Chan, Chee KaiSenden, TimothyJans, David A2015-12-130969-7128http://hdl.handle.net/1885/89119Polylysine (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 α/β 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 α/β, 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 α/β recognition, and gene transfer efficiency, thus underlining the importance of using ply-DNA at the optimal Ly/Nu ratio.Keywords: cell nucleus receptor; DNA; DNA binding protein; karyopherin; nucleotide; polylysine; animal cell; article; atomic force microscopy; cell nucleus; cell ultrastructure; controlled study; electron microscopy; expression vector; gene mutation; gene targeting Atomic force microscopy; Electron microscopy; Importin subunits; Nonviral DNA transfer; Nuclear localization signals20002015-12-12