Fragment ions illustrating characterization of the different isoforms are shown in Figure . While bracketing fragments were not observed at positions 56 and 66 for all three isoforms, w fragments originating from phosphodiester backbone cleavages between each methylation site were observed in each spectrum. Each of the four w fragment ions at positions 51, 60, 62, and 70 (i.e. w27, w18, w16, and w8) shown in Figure illustrate isotopic distributions dependent on inclusion of the modified nucleotide. These fragment ions indicate the presence of a modified nucleotide in the identified fragment when the observed fragment ion in the m6A 56 and m5C 66 variants exhibit lower m/z values than the unmodified let7 precursor. The m6A 56 variant exhibits this m/z difference for only the w27 10-ion, whereas the m5C 66 variant exhibits the characteristic m/z shift in the w27 10-, w18 7-, and w16 6-ions, while no -2 Da shift is observed for the w8 3-ion. The 1 Da discrepancy between the unmodified let7 precursor and the m6A 56 and m5C 66 variants in the w8 3-ion is attributable to a sequence mutation from cytidine to uridine at the 3' terminus, which is also observable at a 3 Da difference in the precursor isotopic distributions, shown in Figure . Nucleo-SAFARI adds to the burgeoning field of nucleic acid mass spectrometry data analysis tools and provides an R-based workflow designed for top-down spectra of increasingly large (25 kDa, 80 nt) nucleic acids. Fragment identification in the m/z domain for DNA and RNA molecules in the positive or negative polarities coupled with detailed results and visualization outputs enable straightforward analysis of nucleic acid MS/MS spectra with support for a variety of synthetic and biological modifications. Moreover, identification of fragments directly in the m/z domain without reliance on deconvolution and deisotoping algorithms enables direct evaluation of fragment ion abundances and accurate consideration of non-canonical heteroatoms. This approach is being utilized to continue investigations into the fragmentation propensities of various oligonucleotides, increasing the depth to which top-down mass spectrometry methods can characterize modified nucleic acids and distinguish positional isomers of specific covalent modifications based on their fragmentation trends.