A means to control DNA compaction with light illumination has been developed using the interaction of DNA with a photoresponsive cationic surfactant. The surfactant undergoes a reversible photoisomerization upon exposure to visible (trans isomer, more hydrophobic) or UV (cis isomer, more hydrophilic) light. As a result, surfactant binding to DNA and the resulting DNA condensation can be tuned with light. Dynamic light scattering (DLS) measurements were used to follow lambda-DNA compaction from the elongated-coil to the compact globular form as a function of surfactant addition and light illumination. The results reveal that compaction occurs at a surfactant-to-DNA base pair ratio of approximately 7 under visible light, while no compaction is observed up to a ratio of 31 under UV light. Upon compaction, the measured diffusion coefficient increases from a value of 0.6 x 10(-8) cm2/s (elongated coil with an end-to-end distance of 1.27 microm) to a value of 1.7 x 10(-8) cm2/s (compact globule with a hydrodynamic radius of 120 nm). Moreover, the light-scattering results demonstrate that the compaction process is completely photoreversible. Fluorescence microscopy with T4-DNA was used to further confirm the light-scattering results, allowing single-molecule detection of the light-controlled coil-to-globule transition. These structural studies were combined with absorbance and fluorescence spectroscopy of crystal violet in order to elucidate the binding mechanism of the photosurfactant to DNA. The results indicate that both electrostatic and hydrophobic forces are important in the compaction process. Finally, a DNA-photosurfactant-water phase diagram was constructed to examine the effects of both DNA and surfactant concentration on DNA compaction. The results reveal that precipitation, which occurs during the latter stages of condensation, can also be reversibly controlled with light illumination. The combined results clearly show the ability to control the interaction between DNA and the complexing agent and, therefore, DNA condensation with light.

Photoresponsive catanionic vesicles have been developed as a novel gene delivery vector combining enhanced cellular uptake with phototriggered release of vesicle payload following entry into cells. Vesicles with diameters ranging from 50 to 200 nm [measured using cryo-transmission electron microscopy (TEM) and light-scattering techniques] form spontaneously, following mixing of positively charged azobenzene-containing surfactant and negatively charged alkyl surfactant species. Fluorescent probe measurements showed that the catanionic vesicles at a cation/anion ratio of 7:3 formed at surfactant concentrations as low as 10 microM of the azobenzene surfactant under visible light (with the azobenzene surfactant species principally in the trans configuration), while 50-60 microM of the azobenzene surfactant is required to form vesicles under UV illumination (with the azobenzene surfactant species principally in the cis configuration). At intermediate surfactant concentrations (ca. 15-45 microM) under visible light conditions, transport of DNA-vesicle complexes occurred past the cell membrane of murine fibroblast NIH 3T3 cells through endocytosis. Subsequent UV illumination induced rupture of the vesicles and release of uncomplexed DNA into the cell interiors, where it was capable of passing through the nuclear membrane and thereby contributing to enhanced expression. Single-molecule fluorescent images of T4-DNA demonstrated that the formation of vesicles with a net positive charge led to compaction of DNA molecules via complex formation within a few seconds, while UV-induced disruption of the vesicle-DNA complexes led to DNA re-expansion to the elongated-coil state, also within a few seconds. Transfection experiments with eGFP DNA revealed that photoresponsive catanionic vesicles are more effectively taken up by cells compared to otherwise identical alkyl (i.e., nonazobenzene-containing and thus nonlight-responsive) catanionic vesicles, presumably because of pi-pi stacking interactions that enhance bilayer rigidity in the photoresponsive vesicles. Subsequent UV illumination following endocytosis leads to further dramatic enhancements in the transfection efficiencies, demonstrating that vector unpacking and release of DNA from the carrier complex can be the limiting step in the overall process of gene delivery.

A means to control lysozyme conformation with light illumination has been developed using the interaction of the protein with a photoresponsive surfactant. Upon exposure to the appropriate wavelength of light, the azobenzene surfactant undergoes a reversible photoisomerization, with the visible-light (trans) form being more hydrophobic than the UV-light (cis) form. As a result, surfactant binding to the protein and, thus, protein unfolding, can be tuned with light. Small-angle neutron scattering (SANS) measurements were used to provide detailed information of the protein conformation in solution. Shape-reconstruction methods applied to the SANS data indicate that under visible light the protein exhibits a native-like form at low surfactant concentrations, a partially swollen form at intermediate concentrations, and a swollen/unfolded form at higher surfactant concentrations. Furthermore, the SANS data combined with FT-IR spectroscopic analysis of the protein secondary structure reveal that unfolding occurs primarily in the alpha domain of lysozyme, while the beta domain remains relatively intact. Thus, the surfactant-unfolded intermediate of lysozyme appears to be a separate structure than the well-known alpha-domain intermediate of lysozyme that contains a folded alpha domain and unfolded beta domain. Because the interactions between the photosurfactant and protein can be tuned with light, illumination with UV light returns the protein to a native-like conformation. Fluorescence emission data of the nonpolar probe Nile red indicate that hydrophobic domains become available for probe partitioning in surfactant-protein solutions under visible light, while the availability of these hydrophobic domains to the probe decrease under UV light. Dynamic light scattering and UV-vis spectroscopic measurements further confirm the shape-reconstruction findings and reveal three discrete conformations of lysozyme. The results clearly demonstrate that visible light causes a greater degree of lysozyme swelling than UV light, thus allowing for the protein conformation to be controlled with light.

The interaction of a light-responsive azobenzene surfactant with bovine serum albumin (BSA) has been investigated as a means to examine photoreversible changes in protein secondary structure. The cationic azobenzene surfactant undergoes a reversible photoisomeriztion upon exposure to the appropriate wavelength of light, with the visible-light (trans) form being more hydrophobic and, thus, inducing a greater degree of protein unfolding than the UV-light (cis) form. Fourier transform infrared (FT-IR) spectroscopy is used to provide quantitative information on the secondary structure elements in the protein (alpha-helices, beta-strands, beta-turns, and unordered domains). Comparing the secondary structure changes induced by light illumination in the presence of the photoresponsive surfactant with previous measurements of the tertiary structure of BSA obtained from small-angle neutron scattering (SANS) allows the three discrete conformation changes in BSA to be fully characterized. At low surfactant concentrations, an alpha-helix --> beta-structure rearrangement is observed as the tertiary structure of BSA changes from a heart-shaped to a distorted heart-shaped conformation. Intermediate surfactant concentrations lead to a dramatic decrease in the alpha-helix fraction in favor of unordered structures, which is accompanied by an unfolding of the C-terminal portion of the protein as evidenced from SANS. Further increases in photosurfactant concentration lead to a beta --> unordered transition with the protein adopting a highly elongated conformation in solution. Each of these protein conformational changes can be precisely and reversibly controlled with light illumination, as revealed through FT-IR spectra collected during repeated visible-light <--> UV-light cycles.