Table 1.

Summary of main techniques for the detection of RNA modifications.

Methods			Modifications Detected	Quantification	Genome Wide	Positional Information	Remarks (Pros/Cons)
Structure Determination	X-ray Cristallography		All modifications	✕ / ✓	✕	✓	Difficult to obtain crystals
	Cryo Electron Microscopy						Heterogeneous resolution
	Nuclear Magnetic Resonance						Size limit
LC/MSMS	Nucleoside analysis	DMRM [34]	Known modifications	✓	✓	✕	Fragmentation pattern and retention time of modifications must be known
		NLS [34]	Various modifications	✓	✓	✕	NLS is less suitable for quantification than DMRM
	Fragment analysis	With a reference (SILNAS/CARD/ SILCARD) [52,53]	Known modifications	✓	✕	✓	Relative quantification can be assessed with reference in vitro RNA
		Without reference (RNase digests) [39]	Known modifications	✕	✕	✓	Determination of base composition and localization by comparing mass-spectrometry results with expected RNase fragments
NGS-based methods	RNA deep-sequencing direct method		A-to-I [54]	✓	✓	✓	To be accompanied by DNA sequencing to distinguish editing events from SNPs
			Methylations [55]	✕	✕		Based on RT stops or misincorporations
	Nanopore RNA sequencing [56,57]		m6A, m5C, A-to-I, Ψ and others	✓	✓		Based on the use of systematic base-calling ‘errors’ caused by the presence of RNA modifications. Software is still in development
	Indirect methods: chemical treatments	ICE-Seq [58,59]	A-to-I	✓	✓		No need of DNA seq
		Bisulfite-Seq [60]	m5C
		Riboxi-Seq [61]	Nm
		RiboMethSeq [62,63]
		Pseudo-Seq [64]	Ψ
		Ψ-Seq
		PSI-Seq [65]
		HydraPsi-Seq [66]
		SLAM-Seq [67]	s4U
		ARM-Seq [68]	m1A, m3C, m1G
		TRAC-Seq [69]	m7G
		AlkAniline-Seq [70]	m7G, m3C, D
	Indirect methods: IP	miCLIP [71]	Methylation	✓	✓
		m6A-Seq [72]	m6A
		meRIP-Seq [73]
		m6A-LAIC-Seq [74]
		Nm-Seq / 2OMe-Seq [75]	Nm
		acRIP-Seq [76]	ac4C
		NAD capture-Seq [77]	5’-NAD cap
Affinity gel electrophoresis	Mercury-sulfur affinity [78]		s2U, s4U	✕	✕	✕	APM treatment (Acrylo-aminophenylmercuric chloride)
	Boronate affinity [79]		NAD- or FAD-modified RNAs	✓	✓	✕	APB treatment (Acryloylaminophenyl boronic acid); fast screening (easy and quick); quantification possible as per intensity of bands

✕ = not available; ✓ = available.