Table 1 Summary of ac4C detection technologies

HPLC-based methods

RP-HPLC

(1) the main nucleosides and the modified nucleosides obtained by enzymolysis of rna can be separated

(2) it does not rely on expensive mass spectrometer detectors

(1) it needs to consume a lot of mobile phase solvent

(2) nucleosides with similar retention times could not be qualitatively and quantitatively analyzed

[17, 18]

UV-HPLC

it can accurately locate the position of the ac4C

(1) the signal cannot be amplified, and the sensitivity is limited

(2) limit utility and throughput

[7]

HPLC conjugated with MISPE

the endogenous pyrimidine nucleosides were selectively extracted from urine, which improved the sensitivity of HPLC analysis results in the subsequent detection process

It is necessary to process the sample under the condition of ph = 10, but AC4c will be hydrolyzed into c under this alkaline condition, and the analysis of AC4c cannot be realized

[21]

mung bean nuclease cleavage coupled to UV-HPLC

ac4C can be localized to specific sites in cellular rna

it suffers from poor sensitivity due to a lack of signal amplification and requires the synthesis of tiling oligonucleotides, limiting throughput

[20]

HPLC conjugated with MS

LC-MS/HPLC-MS

high precision, high sensitivity, high selectivity

complex operation steps

[13]

Antibody-based methods

acRIP-seq

it can generate thousands of ac4C-enriched transcribed regions

(1) the reads may be biased by the affinity of mRNA and the antibody

(2) it cannot provide a base-resolution ac4C map at the transcriptome level

[12, 22]

Borohydride reduction-based methods

borohydridebased reduction

The nucleotide resolution of AC4c can be quantitatively detected

The selectivity is not high, and control tests should be designed in combination with the unstable hydrolysis properties of AC4c in practical applications to further determine the site of AC4c

[7]

borohydridebased Sanger sequencing

It can sensitively detect a single ac4C site using PCR amplification

it is unable to analyze ac4C in RNAs with densemodified nucleotides

[7]

Computational methods for ac4C site prediction

PACES

good performance

(1) only moieties that are likely to undergo acetylation can be predicted, but the exact location of acetylation cannot be predicted

(2) repeated cxx moieties remain ambiguous and the exact form of ac4c acetylation needs to be further studied

(3) cross-tissue or cross-species predictions cannot be made due to the limitations of available data

[23]

XG-ac4C

outperforms the most advanced methods in both cross-validation and independent testing

[24]