The label-free detection of oligonucleotides of 12-14 bases at a concentration of 10 −7 M (double helix) was achieved by using surface enhanced Raman scattering (SERS) and spermine as the aggregant of Ag sol. The wavenumbers of the produced SERS spectra of DNA are similar to those in the corresponding normal Raman spectra of free DNA, allowing the detailed assignment of the vibrational modes. The conformation of the adsorbed DNA, the adsorption geometry, and a molecular model of the interactions among DNA, spermine, and Ag nanoparticle are derived from the SERS spectra. The results show that the protonated amine groups of spermine interact with phosphodioxygens of DNA and N7s of dA and dG from the major groove. The protonated amines are also attracted to the negatively charged Ag surface and thus induce the adsorption of DNA on the metal surface. The DNA remains mostly in the B-conformation with a variation in the C5 -O dihedral angle. DNA lies flat with the bases perpendicular to the metal surface.

Cobra CTX A3, the major cardiotoxin (CTX) from Naja atra, is a cytotoxic, basic β-sheet polypeptide that is known to induce a transient membrane leakage of cardiomyocytes through a sulfatide-dependent CTX membrane pore formation and internalization mechanism. The molecular specificity of CTX A3-sulfatide interaction at atomic levels has also been shown by both nuclear magnetic resonance (NMR) and X-ray diffraction techniques to reveal a role of CTX-induced sulfatide conformational changes for CTX A3 binding and dimer formation. In this study, we investigate the role of sulfatide lipid domains in CTX pore formation by various biophysical methods, including fluorescence imaging and atomic force microscopy, and suggest an important role of liquid-disordered (ld) and solid-ordered (so) phase boundary in lipid domains to facilitate the process. Fluorescence spectroscopic studies on the kinetics of membrane leakage and CTX oligomerization further reveal that, although most CTXs can oligomerize on membranes, only a small fraction of CTXs oligomerizations form leakage pores. We therefore suggest that CTX binding at the boundary between the so and so/ld phase coexistence sulfatide lipid domains could form effective pores to significantly enhance the CTX-induced membrane leakage of sulfatide-containing phosphatidylcholine vesicles. The model is consistent with our earlier observations that CTX may penetrate and lyse the bilayers into small aggregates at a lipid/protein molar ratio of about 20 in the ripple P(β)' phase of phosphatidylcholine bilayers and suggest a novel mechanism for the synergistic action of cobra secretary phospholipase A2 and CTXs.

Previous nanoscale investigations of the gel-state membrane surface structure under the action of phospholipase A(2) (PLA(2)) suggest that single enzymes at work scoot on the membrane surface from the observed defects, which creates nanosized channels oriented along the lipid crystal-packing structure. To date, however, there have been no reports of direct observation of PLA(2) at the single-molecule level focusing on how the enzymes interact with the defects. Herein, we report a single-molecule fluorescence microscopy study on the action of enzymatically active rhodamine B-labeled cobra PLA(2) on a supported lipid membrane with visible packing defects on a glass substrate. Working with a gel-state phospholipid bilayer, the low-activity period (lag phase) of PLA(2) action is followed by the burst binding of PLA(2) molecules from aqueous solution on a few newly created active sites. These active sites are distinguished by a spatial resolution of approximately 40 nm, which is below the diffraction limit. The rate of active-site propagation as reflected by new PLA(2) binding on the membrane surface is estimated to be approximately 5 nm min(-1). This rate is about two orders of magnitude slower than the propagation rate of hydrolyzed channels estimated by AFM studies on bee venom PLA(2) on a similar membrane surface. This direct observation of PLA(2) molecules allows the visualization of different PLA(2) binding modes on the membrane surface and on the membrane boundary.

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