Nanoflow online desalting provides a new avenue for top-down analysis of nucleic acids. Drawing from the methodologies employed on microflow platforms, this nanoflow platform provided desalting of a range of oligonucleotide species comparable to that observed at higher flow rates. Additionally, implementation of peak parking allowed a higher number of spectral averages for each run, enabling collection of improved data without detrimental effects on the observed desalting or analyte signal. The nanoflow set-up offered a 10X reduction in sample usage compared to microflow counterparts, making this method suitable for analysis of low-quantity biological samples such as isolated tRNAs. Tandem MS analysis of a yeast phenylalanine tRNA yielded high sequence coverage based on CID, 213 nm UVPD, and a-EPD, with fragment ion assignments facilitated isotopic distribution matching in the m/z domain. Preliminary MS1 results obtained for a G-rich sequence suggested preservation of a G-quadruplex structure as evidenced by the specific retention of three potassium ions, suggesting retention of secondary structure through online desalting despite the high acetonitrile and basic content of the mobile phases.