An example of fragment identification results from Nucleo-SAFARI executed on a UVPD mass spectrum of a synthetic, methylated (m5C 66) let7 miRNA precursor (78 nt, 25 kDa) is shown in ) with an m/z error tolerance of 10 ppm and an intensity tolerance of 30%. These parameters resulted in identification of 127 fragment ions, yielding a sequence coverage of 88.3% and a P-score of 1.03e-180. This oligonucleotide contains a 5-methylcytidine nucleobase at position 66 incorporated with a deoxyribose sugar. In this spectrum, well-resolved w65 and w66 fragments were identified (displayed in insets) that bracket the modified residue and confirm its location in the let7 precursor sequence. These fragment ions and the modified residue are highlighted by red boxes in Figure . The types of fragment ions identified by Nucleo-SAFARI agree with the expected preferential production of d and w ions noted previously for analysis of RNA by UVPD. The sum intensity of these fragment ions likewise followed established trends, where d and w fragments were not only the most numerous, but also the highest total abundance, summarized in Figure . Annotated MS/MS spectra and sequence maps for the m6A 65 let7 precursor and unmodified let7 precursor can be found in Figures and. A critical application of top-down MS for analysis of nucleic acids is differentiation of positional isomers, a particularly critical aspect when localizing covalent modifications. UVPD mass spectra were collected for three variants of the synthetic let7 precursor RNA, including an unmodified species and two methylated isomers m6A 56 and m5C 66. These oligonucleotides contained deoxyribose sugars at the methylated nucleobases, resulting in only a 2 Da mass difference.