Table 4.

Techniques to detect small RNA modifications

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	[206, 207]
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	[210, 211]
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	[212, 213]
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]