Archives of Biochemistry and Biophysics 454 (2006) 146–154 www.elsevier.com/locate/yabbi 0003-9861/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.08.015 N-terminal acylation of the SV40 nuclear localization signal peptide enhances its oligonucleotide binding and membrane translocation eYciencies Ilona Laczkó a,¤ , György Váró a , Sándor Bottka b , Zoltán Bálint a , Eszter Illyés c , Elemér Vass c , Jean-Remy Bertrand d , Claude Malvy d , Miklós Hollósi c a Institute of Biophysics, Biological Research Center, Szeged, Hungary b Institute of Plant Biology, Biological Research Center, Szeged, Hungary c Department of Organic Chemistry, Eötvös Lóránd University, Budapest, Hungary d Institute Gustave Roussy, CNRS, Villejuif, Paris, France Received 14 June 2006, and in revised form 9 August 2006 Available online 31 August 2006 Abstract Octanoyl and palmitoyl groups were coupled to the N-terminus of an analog of the SV40 nuclear localization signal peptide, SV 126– 133 (Ser 128 ), to study the eVect of the fatty acid chain length on the complex formation with a single-stranded antisense oligodeoxynucleo- tide (ODN) and on the cellular uptake of the complex. The strongest binding aYnity was observed for the palmitoylated peptide, indicat- ing the better accessibility of the positively charged lysyl and arginyl side-chains to the phosphate groups due to the turn structures stabilized by the palmitoyl group. On increase of the peptide to ODN molar ratio (r M ), gradual unstacking of the bases was observed, the maximal rate being reached at r M D 10. At r M > 10 restacking of the nucleotide bases was detected and the ODN was completely encapsu- lated in a liposome-like structure made up of palmitoylated peptides. Cell translocation experiments revealed a highly eYcient cell trans- port of the ODN by palmitoylated SV40 peptide at r M > 10.  2006 Elsevier Inc. All rights reserved. Keywords: Antisense oligonucleotide; Acylated peptides; Attenuated total reXection FTIR; Atomic force microscopy; Cell translocation; Circular dichro- ism; EpiXuorescence; Nuclear localization signal peptide The application of antisense oligodeoxyribonucleotides (ODNs) 1 modulating gene expression is a promising approach in medicinal therapeutics. The major limitation of all nonviral nucleic acid- and ODN-based techniques is the low eYciency of membrane translocation and targeting (see Ref. [1] and references therein). The general principle of uptake-enhancing chemicals is based on complex formation between positively charged carriers (lipids, proteins, pep- tides, dendrimers and other polymers) and negatively charged ODN molecules. The most noticeable improve- ments have been achieved in the Weld of artiWcial lipid- based ODN delivery systems. These are probably the most commonly used methods to date [2–4]. The peptide-linked fatty acids obviously play a multiple role in modulating the membrane penetrability and cellular activity of the ODN. Their primary role is in anchoring peptide–ODN com- plexes to biomembranes. The membrane perturbation activities of acylated peptides depend strongly on the chain length of the acyl moiety: acyl chains shorter than those of * Corresponding author. Fax: +36 62 433133. E-mail address: laczko@nucleus.szbk.u-szeged.hu (I. Laczkó). 1 Abbreviations used: ODN, oligodeoxynucleotide; NLS, nuclear locali- zation signal; GFP, green Xuorescent protein; siRNA, small interfering RNA; CD, circular dichroism; ATR-FTIR, attenuated total reXection- Fourier transform infrared; AFM, atomic force microscopy; EM, epiXuo- rescence microscopy; DMEM, Dulbecco’s modiWed Eagle’s medium; TFA, triXuoroacetic acid; HOBt, 1-hydroxybenztriazole; DIC, diisopro- pylcarbodiimide; DIEA, N,N'-diisopropylethylamine; HF, hydrogen Xuoride; TFE, triXuoroethanol; DCM, dichloromethane; ESI-MS, electro- spray ionization mass spectrometry.