Quantitative detection of 2'-O-methylated residues in
non-coding RNAs using DNAzymes and quantitative
RT-PCR

Valentyne Lebret-Kogey, Lilia Ayadi, Sylvain Maenner, Yuri Motorin, Isabelle Behm-Ansmant, Christelle Aigueperse

<figure><img src="image_1.png" /></figure>

To cite this version:

Valentyne Lebret-Kogey, Lilia Ayadi, Sylvain Maenner, Yuri Motorin, Isabelle Behm-Ansmant, et al.. Quantitative detection of 2'-O-methylated residues in non-coding RNAs using DNAzymes and quantitative RT-PCR. Biochimie, 2025, 237, pp.26-39. 10.1016/j.biochi.2025.07.006. hal-05162861

HAL Id: hal-05162861

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# Quantitative detection of 2'-O-methylated residues in non-coding RNAs using DNAzymes and quantitative RT-PCR

<figure><img src="image_3.png" /></figure>

Valentyne Lebret-Kogey<sup>a,b</sup>, Lilia Ayadi<sup>a,c</sup>, Sylvain Maenner<sup>a</sup>, Yuri Motorin<sup>a,c</sup>, Isabelle Behm-Ansmant<sup>a,**</sup>, Christelle Aigueperse<sup>a,*</sup>

## ARTICLE INFO

**Article history:**

Received 24 February 2025

Received in revised form

24 June 2025

Accepted 5 July 2025

Available online 7 July 2025

Handling Editor: Dr B Friguet

**Keywords:**

RNA modifications

2'-O-Methylation

DNAzyme

Deoxyribozyme

UsnRNA

## ABSTRACT

In recent decades, post-transcriptional RNA modifications have emerged as a critical regulatory network in cellular processes. The emergence of advanced technologies, including next-generation sequencing (e.g., Illumina) and third-generation sequencing (e.g., Nanopore and PacBio), has significantly improved the detection of RNA modifications in both coding (mRNAs) and non-coding RNAs (ncRNAs). However, these powerful and transcriptome-wide approaches often generate a significant number of false positive hits, highlighting the need for complementary methods allowing validation of the RNA modification profile as well as more precise quantification. In this study, we present further optimization of a straightforward and sensitive protocol using RNA-cleaving deoxyribozymes (DNAzymes) to validate 2'-O-methylations. The validity of this protocol was demonstrated by the analysis of two well-characterized 2'-O-methylated adenines in human U1 and U2 small nuclear RNAs (snRNAs). Using siRNA targeting NOP58 and antisense oligonucleotides targeting scaRNA7, we showcased the sensitivity and specificity of our approach in detecting variations in 2'-O-methylation levels. The DAMP-RNA (DNAzyme-Assisted Methylation Profiling of RNA) method, combining the DNAzyme cleavage with RT-qPCR provides a robust and efficient way to investigate known 2'-O-methylation in any cellular RNA regardless of its abundance, yielding valuable insights into the dynamics of RNA modifications. Despite its limitations, this method represents a significant advancement, enhancing clarity and reproducibility in the epitranscriptomics field, paving the way for further exploration in the RNA biology.

© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

One of the most prevalent RNA modifications is the 2'-O-methylation (2'-O-Me, Nm), a backbone modification that adds a methyl group at the 2'-O position of the ribose sugar ring (Fig. 1A) [1,2]. This modification affects the biophysical and biochemical properties of RNA, including protection from nucleases, and also provides structural stabilization and modulation of interaction with proteins or other RNAs. Although the precise functions and molecular mechanisms of 2'-O-methylation's action are still under extensive investigation, accumulating evidence suggests that this

modification affects a wide range of cellular processes, including translation, splicing and innate immunity [3]. The dynamic nature of the 2'-O-Me levels at individual sites underlines its potential in the regulation of gene expression [4-8]. Furthermore, alterations in 2'-O-methylation patterns have been associated with several human diseases, including cancer, suggesting that these modifications could serve as valuable biomarkers for diagnostics and as promising therapeutic targets [9-11].

Given the importance of accurate mapping and quantification of 2'-O-methylation levels at individual RNA sites, there is an urgent need for reliable and straightforward methods [12]. In low-throughput mode, the detection or validation of 2'-O-methylation sites can, in principle, be achieved by various techniques, albeit with highly variable performance and complexity in their experimental implementation. General detection strategies, such as SCARLET (Site-specific Cleavage And Radioactive-Labelling followed by ligation-assisted Extraction and Thin-layer

<footnote><sup>a</sup>Université de Lorraine, CNRS, UMR7365 IMoPA, Nancy, France</footnote>

<footnote><sup>b</sup>ENS de Lyon, Lyon, France</footnote>

<footnote><sup>c</sup>Université de Lorraine, SMP IBSLor, EpiRNA-Seq Core Facility, Nancy, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: isabelle.behm@univ-lorraine.fr (I. Behm-Ansmant), christelle.aigueperse@univ-lorraine.fr (C. Aigueperse).</footnote>
Abbreviations

<table><tr><td>2'-O-Me</td><td>Nm, 2'-O-methylation</td></tr><tr><td>ASOs</td><td>AntiSense Oligonucleotides</td></tr><tr><td>DAMP-RNA</td><td>DNAzyme-Assisted Methylation Profiling of RNA</td></tr><tr><td>DNAzymes</td><td>deoxyribozymes</td></tr><tr><td>LC-MS</td><td>Liquid Chromatography with tandem Mass Spectrometry</td></tr><tr><td>ND</td><td>No-DNAzyme</td></tr><tr><td>NGS</td><td>Next-Generation Sequencing</td></tr><tr><td>RTL-P</td><td>Reverse Transcription at Low deoxy-ribonucleoside triphosphate (dNTP) concentrations followed by polymerase chain reaction (PCR)</td></tr><tr><td>RT-qPCR</td><td>Reverse Transcription followed by quantitative polymerase chain reaction</td></tr><tr><td>SCARLET</td><td>Site-specific Cleavage And Radioactive-Labelling followed by ligation-assisted Extraction and Thin-layer chromatography</td></tr></table>

chromatography) [13], are a powerful way to validate RNA modification sites, but remain extremely complex and time-consuming. In addition, the use of radioactive labelling is often precluded for safety reasons. Other more specific methods for the detection of 2'-O-Me residues are based either on the resistance of the RNA phosphodiester bond to cleavage in the presence of a 2'-O-Me group, or on the ability of reverse transcriptase (RT) to pause/abort cDNA synthesis at 2'-O-methylated nucleotides. In the first case, the detection of the resistant sites is usually done by primer extension combined or not to quantitative PCR or, very rarely, by direct analysis of end-labelled RNA [14-16]. In the second approach, the detection of the RT pause sites is done by primer extension which is performed at low dNTP concentration ([dNTP]). When low [dNTP] primer extension is coupled to quantitative PCR (RTL-P protocol), it provides the opportunity for relative quantification [17]. While the RTL-P method has the advantage of allowing positive identification of 2'-O-methylations, it also has several limitations. Firstly, it is not always reliable due to stable RNA structures that can also stop RT in a [dNTP]-dependent manner. Moreover, highly modified regions of RNA are not suitable for analysis and natural RT-arresting residues also preclude the use of this protocol.

With the advent of massive parallel sequencing, at least five independent protocols for next-generation sequencing (NGS)-based detection of 2'-O-Me residues have been developed. The most widely used technique for this purpose remains the Illumina sequencing-based RiboMethSeq protocol, which uses deep sequencing to measure the protection conferred by 2'-O-methylation against alkaline cleavage [6,18-20]. Despite its global performance, this method is susceptible to capturing a substantial number of false positives due to RNA structural complexity and highly irregular cleavage in certain highly structured regions. Moreover, RiboMethSeq requires a fairly high sequencing depth, and is currently limited to abundant (or at least highly enriched) RNAs [5,12]. Machine learning algorithms for the analysis of RiboMethSeq data can also be employed to ascertain the identification of the modified residues, but independent orthogonal validation is still highly recommended [21]. The second NGS-based approach for mapping 2'-O-Me residues (termed 2OMe-seq) exploits RT pausing at low [dNTP] [22], and suffers from the same drawbacks as its low-throughput version. Despite this, the 2OMe-seq protocol has been used for orthogonal validation of

RiboMethSeq data in the context of exhaustive mapping of rRNA 2'-O-Me residues in *A. thaliana* [23,24]. The third proposed approach exploits the resistance of the 3'-terminal 2'-O-Me residues to periodate (IO4) oxidation, thereby maintaining their competence for specific ligation of the 3'-adapter (NmSeq/OxiSeq) [25,26]. This approach requires very large amounts of the input material and has not been implemented in a low-throughput version (not coupled to NGS). The fourth approach is Nm-Mut-seq, a mutation signature-based Nm mapping method that uses a custom RT that introduces mutations at Am-, Cm- and Gm-modified sites, but its limitation is the lack of detection of modifications at the Um site. [27]. The last approach has been described more recently and takes advantage of the sensitivity to RNA ribose methylation of the helicase and hydrolase activities of *Mycoplasma genitalium* (MgR) RNase R on single-stranded RNA. MgR digestion has been combined with NGS to develop NJU-seq (Nm-Judged-Universally sequencing). This method is powerful, but also produces false positives [16].

Recent promising advances in nanopore direct RNA sequencing, which detects modification-induced changes in electric current signals, alongside with machine learning approaches, have enabled the identification of 2'-O-methylation sites in low abundant RNAs, such as messenger RNAs (mRNAs) [28]. The combination of nanopore sequencing with RiboMethSeq analysis has recently been used to validate *D. melanogaster* rRNA 2'-O-methylation sites [29]. Despite these achievements, the limited size of the training dataset and high background signals still preclude the massive use of direct RNA nanopore sequencing for reliable RNA modification analysis [28,30], and valuable data are only obtained by the systematic inclusion of unmodified control RNA species of identical sequence.

Taken together, despite major advances in high-throughput RNA modification analysis, studies of RNA 2'-O-methylation cannot rely exclusively on high-throughput detection techniques, which introduce varying degrees of error and need to be validated with at least one reliable and low-throughput method [5]. Considering these points, we have further investigated and optimized an alternative approach based on the use of specific deoxyribozymes (DNAzymes). These synthetic single-stranded DNA molecules are capable of cleaving RNA at a specific site. Two structurally distinct families of DNAzymes with different constraints on the sequence of the cleavage site have been described in the literature, referred to as DNAzymes of families 10-23 and 8-17 [31]. The properties of DNAzymes have been exploited to develop various analytical techniques, including biosensors for environmental and clinical applications, purification of RNA fragments, as well as imaging cancer markers [32,33]. Each DNAzyme consists of a catalytic loop flanked by 5' and 3' sequences that hybridize to the RNA substrate via complementary Watson-Crick base pairing (Fig. 1B and C). The catalytic core of DNAzyme carries out the cleavage reaction, which is similar to that of RNA nuclease and requires a free 2'-OH moiety in the ribose ring. Thus, DNAzyme cleavage is a simple and effective approach for assessing the 2'-O-methylation status of RNAs. In the past, DNAzyme cleavage has been already used to determine whether a given rRNA or tRNA site is indeed 2'-O-methylated [34-37]. However, the described methods required radioactive labelling or were limited by the analysis of RNA cleavage on denaturing polyacrylamide gel electrophoresis (PAGE).

In this work, we present the detailed DAMP-RNA (DNAzyme-Assisted Methylation Profiling of RNA) protocol for the use DNAzymes to detect 2'-O-Me residues in snRNAs, with a framework that is theoretically adaptable for the validation of numerous 2'-O-methylation sites. To enhance sensitivity and broaden the applicability of our approach, we optimized the protocol and integrated
quantitative RT-PCR, extending the analysis to the majority of RNA types. We also refined the protocol for use with the two major DNAzyme families, 10–23 and 8–17. Proof-of-concept experiments demonstrated that the DNAzyme cleavage combined with quantitative RT-PCR can effectively detect two representative and well-characterized 2'-O-methylated sites, the adenine at position 70 (Am70) and the adenine at position 30 (Am30) in human U1 and U2 snRNAs, respectively. Interestingly, experimental knockdown (KD) of NOP58 and scaRNA7 demonstrated the sensitivity of our technique in detecting reductions in 2'-O-methylation levels on cellular U snRNAs. Our data emphasize that the DAMP-RNA method is a straightforward, rapid and inexpensive validation tool that is sufficiently reliable to detect variations in modification levels at RNA-specific sites. This makes it well-suited for investigating 2'-O-methylation variation in diverse biological contexts and across a wide range of RNA species, including those with low expression levels.

## 2. Materials and methods

### 2.1. Cell culture

HeLa S3 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich) at 37 °C in a humidified atmosphere containing 5 % CO₂, following standard laboratory protocols. The culture medium was supplemented with 2 mM L-glutamine, penicillin/streptomycin, and 10 % fetal calf serum. Total RNA was extracted from the cells using TRIzol (Invitrogen) according to the manufacturer's instructions.

### 2.2. RNA interference

Small interfering RNAs (siRNAs) targeting the mRNA encoding the NOP58 protein (siNOP58) (Invitrogen) [38] and the negative control firefly luciferase siRNAs (siCtr) (Eurofins Genomics) [39] were transfected into HeLa S3 cells at a final concentration of 25 nM using the calcium phosphate transfection method [40]. The target RNA sequences are provided in Supplementary Table S1. To deplete scaRNA7 transcripts, HeLa S3 cells were transfected with 25 nM of scaRNA7 GapmeR AntiSense Oligonucleotides (ASOs) (scaRNA7 ASO) [41] or the negative control NC-ASO (ASO affinity Plus®, IDT) using the same calcium phosphate transfection protocol. ASOs are single-stranded antisense chemically modified oligonucleotides that catalyze RNase H-dependent degradation of complementary RNA targets. Sequences are provided in Supplementary Table S1.

One day before transfection, HeLa S3 cells were plated in six-well plates or 10 cm dishes at a density of 100,000 cells per well or 500,000 cells per dish. The siRNAs or ASO oligonucleotides were combined with 125 mM CaCl₂ and Hepes buffer saline (HBS) pH 7.05 (25 mM Hepes, 0.14 M NaCl, 5 mM KCl, 0.75 mM Na₂HPO₄), pipetted to mix, and incubated for 20 min at room temperature before addition to each well. The cells were incubated with the transfection mixture for 6 h, after which the medium was changed. A second transfection was performed 48 h after the first, and the RNA and proteins were extracted 24 h after the second transfection.

### 2.3. *In vitro* transcription

Templates for *in vitro* transcription of human U1 and U2 snRNAs were produced by PCR amplification of U1 and U2 coding sequences using the Phusion® DNA Polymerase (Thermo Fisher Scientific) and the pUC19-U1 and pUC19-U2 plasmids as templates (generously gift from G. Dreyfuss) (Supplementary Table S1). PCR

templates were purified using the GeneJET Extraction Kit (Thermo Fisher Scientific). *In vitro* transcription was performed using MEGAshortscript™ T7 (Thermo Fisher Scientific) according to the manufacturer's instructions. *In vitro* transcribed RNAs were purified using the Monarch RNA Cleanup Kit (New England Biolabs France). RNA concentration was determined using a NanoDrop™ 2000 spectrophotometer and RNA integrity was verified by PAGE analysis.

### 2.4. Synthesis of 2'-O-methylated RNA

Short synthetic U1 snRNA oligonucleotides, with or without 2'-O-methylation at position 22, equivalent to position 70 in the native U1 snRNA, were obtained from IDT. The sequences are listed in Supplementary Table S1. Prior to DNAzyme treatment, the two RNA oligonucleotides were mixed together at different proportions, in order to establish a linear relationship between the level of the 2'-O-Me and the level of uncleaved RNA detected in the DAMP-RNA protocol.

### 2.5. DNAzyme design

The binding arms of DNAzyme 10–23 and DNAzyme 8–17 are 12 and 20 nucleotides in length, respectively. The DNAzyme catalytic sequence is flanked by reverse complementary sequences. The sequences of the DNAzymes used in this work are provided in Fig. 2C. DNAzymes were obtained from Sigma as desalted oligonucleotides.

### 2.6. DNAzyme digestion

The cleavage protocol was adapted from established methods for each DNAzyme family [42,43]. In this procedure, RNA (2 pmol of transcript or 500 ng of cellular RNA) was combined with DNAzyme or No-DNAzyme oligonucleotides (10–160 pmol) and 4 µl hybridization buffer (200 mM Tris-HCl pH8; 750 mM NaCl) in a 15 µl reaction. Note that comparable results were obtained using as little as 10 ng and 50 ng of input RNA, at positions U1-G81 and U1-Am70, matching those from 500 ng (data not show), indicating that smaller RNA quantities can also be effectively used. To anneal DNAzyme to the target site, the solution was heated at 75 °C for 5 min to facilitate the separation of the strands, followed by a 5-min incubation on ice. After addition of RNase inhibitor (1 µl of Ribolock, Thermo Fisher Scientific) DNAzyme cleavage was initiated by adding a solution of MgCl₂ and MnCl₂ to a final concentration of 10 mM each in 20 µl reaction, and the mixture was incubated at 37 °C for 2 h. Nucleic acids were precipitated with ethanol using AcNH₄⁺ and Glycoblue, dried and resuspended in 20 µl of water. To remove DNAzyme oligonucleotides, 8 µl of RNA were digested with the RapidOut DNA Removal Kit (Thermo Fisher Scientific) at 37 °C for 30 min in a 10 µl reaction volume. In a next step DNase I is safety removed using DNase Removed Reagent (DRR) without potential RNA damaging steps such as heating or organic extraction.

### 2.7. PAGE analysis

A 4 µl aliquot from the DNAzyme reaction post-DNase treatment was mixed with RNA loading dye (New England Biolabs), heated at 95 °C for 5 min, and subsequently loaded on a 10 % denaturing urea (7 M) gel. The gels were stained with SYBR™ Gold nucleic acid gel stain (Thermo Fisher Scientific) for 30 min and scanned using Typhoon 9400 scanner (GE healthcare). Cleavage efficiencies were analyzed using ImageQuant TL software (GE Healthcare) based on the intensities of the bands corresponding to
A

<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /></figure>

C

<figure><img src="image_6.png" /></figure>
**Fig. 1.** The methylation of ribose at 2'-OH group prevents cleavage by DNAzyme. **A.** Chemical structure of RNA 2'-O-methylated nucleotide. The -H atom at the 2' position in the ribose moiety is replaced by a -CH3 group. **B.** The 8-17 DNAzymes preferentially cleave at NG dinucleotide junctions. The catalytic core consists of 14 nucleotides forming a stem-loop structure. The 2'-hydroxyl group attacks the adjacent phosphodiester bond, resulting in the formation of 2'-3'-cyclic phosphate and 5'-hydroxyl termini. **C.** The 10-23 DNAzymes cleave junctions between a purine (R) and a pyrimidine (Y). The 2'-hydroxyl group attacks the adjacent phosphodiester bond, leading to the formation of 2'-3'-cyclic phosphate and 5'-hydroxyl termini.

the full-length RNA and the longer cleaved product.

## 2.8. RT-qPCR

Given that the RT step can introduce technical variability, we recommend using high-quality reverse transcriptase for these reactions (Supplementary Fig. S1C). For each DNAzyme-treated sample, a reverse transcription (RT) reaction was performed using both gene-specific reverse primers for snRNA and GAPDH to ensure optimal efficiency of the reaction (Supplementary Table S1). After removal of DNAzyme with DNase, 10 µl of RNAs with primers and dNTP were incubated at 65 °C for 5 min and cooled on ice. RNAs were reverse transcribed into cDNA using SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific) for 40 min at 55 °C, and the reaction was inactivated by heating at 70 °C for 15 min. To limit the risk of reduced reverse transcription efficiency due to RNA 2D structure, reverse transcription reactions were performed at 55 °C. Quantitative PCR was performed with diluted cDNA, forward and reverse primers (Supplementary Table S1), and iTaq™ Universal SYBR Green Supermix (Biorad) on a QuantStudio thermocycler (Thermo Fisher Scientific) following the manufacturer's guidelines.

The relative standard curve method was employed for quantification. PCR efficiency exceeds 90 % for both *in vitro* and cellular RNAs, demonstrating robust performance across sample types. Normalization was achieved by dividing the average relative quantities of U1 and U2 snRNAs determined in the DNAzyme or No-DNAzyme conditions by the average GAPDH reference value in the corresponding samples. The amount of residual RNA was calculated by dividing the DNAzyme condition by the No-DNAzyme condition, where a value of 1 indicates intact RNA and a value of 0 indicates no residual intact RNA.

## 2.9. UsnRNA enrichment by RNA-immunoprecipitation

Eight 10 cm dishes are required for each condition (siCtr and siNOP58). Cells were harvested in PBS and after centrifugation the pellet was resuspended in 1 ml HNTG lysis buffer (20 mM Hepes pH7.9, 150 mM NaCl, 1 % Triton, 1 mM MgCl₂, 1 mM EDTA, 10 % glycerol, supplemented with Thermo Scientific™ Halt™ Protease and Phosphatase Cocktail). Cells were incubated for 20 min on ice and centrifuged for 10 min at 12,000 g at 4 °C. For each condition, 80 µl of Dynabeads™ Protein G (Thermo Scientific™) were washed 3 times in HNTG buffer and then incubated with or without 50 µl of Y12 antibody (10 µg) (ab3138, Abcam) for 3h at 4 °C on a wheel. At the same time, the extracts were pre-cleared on beads for 1h at 4 °C. After three washes, the beads were incubated with the cell extracts on a wheel for 2 h at 4 °C. After three washes, for the final wash, the beads were separated into 2 tubes, one for RNA extraction and the other for protein extraction. RNA was extracted by adding 500 µl Trizol to the beads. The proteins were extracted with 20 µl of NuPAGE™ LDS Sample Buffer (Thermo Scientific™) supplemented by 100 mM DTT. The control for immunoprecipitation experiment is shown in Supplementary Fig. S2.

## 2.10. Analysis of RNA 2'-O-methylation by RiboMethSeq

RNA-Immunoprecipitation from human HeLa S3 cells was subjected to random fragmentation by alkaline hydrolysis in

50 mM sodium-bicarbonate buffer at pH 9.2 and 96 °C for 10 min. The reaction was stopped by ethanol precipitation using 0.3 M sodium acetate, pH 5.2 and Glycoblue. After centrifugation, the RNA pellet was washed with 80 % ethanol and resuspended in nuclease-free water. RNA fragments were end-repaired as previously described [20] and purified using RNeasy MinElute Cleanup kit (Qiagen) according to the manufacturer's recommendations, except that 675 µL of 96 % ethanol were used for RNA binding step. Elution of purified RNA fragments was performed in 9 µL of nuclease free water. RNA fragments were converted to library using the NEBNext® Small RNA Library Prep Set for Illumina® (NEB ref E7330S, USA) following the manufacturer's recommendations. DNA library was quantified using a fluorometer (Qubit 2.0 fluorometer, Invitrogen, USA) and qualified using a High Sensitivity DNA chip on Agilent Bioanalyzer 2100. Libraries were multiplexed and subjected for high-throughput sequencing on an Illumina HiSeq1000 instrument with a 50 bp single-end read mode.

High quality raw sequencing reads (>Q30) were subjected to trimming using Trimmomatic v0.39 [44] with the following parameters: MINLEN:08, STRINGENCY:7, AVGQUAL:30, trimmed reads were further processed to keep only short reads with fragmentation-defined 3'-extremities. Selected trimmed reads were aligned to the human snRNA reference sequence (NR_004400.1 and NR_002716.3 and Supplementary Table S2) using bowtie2 v2.4.4 [45] in end-to-end mode (-no-unal -no-1mm-upfront -D 15 -R 2 -N 0 -L 10 -i S,1.1.15 as other bowtie2 parameters), only uniquely mapped reads in positive orientation were retained for further analysis. Reads' extremities (5'- and 3'-) were counted for each RNA position in the reference and cumulative 5'/3'-protection profile was established as described [20,46], in R/R-studio environment. Specific RiboMethSeq scores (mean, A, B and C = MethScore, [18,46]) were calculated in ± 2 nt interval for all RNA positions and extracted for positions of known Nm residues in target RNA. Quantification of 2'-O-methylated residues was done using MethScore (previously called scoreC [18] or scoreC2 [46]). This score is used for quantification of the modification level since it keeps linear calibration curve between protection and molar ratio of 2'-methylated residue in RNA.

## 2.11. Availability of datasets

Raw RiboMethSeq data are available at the European Nucleotide Archive (https://www.ebi.ac.uk/ena browser/home) under the accession number PRJEB85526.

## 2.12. Western blot analysis

Proteins were extracted from the cells by incubation for 30 min on ice in lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 % lgepal, 1 mM EDTA and Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific)). Soluble proteins were recovered in the supernatant after centrifugation at 16,000 rpm for 15 min. Proteins were separated by SDS-PAGE gels and transferred to a nitrocellulose membrane (Bio-Rad). The membranes were incubated for 1 h in a solution of 5 % milk in PBS-T (PBS with 0.05 % (v/v) Tween-20), followed by incubation for 1 h with primary antibodies against Anti-beta Actin (Abcam, ab170325) or Anti-NOP58 (Abcam, ab155969) diluted in PBS-T with 3 % BSA (Bovine
<figure><img src="image_7.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>C</figcaption></figure>

<table><thead><tr><th>name</th><th>category</th><th>sequence 5'→3'</th><th>U snRNA</th><th>Target</th><th>DNAzyme family</th></tr></thead><tbody><tr><td>D-Am70</td><td>DNAzyme</td><td>AGCACATCCGGAGGGCTAGCTACAACGAGCAATGGATAAGC</td><td>U1</td><td>Am-C</td><td>10-23</td></tr><tr><td>ND-Am70</td><td>No-DNAzyme</td><td>AGCACATCCGGAGTGCAATGGATAAGC</td><td>U1</td><td></td><td></td></tr><tr><td>D-G81</td><td>DNAzyme</td><td>TCGCAGGGGTCAAGGGCTAGCTACAACGAAACATCCGGAGTGC</td><td>U1</td><td>G-C</td><td>10-23</td></tr><tr><td>ND-G81</td><td>No-DNAzyme</td><td>TCGCAGGGGTCAACGACATCCGGAGTGC</td><td>U1</td><td></td><td></td></tr><tr><td>D-Am30</td><td>DNAzyme</td><td>GATAAGAACAGATACTACATCCGAGCCGGACGATGATCTTAGCCAAAAGGCCG</td><td>U2</td><td>Am-G</td><td>8-17</td></tr><tr><td>ND-Am30</td><td>No-DNAzyme</td><td>GATAAGAACAGATACTACACTTGATCTTAGCCAAAAGGCCG</td><td>U2</td><td></td><td></td></tr><tr><td>D-A51</td><td>DNAzyme</td><td>AGGACGTATCAGATATTAAAATCCGAGCCGGACGAGATAAGAACAGATACTACA</td><td>U2</td><td>A-G</td><td>8-17</td></tr><tr><td>ND-A51</td><td>No-DNAzyme</td><td>AGGACGTATCAGATATTAAACTGATAAGAACAGATACTACA</td><td>U2</td><td></td><td></td></tr><tr><td>D-U156</td><td>DNAzyme</td><td>CCTGGAGGTACTGCAATACTCCGAGCCGGACGAGGTTCGATGCGTGGAGTGGAC</td><td>U2</td><td>U-G</td><td>8-17</td></tr><tr><td>ND-U156</td><td>No-DNAzyme</td><td>CCTGGAGGTACTGCAATAACCAGGTCGATGCGTGGAGTGGAC</td><td>U2</td><td></td><td></td></tr></tbody></table>
**Fig. 2.** Schematic illustration of the DNAzyme-Assisted Methylation Profiling of RNA (DAMP-RNA) methodology. **A.** Experimental strategy of the DAMP-RNA methodology. RNA substrates are incubated with either the oligonucleotide DNAzyme (left) or a control No-DNAzyme (right) to allow hybridization. In the left panel, unmodified RNA is selectively cleaved at the target site by the DNAzyme, while the 2'-O-methylated RNA remains intact. The right panel serves as a control, where RNA is treated with a non-functional DNAzyme (No-DNAzyme), which is an oligonucleotide with the reverse complement sequence of the target but lacking the catalytic site. The uncleaved RNA (residual RNA) is quantified by reverse transcription (RT) using gene-specific primers, followed by quantitative PCR (qPCR). **B.** Schematic representation of the DNAzyme design used in this study. The 10-23 DNAzyme cleaves the A70-C71 junction. The 8-17 DNAzyme cleaves the A30-G31 junction. Flanking sequences adjacent to the catalytic core direct cleavage specificity. The presence of a 2'-O-methylated adenine (Am) prevents DNAzyme-mediated cleavage in both cases. **C.** DNAzyme and No-DNAzyme sequences. The sequence corresponding to the DNAzyme catalytic core is shown in bold and underlined.

Serum Albumin). After three washes in PBS-T, the membranes were incubated with anti-rabbit IgG or anti-mouse IgG conjugated to Horseradish Peroxidase (HRP) (A16072 and A16104, novex by life technologies) for 45 min at room temperature, washed three times and incubated with ECL™ Prime Western Blotting Detection Reagent (GE Healthcare). The chemiluminescence signal was detected using a Fusion Solo Gel Imager (Vilber Lourmat).

## 2.13. Statistical analysis

Statistical analysis was performed using GraphPad Prism 10 (GraphPad Software, USA). The statistical difference between two group was assessed using the Mann-Whitney test, with statistical significance defined as p-values less than 0.05.

# 3. Results

## 3.1. Working model: detection of 2'-O-Me residues in human UsnRNAs

As a model system, we focused on the analysis of the U-rich small nuclear RNAs (UsnRNAs). UsnRNAs associate with specific set of proteins to form uridine-rich small nuclear ribonucleoproteins (UsnRNPs), which assemble sequentially on Pol II primary transcripts to form the spliceosome, the large ribonucleoprotein complex that catalyzes the splicing process [47,48]. Like the majority of cellular RNAs, UsnRNAs are known to contain numerous 2'-O-methylated nucleotides [6,49,50]. Analysis of the U2 snRNA has shown that 2'-O-methylation contributes to spliceosome assembly by remodeling branchpoint sequences and stabilizing the U2 and U6 snRNA interaction, thereby facilitating efficient pre-mRNA splicing [14,51-53]. 2'-O-methylation of snRNA is mediated by a family of box C/D ribonucleoproteins (RNPs), which assemble around a 'guide' RNA that specifies the substrate RNA sequence via base complementarity. For instance, methylation of A70 in U1 snRNA relies on the guide RNA scaRNA7, whereas methylation of A30 in U2 snRNA is mediated by scaRNA9. Box C/D RNPs consist of four core proteins: the methyltransferase fibrillarin, the RNA-binding protein 15.5K, and a heterodimer consisting of the closely related proteins NOP56 and NOP58. Based on the localization of box C/D RNPs, the complex can be subdivided into small nucleolar snoRNPs and small Cajal body-specific scaRNPs [49,54,55].

## 3.2. Application of DNAzyme cleavage for detection and validation of 2'-O-Me residues in UsnRNAs

We developed an experimental protocol combining DNAzyme cleavage with quantitative RT-PCR assays (DAMP-RNA methodology depicted in Fig. 2A) to analyze model 2'-O-methylations: adenine 70 in human U1 snRNA (U1-Am70) and adenine 30 in human U2 snRNA (U2-Am30). DNAzyme was designed to specifically cleave the RNA phosphodiester bond downstream of the U1-Am70 or U2-Am30 residue (Fig. 2B). Upon incubation of RNA with DNAzyme, 2'-O-Me-containing RNA is protected from cleavage and can thus be further amplified by RT-qPCR, while the unmodified

counterpart is cleaved and thus lost in the amplification.

To minimize potential artefacts from the DNAzyme oligonucleotides, we also designed inactive DNAzyme, referred to as No-DNAzyme (ND), which contains only the DNAzyme arms allowing perfect hybridization to the target but does not include the catalytic cleavage sequence (Fig. 2A and C). In the No-DNAzyme control reactions, both modified and unmodified RNAs were amplified, allowing normalization. To ensure the robustness of the DNAzyme cleavage, we included control DNAzymes targeting unmodified nucleotides in the same snRNAs, specifically guanine 81 in U1 snRNA (U1-G81), adenine 51 and uridine 156 in U2 snRNA (U2-A51 and U2-U156, respectively).

For a comprehensive evaluation of the method's efficiency, two types of DNAzymes were used to target selected modified nucleotides. The 10-23 DNAzyme, which is able to cleave any purine-pyrimidine junction [31], was selected to target the Am70-C71 and G81-C82 nucleotide junctions on the U1 snRNA, designated as D-Am70 and D-G81, respectively. Whereas DNAzymes, derived from the 8-17 DNAzyme family, known for preferential cleavage of purine-purine contexts [42,56,57], were designed to target the Am30-G31, A51-G52 and U156-G157 dinucleotide sequences in U2 snRNA, named D-Am30, D-A51 and D-U156, respectively (Fig. 2C).

## 3.3. Specific U1-Am70 and U2-Am30 DNAzymes efficiently cleave respective unmodified RNA at the expected site

The first stage of method optimization involved assessing the efficiency of DNAzyme-mediated cleavage for unmodified U1 and U2 snRNAs. The corresponding unmodified RNA transcripts were produced by *in vitro* T7 transcription, as described in M&M. A wide variety of reaction conditions have been described for DNAzyme cleavage, including the use of heavy metal ions (e. g. Mn²⁺ and Pb²⁺) as cofactors, in addition to Mg²⁺ and the application of cycling conditions to stimulate multiple turnover catalysis [56,58,59]. In our hands, extended incubation at moderate temperature (37 °C, 2h) resulted in better RNA integrity compared to PCR-like cycling conditions (10x (80 °C, 30sec-temperature decrease 0.5 °C/s to 25 °C, 25 °C, 5 min)) (Fig. S1A). We adopted this as the basis for our approach and used 2h-incubation at 37 °C for subsequent experiments. Transcribed U1 and U2 snRNAs were then treated with either active DNAzyme or No-DNAzyme oligonucleotide in the same buffer conditions for the two families (10-23 and 8-17), followed by DNase treatment. The cleavage fragments were separated by denaturing polyacrylamide gel electrophoresis (PAGE) and visualized by SYBR™ Gold staining. As shown in Fig. 3A, the expected cleavage products generated by active DNAzymes specific for Am70, G81, Am30 and A51 were observed. All tested DNAzymes exhibited high efficiency and specificity, with quantifiable cleavage efficiencies greater than 96 %. In contrast, control treatments with the inactive No-DNAzyme oligonucleotides (ND-Am70, ND-G81, ND-Am30 and ND-A51) showed no significant effect on RNA integrity (Fig. 3). These data confirmed that optimized cleavage conditions are suitable for both 10-23 and 8-17 DNAzymes classes. Cleavage specificity was also assessed by competition between active DNAzymes and their ND counterparts. As shown in Fig. S1B, the addition of No-DNAzyme targeting the same region as DNAzyme inhibited
<figure><img src="image_9.png" /><figcaption>**Fig. 3.** **High efficiency cleavage of U1 and U2 snRNAs by DNAzymes.** A. *In vitro* transcribed U1 or U2 snRNAs were incubated with specific DNAzymes, and the cleavage products were separated by polyacrylamide gel electrophoresis (PAGE). The 10–23 DNAzymes D-Am70 and D-G81 cleave U1 snRNA into fragments of 94 and 70 nucleotides, and 83 and 81 nucleotides, respectively, in contrast to the No-DNAzymes ND-Am70 and ND-G81, which exhibit no cleavage activity. The 8–17 DNAzyme D-A51 cleaves U2 snRNA into fragments of 137 and 51 nucleotide, unlike the No-DNAzymes ND-Am30 or ND-A51. For the 8–17 D-Am30 DNAzyme, the short fragment (<30 nt) is not detected from the gel, revealing only one visible fragment (158 nt). Arrows indicate the cleavage products generated by the DNAzymes. Band intensities were quantified, and calculated cleavage efficiency is indicated at the bottom of the gel (NC: Not Calculated). B. Correlation between the cleavage efficiency by DAMP-RNA and the level of 2'-O-methylation of the substrate. Synthetic RNAs with and without 2'-O-Me were mixed to obtain a known ratio of 2'-O-Me. The efficiency of DNAzyme cleavage is illustrated by the quantification by RT-qPCR of the residual amount of RNA, where a value of 1 indicates no cleavage (intact 2'-O-methylated RNA) and 0 indicates complete RNA cleavage. The black dots indicate the mean and the error bars show the standard deviation for three replicates, normalized against No-DNAzyme RNA levels. The red line shows the linear regression and the correlation equation is $Y = 0.009168X + 0.09063$ with a correlation coefficient ($R^2$) of 0.98.</figcaption></figure>

cleavage at the corresponding site, whereas a No-DNAzyme targeting a different RNA region did not affect cleavage efficiency. To achieve coupling of DNAzyme cleavage to a quantitative RT-PCR protocol (RT-qPCR), we tested two widely used RT enzymes (MML-V and SuperScript III) at the cDNA synthesis step in the competition experiment shown in Fig. S1B. Only the SuperScript III enzyme showed a linear-related correlation between uncleaved residual RNA quantified on the gel and the RT-qPCR signal (Fig. S1C), while the signal obtained with the MML-V remained almost constant, irrespective to the amount of uncleaved RNA. This may be related to the lower processivity of MML-V on highly structured snRNA and the higher temperatures used for the SuperScript III enzyme. Therefore, a 2-h incubation with DNAzyme at 37 °C, combined with the use of the SuperScript III enzyme is likely to provide optimal conditions for the DAMP-RNA approach.

To ensure that the method provides accurate quantitative measurements of the modification rate, we created a standard curve using a set of samples with known RNA methylation levels. To this goal, we mixed together short synthetic U1 snRNA oligonucleotides with and without 2'-O-methylation at position 22, corresponding to position 70 in native U1, to mimic different levels of 2'-O-methylation prior to DNAzyme cleavage. As shown in Fig. 3B, the levels of U1 snRNA detected by RT-qPCR exhibit an excellent linear relationship between the cleavage efficiency and methylation levels, as evidenced by a correlation coefficient ($R^2$) of 0.98, indicating appropriate model accuracy.

The high cleavage efficiency observed across different target sequences in U1 and U2 snRNAs, and the clear calibration range between unmodified and fully modified RNAs, ensures that quantitative analyses accurately reflect the presence of 2'-O-methylation rather than variations in DNAzyme cleavage efficiency.

### 3.4. DAMP-RNA protocol efficiency distinguishes between modified and unmodified positions in cellular snRNAs

We then investigated whether our experimental conditions could effectively distinguish between unmethylated and methylated positions within naturally modified human snRNAs. To this end, total RNA fractions from HeLa S3 cells were treated with DNAzymes specific for Am70 and G81 in U1 snRNA or Am30 and

A51 in U2 snRNA. Following DNAzyme treatment, the amount of intact residual snRNAs was quantified by RT-qPCR with RNA levels normalized to GAPDH to account for variations in RNA input, and to No-DNAzyme as a reference for total snRNA levels. To enhance the differential cleavage efficiency for unmethylated and methylated RNA, we tested different concentrations of DNAzyme (Fig. 4). Our data showed that with DNAzyme D-G81, which targets an unmethylated site, the amount of residual U1 snRNA detected after treatment decreased progressively at high concentrations of DNAzyme (Fig. 4A). This decrease reflects the efficiency of DNAzyme to cleave U1 snRNA, which reaches a plateau level at ~80 pmol of DNAzyme for 500 ng of cellular RNA. Note that approximately 10 % of the RNA remains intact, probably due to incomplete cleavage and measurement variability. In contrast, the amount of residual U1 snRNA remained consistently high (close to 90 %) after treatment with DNAzyme D-Am70, indicating a low cleavage efficiency at the Am70 site (Fig. 4A). This significant level of protection from DNAzyme cleavage at U1-Am70 in HeLa S3 cells is consistent with the level of 2'-O-methylation reported in previous studies. Indeed, other reports indicated that the U1-Am70 is highly methylated in human cells, with methylation levels ranging from 70 % to 90 %, depending on the detection method and the cell line used [6,41]. We proceeded in the same way with the U2 snRNA (Fig. 4B). Interestingly, compared to U1 snRNA, smaller quantities of DNAzyme (40 pmol instead of 160 pmol) are required to cleave U2 snRNA with maximum efficiency. This is likely due to U1 being significantly more abundant than other snRNAs in higher eukaryotes.

In conclusion, our improved DNAzyme cleavage protocol, coupled with quantitative RT-PCR (DAMP-RNA protocol) is an effective method for assessing the protection status at specific 2'-O-Me positions within cellular RNAs and for validating 2'-O-Me positions detected by alternative approaches.

### 3.5. The DAMP-RNA protocol allows quantitative measurement of the 2'-O-Me levels in RNA

The next step was to evaluate the ability of the DAMP-RNA protocol to detect changes in the level of 2'-O-methylation at specific snRNA positions. The 2'-O-methylation of mammalian U1-
<figure><img src="image_10.png" /><figcaption>**Fig. 4.** Assessment of DNAzyme activity on endogenous U1 and U2 snRNAs. Total RNA was extracted from HeLa S3 cells and treated with a DNAzyme that was either specific for a 2'-O-methylated position or an unmethylated position. The residual snRNA level (uncleaved) was quantified by RT-qPCR and expressed as the ratio of snRNA in the DNAzyme-treated sample to the snRNA in the No-DNAzyme-treated sample, normalized against GAPDH levels. A. For the U1 snRNA, the target positions are Am70 and G81 with increasing DNAzyme amounts (10–160 pmol). B. For the U2 snRNA, the target positions are Am30 and A51 with increasing DNAzyme amounts (0.5–40 pmol).</figcaption></figure>

Am70 and U2-Am30 snRNAs relies on box C/D snoRNPs; therefore, the depletion of NOP58, an essential component of box C/D snoRNPs in cells, results in a reduced number of active snoRNP particles and must therefore affect the level of modification at these sites.

To achieve depletion of NOP58 in *cellulo*, we transfected HeLa S3 cells with siRNA targeting NOP58, which resulted in efficient knockdown, as demonstrated by Western blotting with anti-NOP58 antibody (Fig. 5A). Total RNA fractions, extracted from control and siRNA-transfected cells were subjected to the DAMP-RNA protocol as described in the previous sections. At the unmethylated positions U1-G81 and U2-A51, both U1 and U2 snRNAs were equally cleaved by specific DNAzyme treatment, leaving a residual RNA level of approximately 10%, as previously observed (Fig. 5B). In contrast, at the 2'-O-Me positions U1-Am70 and U2-Am30, both UsnRNAs showed a high level of protection, close to 91% for U1-Am70 and 89% for U2-Am30. While NOP58 knockdown did not affect cleavage at the two unmethylated positions, cleavage efficiency at the U1-Am70 and U2-Am30 was significantly increased, with a concomitant decrease in the residual snRNA levels detected by RT-qPCR (40% and 21% for U1 and U2, respectively). This is consistent with a detectable decrease in the 2'-O-Me levels at the both tested sites.

### 3.6. 2'-O-Me levels measured by DAMP-RNA are consistent with RiboMethSeq results

To compare the data obtained by the DAMP-RNA protocol with independent orthogonal quantification, we performed 2'-O-Me analysis of human snRNAs using the quantitative RiboMethSeq protocol. In order to enrich target snRNAs and thus drastically reduce the sequencing depth required for reliable analysis, we developed an immunoprecipitation assay using the Y12 antibody directed against the spliceosomal core Sm proteins present in U1 and U2 snRNPs. Enriched UsnRNP fractions were isolated from human HeLa S3 cells and UsnRNAs extracted from those purified fractions were subjected to RiboMethSeq analysis. As anticipated, the decrease in NOP58 had no effect on RNA cleavage by DNAzymes targeting non-methylated positions (U1-G81 and U2-A51) (Fig. 5C). On the other hand, the knockdown of NOP58 resulted in a substantial decrease of about 25% in the measured methylation levels at positions U1-Am70 and U2-Am30, which is in good agreement with the reduction also observed by DAMP-RNA.

### 3.7. Specific depletion of snoRNA guide RNA leads to detectable reduction of the target snRNA upon DNAzyme cleavage

The high sensitivity of the DAMP-RNA protocol also allowed us to demonstrate the involvement of a specific guide snoRNA in methylation at its target site. Considering that methylation of human U1-Am70 is scaRNA7 guide-dependent [6,60], we performed a scaRNA7 knockdown in human HeLa S3 cells using an antisense oligonucleotide (ASO). As shown in Fig. 5D, our experimental conditions enabled a reduction of the cellular level of scaRNA7 by more than 80%. Similar to the NOP58 knockdown described above, the cleavage by DNAzymes targeting the unmethylated control positions U1-G81 and U2-A51 was highly efficient and unaffected by scaRNA7 depletion (Fig. 5E). In contrast, as expected, scaRNA7 depletion resulted in a 60% reduction in residual U1 snRNA levels after treatment with the DNAzyme targeting U1-Am70. However, no effect was observed on DNAzyme cleavage efficiency at U2-Am30, a modification that relies on a different scaRNA guide (scaRNA9) [47]. These observations allowed the DAMP-RNA protocol to unambiguously assign the scaRNA7 guide to the U1 snRNA modification at Am70, with the same sensitivity as previously demonstrated by quantitative liquid chromatography (LC)-with tandem mass spectrometry (MS)-based analysis (Supplementary Table S3) [41].

## 4. Discussion

Collectively, our results demonstrated the efficacy of the DAMP-RNA method in detecting site-by-site variations in 2'-O-methylation level in RNA, yielding comparable results to those obtained by RiboMethSeq or LC-MS. High-throughput RNA profiling has revealed the extensive landscape of RNA 2'-O-methylations, present in tRNAs, rRNAs, snRNAs and mRNAs/lncRNAs [6,20,22,25,27,61]. Despite these outstanding achievements, all deep sequencing-based methods available for 2'-O-Me profiling suffer from multiple false positive (and also false negative) hits, reducing the reliability of such global profiling. The RiboMethSeq protocol measures the protection of a given RNA phosphodiester bond to alkaline cleavage at high temperature. To achieve this, the RNA is first randomly fragmented at high pH and high temperature, the random fragments are 3'-dephosphorylated and then 5'-phosphorylated to make them compatible with direct adapter ligation, and subjected to library preparation using an appropriate small RNA kit (here from NEB). Sequencing is performed on the
<figure><img src="image_11.png" /><figcaption>**Fig. 5.** Site-specific analysis of 2'-O-methylation of U1 and U2 snRNAs after NOP58 or scaRNA7 knockdown. **A.** Western blot analysis showing the efficiency of NOP58 knockdown in HeLa S3 cells. **B.** Analysis by the DAMP-RNA methodology of the 2'-O-methylation status of the U1 and U2 snRNAs in HeLa S3 cells depleted for NOP58. The analyzed positions include methylated U1-Am70 and U2-Am30 positions, as well as unmethylated U1-G81 and U2-A51 positions, in control cells (siCtr, black) or in cells depleted for NOP58 (siNOP58, purple). The efficiency of DNAzyme cleavage is illustrated by the residual amount of RNA, where a value of 1 indicates no cleavage (intact 2'-O-methylated RNA) and 0 indicates complete RNA cleavage. Each data point represents an independent experiment, normalized against GAPDH mRNA levels and No-DNAzyme snRNA levels (n = 4 or 5). Statistical significance was assessed using the Mann-Whitney test with p < 0.05. **C.** Analysis by RiboMethseq of the 2'-O-methylation status of the U1 and U2 snRNAs in HeLa S3 cells depleted for NOP58. The calculated protection level at methylated U1-Am70 and U2-Am30 and unmethylated U1-G81 and U2-A51 positions is indicated in relation to the presence (siCtr, black) or absence (siNOP58, purple) of NOP58. **D.** The efficiency of scaRNA7 knockdown by ASO was monitored through RT and quantitative PCR, with values derived from four independent experiments and normalized against GAPDH mRNA levels and scaRNA levels in the NC-ASO condition. **E.** Analysis by the DAMP-RNA methodology of the 2'-O-methylation status of the U1 and U2 snRNAs in HeLa S3 cells depleted for scaRNA7. The analyzed positions include methylated U1-Am70 and U2-Am30 positions, as well as unmethylated U1-G81 and U2-A51 positions, in control cells (NC ASO, black) or in cells depleted for scaRNA7 (scaRNA7 ASO, green). The efficiency of DNAzyme cleavage is illustrated by the residual amount of RNA, where a value of 1 indicates no cleavage (intact 2'-O-methylated RNA) and 0 indicates complete RNA cleavage. Each data point corresponds to an independent experiment, normalized against GAPDH mRNA levels and No-DNAzyme snRNA levels (n = 4). Statistical significance was determined using the Mann-Whitney test with p < 0.05.</figcaption></figure>

Illumina machine in single read mode and fragments with both
defined 5'- and 3'-ends are retrieved and mapped to the reference.
Counting both 5' and 3'-ends allows the definition of the RNA
cleavage/protection profile, with drop-in coverage (gap in the
profile) appearing at 2'-O-Me nucleotides. Despite the elegance of
this method, background noise persists due to variable ligation
efficiency, unintended RT pauses during primer extension, and
partial resistance to alkaline cleavage encountered at particular
RNA secondary or tertiary structures. All these uncontrollable
parameters accentuate the irregular RNA cleavage, with arbitrarily
protected residues, which can be interpreted as partial 2'-O-Me
modification. Direct RNA sequencing using nanopores is an
emerging technology with significant potential but requires
further optimization to ensure consistent reliability [28]. Bioin-
formatics pipelines for RNA modification detection focus on
improving denoising techniques to enhance signal clarity, utilizing
nanopore sequencing platforms to accurately identify modifica-
tions, and refining statistical thresholds to ensure robust and ac-
curate modification calls. While this approach has the potential to
detect a wide range of RNA modifications, current methods are
limited to quantifying a subset in parallel, including Ψ, Nm, m6A,
m7G, m5C, and 5-hydroxymethylcytosine (hm5C) [62]. However,
the detection of 2'-O-methylation (2'-O-me) remains beyond the
capabilities of existing techniques.

Considering these points, alternative (mainly low-throughput)
validation approaches are often used to confirm at least a subset of
2'-O-Me sites found by high-throughput screening. These alter-
natives include: low [dNTP] primer extension coupled with RT-
qPCR (RTL-P) [17,63], the use of RNase H cleavage to test for 2'-
O-Me resistance to cleavage [15,16,64] and, possibly other more
general techniques such as SCARLET [13], as well as LC-MS(MS)

analysis [65,66]. The RTL-P method, which exploits the tendency
of certain reverse transcriptases (RTs) to halt primer extension at
low dNTP concentrations in the presence of 2'-O-methylation,
provides a semi-quantitative measure of the 2'-O-methylation
fraction. However, RTL-P is not suitable for studying modifications
in clusters, since it fails to distinguish individual 2'-O-Me residues
in highly modified RNA regions. In addition, non-specific low
[dNTP]-induced RT stops related to RNA structural elements may
also be detected as potential 2'-O-Me residues, which can lead to
misinterpretation [67]. The use of RNase H to analyze the 2'-O-Me
state of an RNA is a sensitive approach based, on the inhibition of
RNA cleavage by the presence of 2'-O-Me. However, this method
requires synthetic chimeric RNA-DNA oligonucleotides and re-
quires numerous controls to avoid non-specific degradation. Other
possible, and more general methods, such as SCARLET, use large
amounts of RNA starting material and radioactive labeling to
achieve the required sensitivity, while LC-MS(MS) analysis re-
quires high purity RNA samples for reliable analysis and does not
provide sequence-specific information on the location of the
modification.

Since the practical implementation of all these options is not
trivial, we still need alternative experimental approaches to (i)
validate the presence of 2'-O-methylation at a given RNA site and
(ii) evaluate the stoichiometry of these modifications, especially
under varying physiological conditions or in pathological contexts.
Our DAMP-RNA methodology proposes DNAzyme cleavage,
coupled with quantification of the cleavage efficiency by RT-qPCR,
as a possible alternative approach for robust measurement of 2'-O-
methylation levels at specific positions in RNAs.

Our results show that the DAMP-RNA method, is both sensitive
and specific enough to detect changes in the levels of 2'-O-
methylation in UsnRNAs following the knockdown of a C/D box RNP protein or a corresponding scaRNA guide. The main technical challenge is to ensure that the DNAzyme efficiently cleaves the RNA at the expected site, this validation step requires *in vitro* RNA transcript with the same sequence. Despite the ease of implementation of this method, several limitations warrant consideration: (i) Optimal cleavage by the DNAzyme is essential for accurate interpretation of the presence or the absence of a modification. Although the 8-17 DNAzyme and its variants can cleave all dinucleotides, they exhibit a stronger preference for purine-purine sequences [42,56,57,68]. Consequently, the Y/C and N/U combinations are poor substrates and only a subset of the modified positions in RNA can be studied. (ii) The position of the methylated base is also a limitation, as the size of the DNAzyme arms restricts the ability to study modifications at the ends of RNAs or on very short RNAs, such as miRNAs.

Nevertheless, the accuracy and sensitivity of DAMP-RNA are significant advantages for studying methylation plasticity at a given site, making it an ideal tool for understanding the regulation of modifications and characterizing their functions. There are no significant methodological barriers to adapting this method to detect 2'-O-methylation in other (even low abundant) RNA species. The analysis is straightforward, fast, and does not require radioactive labeling or expensive reagents. This method provides clear and reproducible results, facilitating the study of multiple 2'-O-methylations within the same RNA, and agrees well with results obtained by other quantitative orthogonal techniques, such as RiboMethSeq and LC-MS-based methods (Fig. 5B and C, Supplementary Table S3). It is noteworthy that DAMP-RNA can be adapted to study other modifications, such as pseudouridines, which may interfere with the binding or action of DNAzymes [34]. In addition, new families of DNAzymes have been identified that can discriminate the presence of N6-methyladenosine [69-71].

## 5. Conclusion

In conclusion, while the DAMP-RNA method provides a straightforward approach to validate novel 2'-O-methylation sites detected by high-throughput methods across a wide range of RNAs and assess the potential modulations they may undergo, it can also be applied to various other RNA modifications. This versatility makes DAMP-RNA a relevant tool for studying the dynamics of known (or, at least, previously mapped) RNA modifications in both physiological and pathological conditions, providing insights into their role in the development of diseases such as cancer.

### CRediT authorship contribution statement

**Valentyne Lebret-Kogey:** Visualization, Investigation. **Lilia Ayadi:** Writing - review & editing, Investigation. **Sylvain Maenner:** Writing - review & editing. **Yuri Motorin:** Writing - review & editing, Formal analysis. **Isabelle Behm-Ansmant:** Writing - review & editing, Project administration, Funding acquisition, Conceptualization. **Christelle Aigueperse:** Writing - original draft, Visualization, Validation, Methodology, Investigation, Formal analysis.

### Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used ChatGPT in order to help find the acronym. After using this tool, the authors modified and edited the content and takes full responsibility for

the content of the publication.

### Formatting of funding sources

This work was supported by the Lorraine University and the CNRS.

### Declaration of competing interest

None, the authors declare that they have no financial or personal conflicts of interest related to the publication of this manuscript.

### Acknowledgments

We thank Dr. G. Dreyfuss for the generous gift of the pUC19-U1 and pUC19-U2 plasmids used in this work.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biochi.2025.07.006.

## SUPPLEMENTARY MATERIAL

A. Optimization of the incubation conditions. One picomole of *in vitro* transcribed U2 snRNA has been incubated with 400 pmol of DNAzyme D-Am30 or D-A51 in reaction buffer (400 mM NaCl, 100 mM KCl, 50 mM Hepes pH7.0, 7.5 mM MgCl₂, 7.5 mM MnCl₂) for 2h at 37 °C or 10 cycles (denaturation for 30s at 80 °C, temperature decrease 0.5 °C/s to 25 °C, and cleavage for 5 min at 25 °C). Cleavage yields were analyzed by polyacrylamide gel electrophoresis (PAGE).

B. Assessment of the specificity of the DNAzyme cleavage by competition experiment. Two picomoles of *in vitro* transcribed U2 snRNAs were treated with 10 pmol of DNAzyme D-U156 in reaction buffer (400 mM NaCl, 100 mM KCl, 50 mM Hepes pH7.0, 7.5 mM MgCl₂, 7.5 mM MnCl₂) for 2h at 37 °C (lanes 2 to 6). Addition of the No-DNAzyme oligonucleotide (10 or 100 pmol) targeting the same position (ND-U156) prevents cleavage (lanes 3 and 4), whereas the addition of a No-DNAzyme targeting another position (ND-Am30) has no effect on DNAzyme D-U156 cleavage efficiency (lanes 5 and 6).

C. Optimization of RT-PCR conditions. For the three DNAzyme-treated samples shown in B (lanes 2, 3 and 4), the amount of residual RNA after cleavage has been determined by RT-PCR using either the MML-V or the SuperScript III reverse transcriptase and compared to the residual RNA amount quantified from the gel shown in Panel B (lanes 2, 3 and 4). Only the SuperScript III enzyme (grey dots) showed a linear-related correlation between uncleaved residual RNA quantified on the gel (open squares) and the RT-qPCR signal, while the signal obtained with the MML-V reverse transcriptase (black dots) remained nearly constant, irrespective to the amount of uncleaved RNA.

Total extracts were produced from HeLa S3 cells depleted for NOP58 (siNOP58) or mock-treated (siCtr). Immunoprecipitations (IPs) were carried out with G-Sepharose beads without (control) or with Y12 antibody reported to recognize Sm proteins. The IP and a fraction of total cell extract (2.5 %, input) were analyzed by SDS-PAGE and Western blot with antibodies against SmD3 protein.
<table><caption>**Supplementary Table S1**
oligonucleotide sequences</caption><thead><tr><th>PCR (Transcription)</th><th>sequence 5'-3'</th><th>Target</th></tr></thead><tbody><tr><td>T7-U1</td><td>AAATTTAATACGACTCACTATAGGGATACTTACCTGGCAGGGG</td><td>U1</td></tr><tr><td>3'-end-U1</td><td>CAGGGGAAAGCGCGAACG</td><td>U1</td></tr><tr><td>T7-U2</td><td>AAATTTAATACGACTCACTATAGGGATCGCTTCTCGGCCTTTTTG</td><td>U2</td></tr><tr><td>3'-end-U2</td><td>GGGTGCACCGTTCCTGGAGGT</td><td>U2</td></tr><tr><td><strong>RT-QPCR</strong></td><td><strong>sequence 5'-3'</strong></td><td><strong>Target</strong></td></tr><tr><td>Fw-U1</td><td>TACCTGGCAGGGGAGATACC</td><td>U1</td></tr><tr><td>Rev-U1</td><td>ATGCAGTCGAGTTTCCCACA</td><td>U1</td></tr><tr><td>Fw-U1 synthetic</td><td>AGGGCGAGGCTTATCCATTG</td><td>U1 synthetic</td></tr><tr><td>Rev-U1 synthetic</td><td>CATTTGGGAAATCGCAGGG</td><td>U1 synthetic</td></tr><tr><td>Fw-U2 (Am30)</td><td>TTCTCGGCCTTTTGGCTAAG</td><td>U2 (Am30)</td></tr><tr><td>Rev-U2 (Am30)</td><td>ATTCCATCTCCCTGCTCCAA</td><td>U2 (Am30)</td></tr><tr><td>Fw-U2 (U156)</td><td>TTGGAGCAGGGAGATGGAAT</td><td>U2 (U156)</td></tr><tr><td>Rev-U2 (U156)</td><td>CGTTCCTGGAGGTACTGCAA</td><td>U2 (U156)</td></tr><tr><td>Fw-GAPDH</td><td>AACAGCCTCAAGATCATCAGC</td><td>GAPDH</td></tr><tr><td>Rev-GAPDH</td><td>GGATGATGTTCTGGAGAGCC</td><td>GAPDH</td></tr><tr><td><strong>siRNA</strong></td><td><strong>sequence 5'-3'</strong></td><td><strong>Target</strong></td></tr><tr><td>siNOP58</td><td>CAAGCAUGCAGCUUCUACCGUUCtt</td><td>NOP58</td></tr><tr><td>siCtr</td><td>CGUACGCGGAAAUACUUCGAtt</td><td>Luciferase</td></tr><tr><td><strong>gapmer ASOs</strong></td><td><strong>sequence 5'-3'</strong></td><td><strong>Target</strong></td></tr><tr><td>NC ASO</td><td>+G*+C*+G*+A*+C*T*A*T*A*C*G*C*C*C*A*+A*+T*+A*+T*+G</td><td>none</td></tr><tr><td>scaRNA7 ASO</td><td>+A*+T*+G*+C*+A*C*T*C*C*A*+A*T*A*T*+A*+G*+C*+A*+T</td><td>scaRNA</td></tr><tr><td><strong>Synthetic RNA</strong></td><td><strong>sequence 5'-3'</strong></td><td></td></tr><tr><td>U1 Am70</td><td>AGGGCGAGGCTTATCCATTGCAmCTCCGGATGTGCTGACCCCTGCGATTTCCCCAAATG</td><td></td></tr><tr><td>U1 A70</td><td>AGGGCGAGGCTTATCCATTGCACTCCGGATGTGCTGACCCCTGCGATTTCCCCAAATG</td><td></td></tr></tbody></table>

ASOs: AntiSense Oligonucleotides.

+: Locked Nucleic Acids base (IDT's Affinity Plus™).

*: phosphorothioate modifications.

<table><caption>**Supplementary Table S2**
UsnRNA reference sequences upstream and downstream of the modified nucleotide used for RiboMetSeq analysis.</caption><thead><tr><th>RNA</th><th>pos</th><th>base</th><th>sequenceUpStream</th><th>modNt</th><th>sequenceDownStream</th><th>fullRegion</th></tr></thead><tbody><tr><td>RNU1</td><td>70</td><td>A</td><td>ATCCATTGC</td><td>Am</td><td>CTCCGGATG</td><td>ATCCATTGCACTCCGGATG</td></tr><tr><td>RNU2</td><td>11</td><td>G</td><td>TCGCTTCTC</td><td>Gm</td><td>GCCTTTTGG</td><td>TCGCTTCTCGGCCTTTTGG</td></tr><tr><td>RNU2</td><td>12</td><td>G</td><td>CGCTTCTCG</td><td>Gm</td><td>CCTTTTGGC</td><td>CGCTTCTCGGCCTTTTGGC</td></tr><tr><td>RNU2</td><td>19</td><td>G</td><td>CGGCCTTTT</td><td>Gm</td><td>GCTAAGATC</td><td>CGGCCTTTTGGCTAAGATC</td></tr><tr><td>RNU2</td><td>25</td><td>G</td><td>TTTGGCTAA</td><td>Gm</td><td>ATCAAGTGT</td><td>TTTGGCTAAGATCAAGTGT</td></tr><tr><td>RNU2</td><td>30</td><td>A</td><td>CTAAGATCA</td><td>Am</td><td>GTGTAGTAT</td><td>CTAAGATCAAGTGTAGTAT</td></tr><tr><td>RNU2</td><td>40</td><td>C</td><td>GTGTAGTAT</td><td>Cm</td><td>TGTTCTTAT</td><td>GTGTAGTATCTGTTCTTAT</td></tr><tr><td>RNU2</td><td>43</td><td>T</td><td>TAGTATCTG</td><td>Tm</td><td>TCTTATCAG</td><td>TAGTATCTGTTCTTATCAG</td></tr><tr><td>RNU2</td><td>44</td><td>T</td><td>AGTATCTGT</td><td>Tm</td><td>CTTTATCAGT</td><td>AGTATCTGTTCTTATCAGT</td></tr><tr><td>RNU2</td><td>60</td><td>T</td><td>AGTTTAATA</td><td>Tm</td><td>CTGATACGT</td><td>AGTTTAATATCTGATACGT</td></tr></tbody></table>

<table><caption>**Supplementary Table S3**
Comparison of the percentages of residual RNA detected by DNAzyme with the percentages of modifications obtained by RiboMethseq and liquid chromatography (LC)-Mass spectroscopy (MS)-based techniques at position Am70 on U1 snRNA.</caption><thead><tr><th>Quantitative methods</th><th>cells</th><th>%</th><th>GFP/NC-ASO</th><th>scaRNA7 ASO</th><th>siCtr</th><th>siNOP58</th><th>references</th></tr></thead><tbody><tr><td>DNAzyme</td><td>HeLa S3</td><td>residual RNA</td><td>86.9 ± 2.9 (a)</td><td>35.6 ± 1.9 (a)</td><td>91.3 ± 0.11 (a)</td><td>54.1 ± 0.07 (a)</td><td>present study</td></tr><tr><td>SILNAS</td><td>HeLa</td><td>modification</td><td>83.9 (b)</td><td>59.6 (b)</td><td></td><td></td><td>Izumikawa et al., 2019</td></tr><tr><td>Peak area</td><td>HeLa</td><td>modification</td><td>87.7 (b)</td><td>48.2 (b)</td><td></td><td></td><td>Izumikawa et al., 2019</td></tr><tr><td>RiboMethseq</td><td>HeLa S3</td><td>%protection level</td><td></td><td></td><td>72.6 ± 0.01 (c)</td><td>52.7 ± 0.02 (c)</td><td>present study</td></tr></tbody></table>

(a) values are obtained as mean ± SEM (n = 4); (b) values are given as a single analysis; (c) values are obtained as mean ± SEM (n = 3).

SILNAS (Stable Isotope–Labelled riboNucleic Acid as an internal Standard), and peak area-based quantification were taken from the Izumikawa publication [41], and values are given as a single analysis. RiboMethseq values are the mean of 3 independent experiments ± standard deviation. DNAzyme values are the mean of 4 or 5 independent experiments ± standard deviation.

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