Complex formation and vectorization of a phosphorothioate oligonucleotide with an amphipathic leucine‐ and lysine‐rich peptide: Study at molecular and cellular levels

Abstract

Optical spectroscopic techniques such as CD, Raman scattering, and fluorescence imaging allowed us to analyze the complex formation and vectorization of a single-stranded 20-mer phosphorothioate oligodeoxynucleotide with a 15-mer amphipathic peptide at molecular and cellular levels. Different solvent mixtures (methanol and water) and molecular ratios of peptide/oligodeoxynucleotide complexes were tested in order to overcome the problems related to solubility. Optimal conditions for both spectroscopic and cellular experiments were obtained with the molecular ratio peptide/oligodeoxynucleotide equal to 21:4, corresponding to a 7:5 ratio for their respective +/- charge ratio. At the molecular level, CD and Raman spectra were consistent with a alpha-helix conformation of the peptide in water or in a methanol-water mixture. The presence of methanol increased considerably the solubility of the peptide without altering its alpha-helix conformation, as evidenced by CD and Raman spectroscopies. UV absorption melting profile of the oligodeoxynucleotide gave rise to a flat melting profile, corresponding to its random structure in solution. Raman spectra of oligodeoxynucleotide/peptide complexes could only be studied in methanol/water mixture solutions. Drastic changes observed in Raman spectra have undoubtedly shown: (a) the perturbation occurred in the peptide secondary structure, and (b) possible interaction between the lysine residues of the peptide and the oligodeoxynucleotide. At the cellular level, the complex was prepared in a mixture of 10% methanol and 90% cell medium. Cellular uptake in optimal conditions for the oligodeoxynucleotide delivery with low cytotoxicity was controlled by fluorescence imaging allowing to specifically locate the compacted oligonucleotide labeled with fluorescein at its 5'-terminus with the peptide into human glioma cells after 1 h of incubation at 37 degrees C.

Low concentration structural dynamics of lanreotide and somatostatin‐14

“…fluorescence, circular dichroism and Raman scattering. We would like to stress the capability of the classical Raman spectra for providing structural information on the short size peptides through the amide (I and III) regions , . Particularly, the ability of the amide III Raman markers to confirm the reliability of the structural information deduced from the amide I (or amide I') region, as confirmed here for lanreotide, should be emphasized.…”

Vibrational Analysis of Amino Acids and Short Peptides in Hydrated Media. II. Role of KLLL Repeats To Induce Helical Conformations in Minimalist LK-Peptides

“…12 For the sake of brevity these peptides are hereafter referred to as the 5-mer, 11-mer, 15-mer, and 19-mer, respectively. Purified peptides containing TFA as counterion were dissolved in phosphate buffer (containing 10 mM of K + and Na + cations, pH or pD 6.8) in PBS (phosphate buffer saline, containing an excess of 100 mM NaCl in solution, pH 7.4), in Tris (pH 7.4), in methanol, as well as in bicomponent mixtures containing the three latter buffers and methanol.…”

Secondary conformation of short lysine‐ and leucine‐rich peptides assessed by optical spectroscopies: Effect of chain length, concentration, solvent, and time

“…[8][9][10][11][12][15][16][17][18] Structural properties of a few KL-minimalist peptides were subject to previous investigations by means of CD and vibrational spectroscopy, [8][9][10][11][12]19,20 with the consideration of chain length, molecular concentration, and ionic force. 20 We can recall here the results obtained from some (LK) n and (LKKL) n peptides, 20 those from the peptides belonging to L i K j (i ¼ 2j) 1,[8][9][10]12 and L n K nþ1 11 families. Among the L i K j (i ¼ 2j) peptides, the 9-mer N ter -KLLLKLLLK (L 6 K 3 ) revealed a very low level of structuring, whereas the 15-mer N ter -KLLKLLLKLLLKLLK (L 10 K 5 ) was shown to be capable of forming an -helical secondary structure.…”

“…1 Spectroscopic investigations (CD and Raman scattering) could emphasize that this peptide adopts an -helix structure in phosphate buffer and in methanol. The capability of cationic vectors, [2][3][4][5] particularly minimalist cationic peptides, 6,7 to condense DNA and to facilitate its in vitro cellular transfection were shown in the framework of antisense therapy.…”

“…Among the minimalist peptides, those formed only by lysine (K: cationic and hydrophilic monomer) and leucine (L: neutral and hydrophobic monomer), were found to be highly efficient. 1,[8][9][10][11][12][13][14][15] This is mainly due to their ability to mimic the behavior of natural peptides as regards their strong lytic activity and their aptitude to perturb cell membranes. [9][10][11][12][13][14][15] On the other hand, it is now well established that the secondary structure of peptides and proteins does influence their interactions with membranes and model membranes.…”