Supramolecular structure and nuclear targeting efficiency determine the enhancement of transfection by modified polylysines

Polylysine (pLy) has been used as a DNA carrier in nonviral gene delivery s ystems because it forms complexes with plasmid DNA via charge interaction, and condenses it into a compact structure. We have recently shown that cros slinking nuclear localization sequences (NLSs) to ply can enhance transfect ion by conferring specific recognition by the cellular nuclear import 'rece ptor: the NLS-binding importin alpha/beta heterodimer. The present study ex amines and correlates for the first time the effect of the lysine/nucleotid e (Ly/Nu) ratio on transfection, recognition by importin alpha/beta, and st ructure as determined using electron microscopy (EM) and atomic force micro scopy (AFM), for pLy-DNA complexes with and without NLSs or mutant versions thereof Intriguingly, we observed two distinct peaks of transfection enhan cement at Ly/Nu ratios of 0.4 and 4.0, attributable to specific NLS recogni tion by importins and DNA compaction, respectively. The results indicate a clear correlation between the pLy-DNA structure, importin alpha/beta recogn ition, and gene transfer efficiency, thus underlining the importance of usi ng pLy-DNA at the optimal Ly/Nu ratio.