We designed a peptide, H5WYG (GLFHAIAHFIHGGWHGLIHGWYG), that permeabilizes cell membrane at a slightly acidic pH but not at neutral pH. Absorbance, fluorescence, and circular dichroism spectra showed that H5WYG undergoes a dramatic conformational change between pH 7.0 and 6.0 that correlates with the protonation of the histidyl residues. Cell permeabilization studies monitored by flow cytometry on living cells showed that H5WYG permeabilizes the cell membrane with a great efficiency at pH 6.4 but was not active at neutral pH; at pH 6.8, the peptide permeabilized 50% of the cells at 20 degrees C within 10 min. H5WYG increased the expression of genes transferred to cells as glycosylated polylysine-DNA complexes, and the transfection efficiency was not impaired in the presence of serum. Therefore, this peptide containing several histidines that become positively charged when the pH decreased to less than 7.0 is a suitable helper for delivering molecules into the cytosol upon either permeabilization of the plasma membrane induced by lowering the extracellular medium to pH 6.4 or permeabilization of the endosomal membrane induced by acidification of endosomes.
The secondary structure of guanine-rich oligodeoxynucleotides has been investigated with fluorescent probes. Intramolecular folding of a telomeric oligonucleotide into a quadruplex led to fluorescence resonance energy transfer (FRET) between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5' and 3' ends of the DNA, respectively. Depending on oligonucleotide length, quenching efficiency varied between 0.45 and 0.72 at 20 degrees C. The conjugation of the dyes to the oligonucleotide had a limited, but significant, influence on the thermodynamics of G-quartet formation. Intramolecular folding was demonstrated from the concentration independence of fluorescence resonance energy transfer over a wide concentration range. Folding of the oligonucleotide was confirmed by UV absorption, UV melting, and circular dichroism experiments. The folding of the G-quartet could be followed at concentrations as low as 100 pM. Fluorescence resonance energy transfer can thus be used to reveal the formation of multistranded DNA structures.
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