Dupont, D. M. | Search bioRxiv

bioRxiv2020

Enhanced Anticoagulation Activity of Functional RNA Origami

Anticoagulants are commonly utilized during surgeries and to treat thrombotic diseases like stroke and deep vein thrombosis. However, conventional anticoagulants have serious side-effects, narrow therapeutic windows, and lack safe reversal agents (antidotes). Here, an alternative RNA origami displaying RNA aptamers as target-specific anticoagulant, is described. Improved design and construction techniques for self-folding, single-molecule RNA origami as a platform for displaying pre-selected RNA aptamers with precise orientational and spatial control, are reported. Nuclease resistance is added using 2-fluoro-modified pyrimidines during in vitro transcription. When four aptamers are displayed on the RNA origami platform, the measured thrombin inhibition and anticoagulation activity is higher than observed for free aptamers, ssRNA-linked RNA aptamers, and RNA origami displaying fewer aptamers. Importantly, thrombin inhibition is immediately switched off by addition of specific reversal agents. Results for ssDNA and ssPNA (peptide nucleic acid) antidotes show restoration of 75% and 95% coagulation activity, respectively. To demonstrate potential for practical, long-term storage for clinical use, RNA origami was freeze-dried, and stored at room temperature. Freshly produced and freeze-dried RNA show identical levels of activity in coagulation assays. Compared to current commercial intravenous anticoagulants, RNA origami-based molecules show promise as safer alternatives with rapid activity switching for future therapeutic applications.

bioengineering↗

bioRxiv2019

Nanopore sequencing of full-length circRNAs in human and mouse brains reveals circRNA-specific exon usage and intron retention

Circular RNA (circRNA) is a poorly understood class of non-coding RNAs, some of which have been shown to be functional important for cell proliferation and development. CircRNAs mainly derive from back splicing events of coding mRNAs, making it difficult to distinguish the internal exon composition of circRNA from the linearly spliced mRNA. To examine the global exon composition of circRNAs, we performed long-read sequencing of single molecules using nanopore technology for human and mouse brain-derived RNA. By applying an optimized circRNA enrichment protocol prior to sequencing, we were able to detect 7,834 and 10,975 circRNAs in human and mouse brain, respectively, of which 2,945 and 7,052 are not currently found in circBase. Alternative splicing was more prevalent in circRNAs than in linear spliced transcripts, and notably >200 not previously annotated exons were used in circRNAs. This suggests that properties associated with circRNA- specific features, e.g. the unusual back-splicing step during biogenesis, increased stability and /or their lack of translation, alter the general exon usage at steady state. We conclude that the nanopore sequencing technology provides a fast and reliable method to map the specific exon composition of circRNA.

genetics↗