A 60 mer DNA was synthesized by IDT (Coralville, IA), while all RNA oligonucleotides (miR145, let7 precursors) were provided by the Xhemalce group (University of Texas at Austin). Sequences and associated monoisotopic masses can be found in Table . Nanoflow on-line desalting was performed as described previously. Briefly, an isocratic gradient (4:1 acetonitrile:water, 10 mM piperidine ~pH 10) at a flow rate of 1 µL/min was utilized to desalt nucleic acid samples with an inhouse packed Waters XBridge Phenyl trap column (5 cm X 100 µm). Oligonucleotides were ionized in the negative mode using an electrospray voltage of 1600 V applied to a stainless-steel tee and pulled fused silica emitter (New Objective, Littleton, MA). Each acquisition utilized 0.5 µL of sample at 10 µM in 4:1 acetonitrile:water All spectra were collected on an Orbitrap Lumos mass spectrometer (Thermo Scientific, San Jose, CA), and averaged and exported using QualBrowser. All MS/MS spectra utilized quadrupole isolation with a width of 2 m/z. CID activation used and NCE values of 25 and a q value of 0.14. 193 nm UVPD activation utilized 1 pulse (2ms), at an energy of 1 mJ, while 213 nm UVPD activation employed 50 pulses (20 ms) at a fixed pulse energy of ~2 µJ per pulse. Fragment identification was carried out in Nucleo-SAFARI (R 4.3.3 "Angel Food Cake") using a 10 ppm m/z error tolerance and 30% intensity error tolerance. A full module diagram of the application is shown in