Table 4 Techniques to detect small RNA modifications
From: Small RNA modifications: regulatory molecules and potential applications
Techniques | Modification | Verified small RNAs | Principle | Characteristics (including advantages and challenges) | References |
|---|---|---|---|---|---|
m6A-individual nucleotide resolution crosslinking and immunoprecipitation (miCLIP- m6A) | m6A | snoRNA | Mapping of m6A residues achieved through the creation of unique signature mutations using m6A-specific antibodies and UV crosslinking | Identifies the exact sites of m6A; Without pretreatment of cells with modified nucleotides; Unbiased identification of m6A residues | [204] |
m5C-individual nucleotide resolution crosslinking and immunoprecipitation (miCLIP-m5C) | m5C | Vault RNA | The specific complex containing NSUN2 and m5C leads to a truncation site during RT-PCR, which can be interpreted as a marker for m5C modification | Identifies the exact sites of m5C | [139] |
Demethylase tRNA sequencing (DM-tRNA-seq) | m1A, m3C, m1G, m22G and m3U | tRNA, rRNA | Use of AlkB demethylase and its engineered mutant as central components to remove m1A, m3C and m1G modifications at the Watson–Crick face in tRNA prior to cDNA synthesis | Use of a modification index (MI) to assess the quantitative nature of each detectable modification site | [205] |
Borohydride Reduction sequencing (BoRed-seq) | m7G | miRNA | RNA fragments that contain certain modifications can be enriched through specific antibody immunoprecipitation | Approach exhibits high specificity but lacks single-nucleotide resolution and is unable to detect methylation in low-abundance RNAs | [168] |
RiboMeth-seq | Nm | rRNA | Nm can be mapped by analyzing the read-end information in sequencing data due to its resistance to alkaline hydrolysis | This method is capable of identifying missing peak regions that relate to Nm locations | |
2′-OMe-seq | Nm | rRNA | Restricting the concentration of either dNTP or Mg2 + during RT reactions leads to halting of RT at Nm sites | A relatively straightforward and sensitive approach with strong specificity; allows identification at single-base resolution and quantitation of 2΄-O-methylated residues | [208] |
Direct m6A Sequencing | m6A | tRNA | Use of KlenTaq DNA polymerase to function as an reverse transriptase, which can result in the incorporation of incorrect nucleotides at m6A sites | Enables the direct detection of m6A sites from untreated RNA sequencing data | [209] |
RNA bisulfite sequencing technology (RNA-BisSeq) | m5C | tRNA, rRNA | Addition of sodium bisulfite deaminates unmethylated cytosines (at acidic pH) or uracil (at basic pH), preserving methylated cytosines | Provides single-nucleotide resolution avoids the requirement of high RNA concentrations for analysis; Unable to react with cytosines that are base-paired; Cannot differentiate between 5-methylcytosine and 5-hydroxymethylcytosine | |
RBS-seq | m5C、Ψ and m1A | tRNA, rRNA | Optimizing bisulfite treatment conditions and concomitant detection of all three modifications within the same RNA | Identification of every modification through a distinct chemical method that facilitates accurate mapping of all three modifications in a single RNA molecule, thereby enabling co-variation analyses | |
Ψ-seq | Ψ | rRNA, tRNA and snRNA | N-cyclohexyl-N′-β-(4-methylmorpholinium)-ethylcarbodiimide (CMC) can label Ψ, leading to the formation of CMC-Ψ adducts that cause RT to halt | Unbiased, quantitative profiling of Ψ across the transcriptome at the single-nucleotide resolution level | [214] |
m7G Mutational Profiling sequencing (m7G-MaP-seq) | m7G | rRNA, tRNA | By reducing sodium borohydride, positions with m7G modifications are transformed into abasic sites which can be directly detected as cDNA mutations | High throughput detection of m7G modifications at single nucleotide resolution | [215] |
AlkAniline-Seq | m7G and m3C | rRNA, tRNA | The resistance of m3C and m7G to NaBH4-aniline treatment and cleavage makes it possible to use selective ligation to enrich modified fragments | Does not adopt traditional RNA sequencing chemistry and depends on a chemical-based method for selectively enriching reads in the resulting libraries | [216] |
Hydrazine-Aniline Cleavage sequencing (HAC-seq) | m3C | tRNA | m3C-modified sites can be selectively cleaved through treatment with hydrazine/aniline, allowing for their mapping through calculation of the cleavage ratio | Unbiased and transcriptome-wide detection of m3C RNA modification at the single-nucleotide level | [217] |
HydraPsiSseq | Ψ | rRNA | Reliant on specific protection from hydrazine/aniline cleavage | Absolute measurements of modification levels; only requires extremely small amounts of RNA | [218] |
LC–MS-based RNA sequencing (2D mass-tR direct RNA sequencing) | Ψ, m5C, etc | Short synthetic RNAs (< 35 nt), tRNA | Introduce a 2D hydrophobic end-labeling strategy into conventional mass spectrometry-based sequencing, which enables the de novo sequencing of RNA mixtures and improves the efficiency of sample utilization | Accurately identifies, locates and quantifies base modifications in both single and mixed RNA samples, with single-base resolution; can directly read the complete sequence; can be applied to samples containing multiple different modifications | [219] |