This research was funded in part by: a National Institutes of Health (NIH)/ National Institute of Child Health and Human Development (NICHD) grant (R00 HD080742) and Washington University School of Medicine start-up funds to R.K., an NIH/NICHD grant (RO1 HD-07857) to B.W.O.M., and a NIH/NICHD grant (R01 HD-042311) to J.P.L. The authors declare no conflicts of interests.
T4 polynucleotide kinase is widely used for 5′-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3′-phosphorylation. Here, we report the in vitro selection of deoxyribozymes (DNA enzymes) capable of DNA oligonucleotide 3′-phosphorylation, using a 5′-triphosphorylated RNA transcript (pppRNA) as the phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3′-phosphorylated DNA substrate terminus by 2-methylimidazole activation of the 3′-phosphate (forming 3′-MeImp) and subsequent splint ligation with a 5′-amino DNA oligonucleotide. Competing and precedented DNA-catalyzed reactions were DNA phosphodiester hydrolysis or deglycosylation, each also leading to a 3′-phosphate but at a different nucleotide position within the DNA substrate. One oligonucleotide 3′-kinase deoxyribozyme, obtained from an N40 random pool and named 3′Kin1, can 3′-phosphorylate nearly any DNA oligonucleotide substrate for which the 3′-terminus has the sequence motif 5′-NKR-3′, where N denotes any oligonucleotide sequence, K = T or G, and R = A or G. These results establish the viabilty of in vitro selection for identifying DNA enzymes that 3′-phosphorylate DNA oligonucleotides.
Autophagy is a ubiquitous cell recycling pathway that delivers cytoplasmic constituents to the lysosome and is essential for normal cellular function. Autophagic activity is up‐regulated under physiological conditions as well as stressful conditions such as nutrient deprivation, oxidative stress, hypoxia, inflammation, and infection. Thus, it is essential to regard the functional importance of the pathway and its components in a given tissue context. Here we review what is known about the involvement of autophagy process during physiological processes in the female reproductive tract and in pregnancy from preimplantation to oocyte function to placental development, parturition, and postpartum remodeling of the uterus; as well as in pathological and adverse events during these processes.
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