Because RNA interference (RNAi) can be applied to any gene, this technique has been widely used for studying gene functions. In addition, many researchers are attempting to use RNAi technology in RNAi-based therapies. However, several challenging and controversial issues have arisen during the widespread application of RNAi including target gene specificity, target cell specificity, and spatiotemporal control of gene silencing. To address these issues, several groups have utilized photochemistry to control the RNA release, both spatially and temporally. In this Account, we focus on recent studies using photocleavable protecting groups, photosensitizers, Hand gold nanoparticles for photoinduced RNAi. In 2005 the first report of photoinduced RNAi used a caged short interfering RNA (siRNA), an siRNA carrying a photocleavable protecting group. Caging groups block the bioactivities of target molecules, but allow for complete recovery of these functions via photoactivation. However, some RNAi activity can occur in these caged siRNAs, so it will be necessary to decrease this "leakage" and raise the RNAi activity restored after irradiation. This technique also uses UV light around 350 nm, which is cytotoxic, but in the near future we expect that it will be possible to use visible and near-infrared light We also examine the application of photochemical internalization (PCI) to RNAi technology, which involves a combination of photosensitizers and light. Instead of inducing RNAi using light, the strategy behind this method was to enhance RNAi using RNA carriers. Many wellknown RNA carriers deliver siRNAs into cells by endocytosis. The siRNAs are trapped in endocytic vesicles and have to be released into the cytoplasm in order to express their activity. To achieve the endosomal escape of siRNAs, PCI technology employed photosensitizers to generate light-dependent reactive oxygen species (ROS) that disrupted the endocytic vesicles. In most studies, RNAi-mediated knockdown of the target gene was detected even without PCI. Recently, a polymer capable of trapping the siRNA in endocytic vesicles controlled RNAi almost entirely by light. CLIP-RNAi uses photosensitizing carrier proteins that can be activated over a wide range of visible light wavelengths. With this method RNA carrier/siRNA complexes are completely trapped within endosomes, and RNAi is controlled strictly by light. Such precise, light-dependent control will open up new possibilities for cellular and molecular biology and therapy. Most recently, gold nanoparticles (AuNPs) conjugated to siRNA have provided temporal and spatial control of RNAi. The light-dependent melting of AuNPs accompanied by a shape transformation induces the release of thiolated siRNAs from AuNPs. In this method, the unique optical properties of the AuNP enable deep penetration of the excitation light into tissues at nearinfrared wavelengths. The development of photoinduced RNAi technology will lead to novel insights into gene functions and selective drug delivery, and many other scientific fields...

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Mutations of human spastin, an AAA (ATPases associated with diverse cellular activity) family protein, cause an autosomal dominant form of hereditary spastic paraplegia, which is characterized by weakness, spasticity and loss of the vibratory sense in the lower limbs. Recently, it has been reported that spastin displays microtubule-severing activity. We also previously reported that Caenorhabditis elegans spastin homologue SPAS-1 displays microtubule severing. However, the detailed molecular mechanism of microtubule severing remains unknown. Here, we describe that SPAS-1 forms a stable hexamer in a concentration-dependent manner and that ATPase activity of SPAS-1 is greatly stimulated by microtubules. Furthermore, MTBD (microtubule-binding domain) of SPAS-1 is essential for binding to microtubules. Taken these results together, we propose that MTBD of SPAS-1 plays a critical role in enrichment of SPAS-1 to microtubules, where SPAS-1 is concentrated and able to form a stable hexamer, subsequently its ATPase activity is stimulated. On the other hand, our mutational analyses revealed that the conserved aromatic and basic amino acid residues in the pore region are important for microtubule severing. We also detected the direct interaction of the extremely acidic C-terminal polypeptide of tubulin with SPAS-1. Consequently, we propose that the central pore residues are important for the recognition of substrates.

Many biologists, biochemists, and biophysicists study giant vesicles, which have a diameter of >1 μm, owing to their ease of characterization using standard optical methods. More recently, there has been interest in using giant vesicles as model systems for living cells and for the construction of artificial cells. In fact, there have been a number of reports about functionalizing giant vesicles using membrane-bound pore proteins and encapsulating biochemical reactions. Among the various methods for preparing giant vesicles, the water-in-oil emulsion transfer method is particularly well established. However, the giant vesicles prepared by this method have complex and heterogeneous properties, such as particle size and membrane structure. Here, we demonstrate the characterization of giant vesicles by imaging flow cytometry to provide quantitative and qualitative information about the vesicle products prepared by the water-in-oil emulsion transfer method. Through image-based analyses, several kinds of protocol byproducts, such as oil droplets and vesicles encapsulating no target molecules, were identified and successfully quantified. Further, the optimal agitation conditions for the water-in-oil emulsion transfer method were found from detailed analysis of imaging flow cytometry data. Our results indicate that a sonication-based water-in-oil emulsion transfer method exhibited a higher efficiency in producing giant vesicles, about 10 times or higher than that of vortex and rumble strip-based methods. It is anticipated that these approaches will be useful for fine-tuning giant vesicle production and subsequent applications.

RNA interference (RNAi) is being widely explored as a tool in functional genomics and tissue engineering, and in the therapy of intractable diseases, including cancer and neurodegenerative diseases. Recently, we developed a photoinducible RNAi method using photosensitizing carrier proteins, named CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi). Novel carrier proteins were designed for this study to establish a highly efficient delivery system for small interfering RNA (siRNA) or short hairpin RNA (shRNA) and to demonstrate light-dependent gene silencing. In addition, the results suggested that the dissociation of the siRNA (or shRNA) from carrier proteins in the cytoplasm is a critical event in CLIP-RNAi-mediated gene silencing.