Riboswitches are regulatory elements in non-coding regions of mRNAs. By binding small ligands, they control the expression of the genes located downstream of the aptamer. The N1 synthetic riboswitch binds aminoglycoside antibiotics from the neomycin family. It consists of a 27-nucleotide-long hairpin with two flexible regions: a bulge and an apical loop. In N1 riboswitch, different aminoglycosides evoke a varying response: ribostamycin and neomycin inhibit the expression of genes in yeast and paromomycin is inactive, even though the two latter ligands differ by only one functional group in ring I. The NMR structures of the ribostamycin and paromomycin complexes with N1 are available. However, the structures of the N1 riboswitch with neomycin and without ligands are not known. To explain how such a small difference between neomycin and paromomycin affects the interactions and dynamics of the N1 riboswitch, we performed all-atom molecular dynamics simulations. Replica exchange method helped increase the sampling. The non-Watson-Crick hydrogen bonds differ between the riboswitch with and without ligands even though the secondary structure is similar. Aminoglycosides active in yeast (neomycin and ribostamycin) display significant stabilization of the apical loop nucleobase A17 upon binding with the riboswitch. In paromomycin complex, the A17 base is only partially stabilized and creates a frequent, but unstable hydrogen bonding with a terminal hydroxyl group in ring I of paromomycin. Surprisingly, this hydrogen bond was not captured in recent NMR experiments. The N1 riboswitch shares several similar patterns with another aminoglycoside binding site in ribosome called A-site. However, A-site does not discriminate between paromomycin and neomycin. Thus, we compare those two aminoglycoside binding sites from the point of view of their sequence, hydrogen bond network, and sodium ion concentration.