New derivatives of aminoglycosides with a side chain 1,2-aminoalcohol at the 5" position of ring III were designed, synthesized, and biologically evaluated. The novel lead structure (compound <b>6</b>), exhibiting substantially enhanced selectivity toward eukaryotic versus prokaryotic ribosome, high readthrough activity, and considerably lower toxicity than the previous lead compounds, was discovered. Balanced readthrough activity and toxicity of <b>6</b> were demonstrated in three different nonsense DNA-constructs underlying the genetic diseases, cystic fibrosis and Usher syndrome, and in two different cell lines, baby hamster kidney and human embryonic kidney cells. Molecular dynamics simulations within the A site of the 80S yeast ribosome demonstrated a remarkable kinetic stability of <b>6</b>, which potentially determines its high readthrough activity.