A recent ground-breaking study suggested that small RNA from mammalian cells can undergo N-glycan modifications (termed glycoRNA)¹. The discovery relied upon a metabolic glycan labeling strategy in combination with commonly used phase-separation-based RNA isolation. Following the reported procedure, here we likewise identify an N-glycosylated species in the RNA fraction. However, our results suggest that the reported RNase sensitivity of the glycosylated species depends on the specific RNA purification method. This suggests the possibility of copurifying unexpected RNase-insensitive N-glycoconjugates during glycoRNA isolation. The co-existence of two independent, yet highly similar molecular entities, complicates biochemical assays on glycoRNA and calls for more specific approaches for glycoRNA analysis. To address this, we propose a control experiment that can help distinguish genuine glycoRNA species from copurified glycoconjugates.

Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O-GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis