Mutations of proteins with dual activities that lead to enhancement of one activity are frequently accompanied by attenuation of the other activity. However, this mutational negative trade-off phenomenon typically only involves the canonical 20 amino acids. To test the effect of non-canonical amino acids on the negative trade-off phenomenon, two bioactivities of HIV-1 Tat-derived peptides were monitored upon changing the Arg side chain length. In contrast to the expected mutational negative trade-off, shortening Arg by one methylene resulted in both higher TAR RNA binding specificity and higher cellular uptake. These results suggest that introducing previously unexploited building blocks, even if the difference is only one methylene, can alter the peptide bioactivity landscape leading to the enhancement of multiple bioactivities.