Three new derivatives of neamine, 3 (NE), 6 (NEA) and 9 (NEL), were synthesized by connecting arginine or lysine to 5-hydroxyl group of neamine using ethylenediamine as a linker. The binding affinities of these derivatives to A site of 16S RNA and TAR RNA indicate that the modification on 5-hydroxyl of neamine by amino acid can enhance the binding affinity of neamine. Compound 9 (NEL) shows some antibacterial activities. These results demonstrate that modification on 5-hydroxyl group of neamine may provide a promising way for the development of potential candidates effectively targeting to RNAs.