Selection of RNA-Cleaving TNA Enzymes for Cellular Mg2+ Imaging.

Catalytic DNA-based fluorescent sensors have enabled cellular imaging of metal ions such as Mg2+ . However, natural DNA is prone to nuclease-mediated degradation. Here, we report the in vitro selection of threose nucleic acid enzymes (TNAzymes) with RNA endonuclease activities. One such TNAzyme, T17-22, catalyzes a site-specific RNA cleavage reaction with a kcat of 0.017 min-1 and KM of 675 nM. A fluorescent sensor based on T17-22 responds to an increasing concentration of Mg2+ with a limit of detection at 0.35 mM. This TNAzyme-based sensor also allows cellular imaging of Mg2+ . This work presents the first proof-of-concept demonstration of using a TNA catalyst in cellular metal ion imaging.

DNA-catalysed alternative RNA splicing.

We report DNA-catalysed alternative RNA splicing in vitro. Using modular DNA catalysts with RNA endonuclease and RNA ligase activities, we show that DNA can modulate RNA structure and activity. Furthermore, we illustrate that such DNA-catalysed reactions can yield, from a common precursor, different splicing isoforms with distinct functions.

Expression of the P2Y2 receptor in the terminal rectum of fetal rats with anorectal malformation.

OBJECTIVE: The expression and distribution of a subtype of purine receptors (P2Y2) in the terminal rectum of fetal rats with anorectal malformations (ARM) were examined to investigate their possible impact on the development of the enteric nervous system (ENS). METHODS: Pregnant Sprague-Dawley rats were randomly divided into a control group (5 rats) and an experimental group (20 rats). The experimental group was treated with ethylene thiourea (ETU). On gestational day 20, the intrauterine fetal rats were collected from both groups of pregnant rats. Sagittal sections of the pelvic perinea were stained with HE. P2Y2 protein and mRNA expression in the terminal recta of the fetal rats in the control group, the ARM group, and the ETU-treated group that exhibited no malformations (the ETU group) were detected by immunohistochemistry, western blot, and qRT-PCR. RESULTS: The fetal rats in the control group showed normal position of the anal opening, with no malformation. The incidence of ARM was 89.2% for the fetal rats in the experimental group. The immunohistochemistry results showed that P2Y2 was expressed in the cytoplasm of the cells in the terminal rectum submucosa and myenteric plexus of the fetus rats in the control group, the ETU group, and the ARM group. The average integrated optical density (IOD) value for the ARM group was significantly lower than the IOD value for the control and ETU groups (186.48 ± 23.03 vs. 493.18 ± 19.70; 186.48 ± 23.03 vs. 479.48 ± 41.71, P<0.01), while the IOD value for the ETU group was comparable to the control group IOD (493.18 ± 19.70 vs. 479.48 ± 41.71, P = 0.360). The western blot and qRT-PCR results showed that the P2Y2 protein and mRNA expressions were significantly lower in the terminal rectum of the fetal rats in the ARM group than in the control and ETU groups (0.28 ± 0.08 vs. 0.51 ± 0.10, 0.28 ± 0.08 vs. 0.48 ± 0.12; 48.91 ± 12.17 vs. 98.03 ± 15.68, 48.91 ± 12.17 vs. 92.53 ± 10.43; P<0.01), while the P2Y2 protein and mRNA levels in the control group were comparable to the ETU group (0.51 ± 0.10 vs. 0.48 ± 0.12, P = 0.494; 98.03 ± 15.68 vs. 92.53 ± 10.43, P = 0.058). CONCLUSION: P2Y2 may participate in and affect the development of ENS in the terminal rectum of fetal rats with ARM.