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However, the physicochemical properties of RNA result in poor ionization efficiencies during electrospray ionization, presenting a major barrier to sensitive MS measurements necessary for low-abundance RNA samples and RNAs with low modification stoichiometries. Here, we report a ligation-based approach to increase ionization efficiencies of RNA oligonucleotides. We show that short (\u223c5 nt), chemically modified DNA oligonucleotides can be enzymatically ligated to RNA to serve as MS signal enhancers. Among a series of signal enhancers appended with various alkyl and alkylimidazolium functional groups, we found that decyl-functionalized derivatives improved MS sensitivity by \u223c15-fold compared to the unlabeled oligonucleotide. When ligated to RNA standards, the decyl-modified signal enhancer increased MS signals by an average of 2.1-fold with the additional benefit of improved retention during liquid chromatography (LC) separations without ion-pairing agents. To apply the ligation-based approach to RNase T1 digests of longer RNAs, a multi-step enzymatic approach was optimized to maximize ligation efficiencies. We then ligated signal enhancers to a yeast transfer RNA (tRNA) digest and observed increased MS signals for numerous sequence-informative digestion products. Importantly, the sequences of RNA oligonucleotides ligated to signal enhancers were readily determined by tandem mass spectrometry with collision-induced dissociation. This ligation-based strategy for enhancing LC-MS\/MS characterization of RNA creates opportunities to measure low-abundance RNA samples and their modifications.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c02458","type":"journal-article","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T03:01:40Z","timestamp":1789441300000},"page":"27996-28005","source":"Crossref","is-referenced-by-count":0,"title":["Enzymatic Ligation\nStrategy to Enhance Electrospray\nIonization Efficiency and Liquid Chromatography\u2500Mass Spectrometry\nof DNA and RNA Oligonucleotides"],"prefix":"10.1021","volume":"98","author":[{"given":"Max D.","family":"Sharin","sequence":"first","affiliation":[{"name":"Tufts University , , , ,","place":["Medford, Massachusetts, United States, 02155"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nina J.","family":"Fitzgerald","sequence":"additional","affiliation":[{"name":"Tufts University , , , ,","place":["Medford, Massachusetts, United States, 02155"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen M.","family":"Kennedy","sequence":"additional","affiliation":[{"name":"Tufts University , , , ,","place":["Medford, Massachusetts, United States, 02155"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabella G.","family":"Park","sequence":"additional","affiliation":[{"name":"Tufts University , , , ,","place":["Medford, Massachusetts, United States, 02155"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0576-7716","authenticated-orcid":true,"given":"Kevin D.","family":"Clark","sequence":"additional","affiliation":[{"name":"Tufts University , , , ,","place":["Medford, Massachusetts, United States, 02155"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,14]]},"reference":[{"issue":"5","key":"2026092909095498100_cit1","doi-asserted-by":"publisher","DOI":"10.1002\/wrna.1651","article-title":"The Potential Versatility of RNA Catalysis","volume":"12","author":"Wilson","year":"2021","journal-title":"WIREs RNA"},{"issue":"1","key":"2026092909095498100_cit2","doi-asserted-by":"publisher","first-page":"20","DOI":"10.1016\/j.tibs.2006.11.007","article-title":"How Ribosomes Make Peptide Bonds","volume":"32","author":"Rodnina","year":"2007","journal-title":"Trends Biochem. 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