The patterns of formation of RNA nanoparticles (NPs) during thermal cycling of bacterial total tRNA in the presence of cations Ca2+, Mn2+, Ni2+, Zn2+, Co2+, and Cu2+ were studied. The optimal conditions for the production of NPs were found, and it was revealed that their size depends on the ratio of the concentrations of Me2+ and tRNA. The concentration of reagents for obtaining NPs of small size (from 5 to 100 nm) was selected. It was shown that tRNA-based nanoparticles can comprise short (20-50 nt) ribooligonucleotides, including aptamers and siRNAs. The stability of NPs during storage in buffer solutions of various composition was studied. It was found that the initial suspensions of NPs are quite stable, but they are rapidly destroyed in PBS buffer (pH 7.4). A simple and effective stabilizer (polyarginine) was found, the additives of which ensure the preservation of nanoparticles in PBS buffer for more than 5 h. Nanoparticles modified with the stabilizer are resistant to blood serum nucleases and can be used for transfection.