Nucleic acid aptamers selected for thrombin binding have previously been shown to possess anticoagulant activity, however problems with rapid renal clearance and short circulation half-life prevented translation to clinical usefulness. Here, we describe a family of self-folding, functional RNA origami molecules bearing multiple thrombin-binding RNA aptamers and showing significantly improved anticoagulant activity. These constructs may overcome earlier problems preventing clinical use of nucleic acid anticoagulants. RNA origami structures were designed in silico and produced by in vitro transcription from DNA templates. Incorporation of 2'-fluoro-modified C-and U-nucleotides was shown to increase nuclease resistance and stability during long-term storage. We demonstrate specific binding to human thrombin as well as high stability in the presence of RNase A and in human plasma, comparatively more stable than DNA. The RNA origami constructs show anticoagulant activity seven-fold greater than free aptamer and higher than previous DNA weave tiles decorated with DNA aptamers. Anticoagulation activity was maintained after at least three months of storage in buffer at 4°C. Additionally, inhibition of thrombin is shown to be reversed by addition of single-stranded DNA antidotes. This project paves the way for development of RNA origami for potential therapeutic applications especially as a safer surgical anticoagulant.
BackgroundSynucleinopathy is any of a group of age-related neurodegenerative disorders including Parkinson's disease, multiple system atrophy, and dementia with Lewy Bodies, which is characterized by α-synuclein inclusions and parkinsonian motor deficits affecting millions of patients worldwide. But there is no cure at present for synucleinopathy. Rapamycin has been shown to be neuroprotective in several in vitro and in vivo synucleinopathy models. However, there are no reports on the long-term effects of RAPA on motor function or measures of neurodegeneration in models of synucleinopathy.MethodsWe determined whether long-term feeding a rapamycin diet (14 ppm in diet; 2.25 mg/kg body weight/day) improves motor function in neuronal A53T α-synuclein transgenic mice (TG) and explored underlying mechanisms using a variety of behavioral and biochemical approaches.ResultsAfter 24 weeks of treatment, rapamycin improved performance on the forepaw stepping adjustment test, accelerating rotarod and pole test. Rapamycin did not alter A53T α-synuclein content. There was no effect of rapamycin treatment on midbrain or striatal monoamines or their metabolites. Proteins adducted to the lipid peroxidation product 4-hydroxynonenal were decreased in brain regions of both wild-type and TG mice treated with rapamycin. Reduced levels of the presynaptic marker synaptophysin were found in several brain regions of TG mice. Rapamycin attenuated the loss of synaptophysin protein in the affected brain regions. Rapamycin also attenuated the loss of synaptophysin protein and prevented the decrease of neurite length in SH-SY5Y cells treated with 4-hydroxynonenal.ConclusionTaken together, these data suggest that rapamycin, an FDA approved drug, may prove useful in the treatment of synucleinopathy.
Edited by Norma AllewellThe transforming growth factor  isoforms, TGF-1, -2, and -3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF- pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-s in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF- monomer, lacking the heel helix, a structural motif essential for binding the TGF- type I receptor (TRI) but dispensable for binding the other receptor required for TGF- signaling, the TGF- type II receptor (TRII), as an alternative therapeutic modality for blocking TGF- signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF- monomers and bound TRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF- signaling with a K i of 20 -70 nM. Investigation of the mechanism showed that the high affinity of the engineered monomer for TRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TRI, enabled it to bind endogenous TRII but prevented it from binding and recruiting TRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF- signaling and may inform similar modifications of other TGF- family members.The transforming growth factor  isoforms, TGF-1, -2, and -3, are small secreted signaling proteins. Their overall structures are similar and consist of two cystine-knotted monomers tethered together by a single inter-chain disulfide bond (1). They coordinate wound healing, modulate immune cell function, maintain the extracellular matrix, and regulate epithelial and endothelial cell growth and differentiation (2). The TGF-s are synthesized as pre-pro-proteins, and after maturation, secretion, and release from their pro-domains (3), the mature homodimeric growth factors (GFs) 3 bind and bring together two single-pass transmembrane receptors, known as TRI and TRII, to form the signaling-competent TRI 2 -TRII 2 heterotetramer (4, 5). TGF- GFs assemble TRI 2 -TRII 2 heterotetramer in a sequential manner, first by binding TRII followed by recruitment of TRI (6, 7). The stepwise assembly of TRII and TRI into a heterotetramer is driven by binding of TRI to a composite TGF-/TRII interface (Fig. 1A) (8, 9).The disruption or dysregulation of the TGF- pathway is responsible for several human diseases. These include connec-
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