CYTOSOLIC AND NUCLEAR DELIVERY OF OLIGONUCLEOTIDES MEDIATED BY AN AMPHIPHILIC ANIONIC PEPTIDE

Antisense oligonucleotides (ODN) were easily introduced into the cytos ol of mammalian cells on permeabilization of the plasma membrane by an amphiphilic anionic peptide. The E5CA peptide (GLFEAIAEFIEGG-WEGLIEGC A) is an E5 peptide analog derived from the N-terminal segment of the HA2 subunit of influenza virus hemagglutinin, This peptide undergoes a conformational change when the pH shifts from neutral to around 6.0, inducing a transient permeabilization of the plasma membrane, In the p resence of the E5CA peptide at pH close to 6.0, fluoresceinylated ODN were rapidly taken up by cells and diffused into the nucleus, The upta ke of ODN was dependent on the E5CA peptide concentration and on the d uration of the incubation at low pH, as shown by confocal microscopy a nd flow cytometry analyses, This procedure is suitable for loading adh erent cells as well as nonadherent cells with single-stranded or doubl e-stranded ODN. Under optimal conditions, a high percentage of cells w ere nuclei loaded, and the viability was not affected, This method mak es use of a well-defined chemical product without the requirement of a ny special equipment, It will be useful to study the interactions of s ingle-stranded or double-stranded ODN used as antisense, antigenes, or decoys.