxidation at the desired site even under highly oxidizing conditions while minimizing off-target effects. Furthermore, the physicochemical properties of the induced RNA loop appear to influence the efficiency and selectivity of oxidative editing. For example, 1-nt bulged loops provide the highest chemical yields to date with minimal off-target oxidation, likely due to their preserved duplex stability. In contrast, internal loops exhibit lower selectivity, highlighting loop geometry as a tunable parameter for optimizing regioselectivity for editing accuracy. Notably, the method's chemoselectivity for guanine (confirmed by mass spectrometry) is consistent with guanine's redox sensitivity. Future work exploring alternative oxidants may expand the scope to oxidative lesions at other natural or modified nucleobases.

[0128] We demonstrated the utility of LOCAL by inducing oxidative damage near the ligand-binding site of a malachite green aptamer, which abolished its fluorescent activity. This initial experiment exemplifies how oxidation of key functional residues impairs RNA activity, with implications for RNA dysfunction in oxidation-driven pathologies. Furthermore, the utility of LOCAL extends beyond oxidation studies—the combination of propargylamine-based bioconjugation and “click” chemistry supports downstream applications such as RNA visualization and affinity-based enrichment. Importantly, its photochemical properties allow fine-tuning of oxidation levels through control of reaction time, enabling the simulation of dose-dependent effects of oxidative damage. Overall, these features highlight the potential of LOCAL as a versatile method for mechanistic studies and as a potentially generalizable biotechnological tool.

Claims

1. A method for site-directed oxidative modification of RNA, characterized in that: The following steps are involved: (a) hybridizing a guide DNA with a target RNA to form an RNA-DNA duplex having a predetermined circular structure, wherein the guide DNA comprises a sequence complementary to the target RNA and introduces at least one nucleotide mismatch at a predetermined position; The target RNA is ≤200nt; The predetermined site is located within the range of at least the sixth nucleotide to at least the sixth-to-last nucleotide in the target RNA sequence; (b) subjecting the duplex formed in step (a) to an oxidation reaction in the presence of an oxidizing agent; The oxidant is Ru(bpy)3 3+ or Eosin B; (c) removing the guide DNA to obtain an RNA product that has undergone specific oxidative modification at the predetermined site; The specific oxidative modification is oxidative modification of guanine.

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

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

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

5. The method for site-directed oxidative modification of RNA according to claim 1, wherein It also includes functional labeling of oxidatively modified RNA through click chemistry, the specific steps are: (1) In the presence of propargylamine, the oxidation product 8-oxoguanosine is covalently linked to the alkyne group; (2) Copper-catalyzed azide-alkyne cycloaddition reaction is used to direct the coupling of functional groups to oxidation sites.

6. The method for site-directed oxidative modification of RNA according to claim 5, wherein: The functional group is selected from biotin, fluorescein or azide groups.

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