Acceleration of the Deamination of Cytosine through Photo-Crosslinking.

Herein, we report the major factor for deamination reaction rate acceleration, i.e., hydrophilicity, by using various 5-substituted target cytosines and by carrying out deamination at high temperatures. Through substitution of the groups at the 5'-position of the cytosine, the effect of hydrophilicity was understood. It was then used to compare the various modifications of the photo-cross-linkable moiety as well as the effect of the counter base of the cytosine to edit both DNA and RNA. Furthermore, we were able to achieve cytosine deamination at 37 °C with a half-life in the order of a few hours.

Bioprinting of hair follicle germs for hair regenerative medicine.

Hair regenerative medicine is a promising approach to treat hair loss. The replication of in vivo tissue configurations and microenvironments, such as hair follicle germs, has been studied to prepare tissue grafts for hair regenerative medicine. However, such approaches should be scalable, because a single patient with alopecia requires thousands of tissue grafts. In this paper, we propose an approach for the scalable and automated preparation of highly hair-inductive tissue grafts using a bioprinter. Two collagen droplets (2 µL each) containing mesenchymal and epithelial cells were placed adjacent to each other to fabricate hair-follicle-germ-like grafts. During three days of culture, the pairs of microgel beads were spontaneously contracted by cell traction forces, whereas the two cell types remained separated, where the densities of the cells and collagen were enriched more than 10 times. This approach allowed us to fabricate submillimeter objects printed with millimeter-order accuracy, facilitating scalable and automated tissue graft preparation. Because of mesenchymal-epithelial interactions, hair microgels (HMGs, i.e., collagen- and cell-enriched microgels) efficiently regenerate hair follicles and shafts when transplanted into the back skin of mice. However, the generated hair shafts mostly remain under the skin. Therefore, we printed microgel beads onto surgical suture guides arrayed on a stage. The microgel beads were contracted along with the suture guides in culture prior to transplantation. The guide-inserted HMGs significantly improved hair-shaft sprouting through the skin, owing to the control of the orientation of the HMGs transplanted into the skin. This approach is a promising strategy to advance hair regenerative medicine. STATEMENT OF SIGNIFICANCE: This study proposes an approach for the scalable and automated preparation of highly hair-inductive grafts using a bioprinter. Two collagen droplets containing mesenchymal and epithelial cells were placed adjacently. Cell traction forces caused the pairs of microgel beads to spontaneously contract in culture. Because of mesenchymal-epithelial interactions, hair microgels (HMGs) efficiently regenerated hair follicles on the back skin of mice. However, the generated hair shafts remained mostly beneath the skin. Therefore, we printed microgel beads onto surgical suture guides arrayed on a stage. The guide-inserted HMGs significantly improved hair-shaft sprouting through the skin owing to the control of the orientation of the HMGs in the skin. This approach represents a promising strategy for advancing hair regenerative medicine.

Ultra-acceleration of Photochemical Cytosine Deamination by Using a 5'-Phosphate-Substituted Oligodeoxyribonucleotide Probe Containing a 3-Cyanovinylcarbazole Nucleotide at Its 5'-End.

Genes are the blueprints for the architectures of living organisms, providing the backbone of the information required for formation of proteins. Changes in genes lead to disorders, and these disorders could be rectified by reversing the mutations that caused them. Photochemical methods currently in use for site-directed mutagenesis employ the photoactive 3-cyanovinylcarbazole (CNV K) nucleotide incorporated in the oligodeoxyribonucleotide (ODN) backbone. The major drawback of this method, the requirement for high temperature, has been addressed, and deamination has previously been achieved at 37 °C but with low efficiency. Here, efficient deamination has been accomplished under physiological conditions by using a short complementary photoactive ODN with a 5'-phosphate group in the -1 position with respect to the target cytosine. It is hypothesized that the free phosphate group affects the microenvironment around the target cytosine by activating the incoming nucleophile through hydrogen bonding with the water molecule, thus facilitating nucleophilic attack on the cytosine C-4 carbon. The degree of deamination observed in this technique is high and the effect of the phosphate group is to accelerate the deamination reaction.