Determination of Penetratin Secondary Structure in Live Cells with Raman Microscopy

摘要

Abstract: Cell penetrating peptides (CPPs) have attracted recent interest as drug delivery tools, althoughnthe mechanisms by which CPPs are internalized by cells are not well-defined. Here, we report a newnexperimental approach for the detection and secondary structure determination of CPPs in live cells usingnRaman microscopy with heavy isotope labeling of the peptide. As a first demonstration of principle, penetratin,na 16-residue CPP derived from the Antennapedia homeodomain protein of Drosophila, was measured innsingle, living melanoma cells. Carbon-13 labeling of the Phe residue of penetratin was used to shift thenintense aromatic ring-breathing vibrational mode from 1003 to 967 cm 1, thereby enabling the peptide tonbe traced in cells. Difference spectroscopy and principal components analysis (PCA) were usednindependently to resolve the Raman spectrum of the peptide from the background cellular Raman signals.nOn the basis of the position of the amide I vibrational band in the Raman spectra, the secondary structurenof the peptide was found to be mainly random coil and u0001-strand in the cytoplasm, and possibly assemblingnas u0001-sheets in the nucleus. The rapid entry and almost uniform cellular distribution of the peptide, as wellnas the lack of correlation between peptide and lipid Raman signatures, indicated that the mechanism ofninternalization under the conditions of study was probably nonendocytotic. This experimental approachncan be used to study a wide variety of CPPs as well as other classes of peptides in living cells.