U-to-C RNA editing by synthetic PPR-DYW proteins in bacteria and human culture cells.

Programmable RNA editing offers significant therapeutic potential for a wide range of genetic diseases. Currently, several deaminase enzymes, including ADAR and APOBEC, can perform programmable adenosine-to-inosine or cytidine-to-uridine RNA correction. However, enzymes to perform guanosine-to-adenosine and uridine-to-cytidine (U-to-C) editing are still lacking to complete the set of transition reactions. It is believed that the DYW:KP proteins, specific to seedless plants, catalyze the U-to-C reactions in mitochondria and chloroplasts. In this study, we designed seven DYW:KP domains based on consensus sequences and fused them to a designer RNA-binding pentatricopeptide repeat (PPR) domain. We show that three of these PPR-DYW:KP proteins edit targeted uridine to cytidine in bacteria and human cells. In addition, we show that these proteins have a 5' but not apparent 3' preference for neighboring nucleotides. Our results establish the DYW:KP aminase domain as a potential candidate for the development of a U-to-C editing tool in human cells.

Contrast enhancement of nanomaterials using phase plate STEM.

Visualizing materials composed of light elements is difficult, and the development of an imaging method that enhances the phase contrast of such materials has been of much interest. In this study, we demonstrate phase-plate scanning transmission electron microscopy (P-STEM), which we developed recently, and its application to nanomaterials. An amorphous carbon film with a small hole in its center was used to control the phase of incident electron waves, and the phase-contrast transfer function (PCTF) was modified from sine-type to cosine-type. The modification of the PCTF enhances image contrast with a spatial frequency below 1 nm-1. The PCTF for P-STEM with a spatial frequency below 1 nm-1 is about three times stronger than that of bright field STEM. The ratio obtained using power spectra is consistent with the result obtained from images of quantum dots. The image contrast of biological materials was also enhanced by P-STEM.

Phase-contrast scanning transmission electron microscopy.

This report introduces the first results obtained using phase-contrast scanning transmission electron microscopy (P-STEM). A carbon-film phase plate (PP) with a small center hole is placed in the condenser aperture plane so that a phase shift is introduced in the incident electron waves except those passing through the center hole. A cosine-type phase-contrast transfer function emerges when the phase-shifted scattered waves interfere with the non-phase-shifted unscattered waves, which passed through the center hole before incidence onto the specimen. The phase contrast resulting in P-STEM is optically identical to that in phase-contrast transmission electron microscopy that is used to provide high contrast for weak phase objects. Therefore, the use of PPs can enhance the phase contrast of the STEM images of specimens in principle. The phase shift resulting from the PP, whose thickness corresponds to a phase shift of π, has been confirmed using interference fringes displayed in the Ronchigram of a silicon single crystal specimen. The interference fringes were found to abruptly shift at the edge of the PP hole by π.