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 systems because it forms complexes with plasmid DNA via charge interaction, and condenses it into a compact structure. We have recently shown that cross-linking nuclear localization sequences (NLSs) to pLy can enhance transfection by conferring specific recognition by the cellular nuclear import 'receptor', the NLS-binding importin alpha/beta heterodimer. The present study examines and correlates for the first time the effect of the lysine/nucleotide (Ly/Nu) ratio on transfection, recognition by importin alpha/beta, and structure as determined using electron microscopy (EM) and atomic force microscopy (AFM), for pLy-DNA complexes with and without NLSs or mutant versions thereof. Intriguingly, we observed two distinct peaks of transfection enhancement at Ly/Nu ratios of 0.4 and 4.0, attributable to specific NLS recognition by importins and DNA compaction, respectively. The results indicate a clear correlation between the pLy-DNA structure, importin alpha/beta recognition, and gene transfer efficiency, thus underlining the importance of using pLy-DNA at the optimal Ly/Nu ratio.