Endosomal Escape of Peptide-Photosensitizer Conjugates Is Affected by Amino Acid Sequences near the Photosensitizer.

Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of peptides and proteins, but CPP fusion peptides and proteins are often transported by endocytosis and trapped in endosomes. Photochemical internalization (PCI) is a method for the endosomal escape of the trapped peptide or protein and release into the cytosol using light and photosensitizers. In PCI, endosomal membranes are thought to be destabilized by singlet oxygen (1O2) photogenerated from photosensitizers localized in endosomes. We previously developed CPP-cargo-photosensitizer (PS) conjugates able to photodependently enter the cytosol via the PCI mechanism. For example, TatU1A-PS (a covalent complex of Tat [CPP], U1A RNA-binding protein [cargo], and PS) can photodependently deliver RNAs into the cytosol, and TatBim-PS (a covalent complex of Tat, Bim [cargo], and PS) can photoinduce apoptosis in mammalian cells. However, for many newly created conjugates, the induction of PCI has been insufficient. We hypothesized that the amino acid linker sequence (XX) adjacent to the photosensitizer is an important determinant of PCI efficiency. In this study, using CPP-cargo-XX-PS platforms, we examined the relationship between PCI efficiency and the linker amino acid sequence near the photosensitizer. We found that hydrophobic FF and LL linkers enhanced the PCI efficiencies of both TatBim-XX-PS and TatU1A-XX-PS. The effectiveness of the linker depended, in part, on both the cargo moiety and the photosensitizer. These results may guide the design of CPP-cargo-PS conjugates conferring broad target functions for PCI and photodynamic therapy.

In-stem molecular beacon targeted to a 5'-region of tRNA inclusive of the D arm that detects mature tRNA with high sensitivity.

Cellular functions are regulated by the up- and down-regulation and localization of RNA molecules. Therefore, many RNA detection methods have been developed to analyze RNA levels and localization. Molecular beacon (MB) is one of the major methods for quantitative RNA detection and analysis of RNA localization. Most oligonucleotide-based probes, including MB, are designed to target a long flexible region on the target RNA molecule, e.g., a single-stranded region. Recently, analyses of tRNA localization and levels became important, as it has been shown that environmental stresses and chemical reagents induce nuclear accumulation of tRNA and tRNA degradation in mammalian cells. However, tRNA is highly structured and does not harbor any long flexible regions. Hence, only a few methods are currently available for detecting tRNA. In the present study, we attempted to detect elongator tRNAMet (eMet) and initiator tRNAMet (iMet) by using an in-stem molecular beacon (ISMB), characterized by more effective quenching and significantly higher sensitivity than those of conventional MB. We found that ISMB1 targeted a 5'- region that includes the D arm of tRNA and that it detected eMet and iMet transcripts as well as mature eMet with high sensitivity. Moreover, the analysis revealed that the formation of the ISMB/tRNA transcript complex required more time than the formation of an ISMB/unstructured short RNA complex. These results suggest that ISMB-based tRNA detection can be a useful tool for various biological and medical studies.

High performance diffuse reflectance circular dichroism spectrophotometer.

A new dual-purpose transmittance circular dichroism (CD) and diffuse reflectance CD (DRCD) universal chiroptical spectrophotometer (UCS-3, J-800KCMFII) was successfully developed to have the capability of measuring DRCD spectra down to 190 nm with high efficiency, based on UCS-2: J-800KCMF. Optical components newly used in UCS-3 are the integrating sphere of optimum size and material to achieve high performance particularly in the shorter wavelength region, a baffle installed to reduce the first specular reflection signals, and a condenser lens to increase light intensity per area for small samples. As a result, UCS-3 has become a very powerful instrument to measure DRCD spectra of powdered samples in situ, with approximately 20 times sensitivity of existing UCS-2. We could show that to achieve similar quality DRCD spectra, only 50 mug of (S)-(+)-1,1(')-binaphthyl-2,2(')-diyl hydrogen phosphate is required on UCS-3, as compared with 1.12 mg for UCS-2.