SWAMNA: a comprehensive platform for analysis of nucleic acid modifications

Abstract

The interest in MS-based analysis of modified nucleic acids is increasing due to the application of nucleic acids in therapeutics. However, there are few available integrated platforms for characterizing nucleic acid modifications. Herein, we report a general mass spectrometry-based SWATH platform to identify and quantify both RNA and DNA modifications, which we call SWATH analysis of modified nucleic acids (SWAMNA). SWAMNA incorporates the search engine, NuMo finder, enabling the analysis of modifications in native and permethylated form. SWAMNA will aid discoveries that provide new insights into nucleic acid modifications.

Figure 1.. Workflows of identifying RNA and DNA modifications using NuMo Finder.

Two search functions, common search and unknown search, were developed. The modifications in Modomics and DNAmod databases were referenced. Unknown modifications were searched based on the feature of ribose and deoxyribose loss in nucleoside fragmentation, and more details about the NuMo Finder can be found in Supplementary Information 2.

Figure 2.. SWAMNA was applied to characterize DNA and RNA modifications in different biological conditions.

(A) and (B) SWAMNA monitored the increasing of 5mC and 5hmC levels in MPNST S462 cells due to the PRC-2 loss. (C) RNA extracted from Adenovirus infected A549 cells were analyzed at 24 and 48 hours post-infection. (D) The modification with significant change was shown and the amount of methylated nucleosides generally increased during adenovirus infection. Asterisks indicate the statistical significance between groups compared using the t-test (*p< 0.05%).

Scheme 1.. The SWAMNA platform for analyzing DNA and RNA modifications.

Cellular DNA and RNA were extracted and purified, then enzymatically digested into mononucleosides using a “one-pot” approach. The resulting digested nucleosides underwent purification through PGC solid-phase extraction and were subsequently subjected to derivatization using permethylation. The zenoSWATH was employed for the analysis, and the NuMo Finder search engine was developed to identify and quantify the modifications present.