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.

Summary Bacterial CRISPR systems provide acquired immunity against invading nucleic acids by activating RNA-programmable RNases and DNases. Cas13a and Cas12a enzymes bound to CRISPR RNA (crRNA) recognize specific nucleic acid targets, initiating cleavage of the targets as well as non-target ( trans ) nucleic acids. Here, we examine the kinetics of single-turnover target and multi-turnover trans -nuclease activities of both enzymes. High-turnover, non-specific Cas13a trans -RNase activity is coupled to rapid binding of target RNA. By contrast, low-turnover Cas12a trans -nuclease activity is coupled to relatively slow cleavage of target DNA, selective for DNA over RNA, indifferent to base identity, and preferential for single-stranded substrates. Combining multiple crRNA increases detection sensitivity of targets, an approach we use to quantify pathogen DNA in samples from patients suspected of Buruli ulcer disease. Results reveal that these enzymes are kinetically adapted to play distinct roles in bacterial adaptive immunity and show how kinetic analysis can be applied to CRISPR-based diagnostics.

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.