Formation of Direction-Controllable Pseudorotaxane and Catenane Using Chemically Cyclized Oligodeoxynucleotides and Their Noncovalent RNA Labeling.

The formation of interlocked structures, such as rotaxane and catenane, enables noncovalent conjugations. We previously confirmed that the chemically cyclized pseudorotaxane-forming oligodeoxynucleotides (prfODNs) with double-tailed parts formed a pseudorotaxane structure with the target DNA and RNA via the slipping process. Here, we report the one-step synthesis of cyclized prfODNs from alkyne-modified ODNs, after which we investigated the properties and mechanism of the slipping process and performed noncovalent RNA labeling with prfODNs. Additionally, the catenane structure was formed by the combination of pseudorotaxane formation with a 5'-end-phosphorylated RNA and enzymatic ligation. The newly synthesized prfODN represents a new tool for achieving the noncovalent conjugation of various functional moieties to RNAs.

Influence of periostin on the development of fibrocartilage layers of anterior cruciate ligament insertion.

BACKGROUND: Periostin (Postn) is thought to play a role in the formation of anterior cruciate ligament (ACL) insertion. However, the influence of Postn on the development of ACL insertion requires further understanding. This study aimed to clarify the influence of Postn on the development of fibrocartilage layers of ACL insertion. HYPOTHESIS: We hypothesized that Postn would influence the development of fibrocartilage layers of ACL insertion. MATERIALS AND METHODS: C57BL/6N wild-type (Postn+/+; n=54) and Postn knockout (Postn-/-; n=54) mice were used in this study. Six animals were euthanized at 1 d and 1, 2, 3, 4, 6, 8, 10, and 12 weeks of age in each group. The chondrocyte number, proliferation, apoptosis, safranin O-stained glycosaminoglycan (GAG) area, type II collagen staining area, tidemark length, and insertion width were evaluated. RESULTS: Chondrocyte proliferation was high up to 2 weeks in Postn+/+, while low at age 1 d; it was, especially lower in Postn-/- than in Postn+/+ at age 1 d and 1 week. Chondrocyte apoptosis was high up to age 8 weeks in Postn+/+ and at 6 weeks in Postn-/-; it was especially higher in Postn-/- than in Postn+/+ at age 1 week. The GAG stained area was thickest for age 1 d to 4 weeks in Postn+/+ and for age 2 to 6 weeks in Postn-/-. The type II collagen staining area in Postn+/+ was thicker than that in Postn-/- at age 6 and 8 weeks. The tidemark length in Postn+/+ was longer than that in Postn-/- from age 8 to 12 weeks. The insertion width in Postn+/+ was longer than that in Postn-/- from age 1 to 3 weeks. DISCUSSION: Postn decreased cell proliferation in the early postnatal phase and influenced the development of the fibrocartilage layer extracellular matrix of ACL insertion in mice. Postn may contribute to the development of methods for regeneration of the ACL insertion. LEVEL OF EVIDENCE: V; controlled laboratory study.

Amphipathic helical peptide-based fluorogenic probes for a marker-free analysis of exosomes based on membrane-curvature sensing.

With increasing knowledge about the diverse roles of exosomes in the biological process, much attention has been paid to develop analytical methods for detection and quantification of exosomes. Immunoassays based on the recognition of exosomal protein markers by antibodies were widely used. However, considering that exosomal protein composition varies with the cell type, the protein markers should be carefully selected for a sensitive and selective analysis of target exosomes. Herein, we developed a new class of exosome-binding fluorogenic probes based on membrane curvature (MC) sensing of amphipathic helical (AH) peptides for exosome analysis without the need to use protein markers on the exosomal membranes. The C-terminal region of apolipoprotein A-I labeled with Nile red (ApoC-NR) exhibited a significant fluorescence enhancement upon selective binding to the highly curved membranes of synthetic vesicles. Circular dichroism (CD) measurements involving 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-2-dioleoyl-sn-glycerol (DOG) vesicles suggested that ApoC-NR recognizes the lipid packing defects in the surface of highly curved membranes via the hydrophobic insertion of the α-helix structure of the ApoC unit. ApoC-NR exhibited a stronger binding affinity for exosome-sized vesicles and a higher MC selectivity compared to all other previously reported peptide probes. ApoC-NR can be used in a simple and rapid "mix and read" analysis of various kinds of exosomes derived from different cell types (limit of detection: -105 particles/µL) without being influenced by the variation in the expression of the surface proteins of the exosomes, which stands in sharp contrast to immunoassays.