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.
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Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra.
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D000818:Animals; D002130:Calcium Phosphates; D002411:Cation Exchange Resins; D004352:Drug Resistance, Microbial / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D028884:Karyopherins; D008055:Lipids; D008163:Luminescent Measurements; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D009355:Neomycin / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D010957:Plasmids; D011107:Polylysine / Q000235:genetics*; D051381:Rats; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D002130:Calcium Phosphates; D002411:Cation Exchange Resins; D028884:Karyopherins; D008055:Lipids; C086724:Lipofectamine; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D011107:Polylysine; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D001616:beta-Galactosidase; D009355:Neomycin; C485829:calcium phosphate, dibasic, anhydrous