RNA origami is a framework for the modular design of nanoscaffolds that can be folded from a single strand of RNA, and used to organize molecular components with nanoscale precision. Design of genetically expressible RNA origami, which must cotranscriptionally fold, requires modeling and design tools that simultaneously consider thermodynamics, folding pathway, sequence constraints, and pseudoknot optimization. Here, we describe RNA Origami Automated Design software (ROAD), which builds origami models from a library of structural modules, identifies potential folding barriers, and designs optimized sequences. Using ROAD, we extend the scale and functional diversity of RNA scaffolds, creating 32 designs of up to 2360 nucleotides, five that scaffold two proteins, and seven that scaffold two small molecules at precise distances. Micrographic and chromatographic comparison of optimized and nonoptimized structures validates that our principles for strand routing and sequence design substantially improve yield. By providing efficient design of RNA origami, ROAD may simplify construction of custom RNA scaffolds for nanomedicine and synthetic biology.

Guanine quadruplexes (G4s) are an important structure of nucleic acids (DNA and RNA) with roles in several cellular processes. RNA G4s require specialized unwinding enzymes, of which only two have been previously identified. We describe the results of a simple and specific mass spectrometry guided method used to screen HEK293T cell lysate for G4 binding proteins. From these results, we validated the RNA helicase protein DDX21. DDX21 is an established RNA helicase, but has not yet been validated as a G4 binding protein. Through biochemical techniques, we confirm that DDX21-quadruplex RNA interactions are direct and mediated via a site of interaction at the C-terminus of the protein. Furthermore, through monitoring changes in nuclease sensitivity we show that DDX21 can unwind RNA G4. Finally, as proof of principle, we demonstrate the ability of DDX21 to suppress the expression of a protein with G4s in the 3΄ UTR of its mRNA.

BackgroundBC200 is a long non-coding RNA expressed at high levels in the brain and elevated in a variety of tumour types. BC200 has a hypothesized role in translational regulation; however, to date the functional role of BC200 in both normal and diseased states remains poorly characterized.MethodsDetailed BC200 expression analyses were performed in tumor cell lines, primary and non-tumorigenic cultured breast and lung cells, and a panel of normal human tissues by quantitative real-time PCR and confirmed by northern blot. Subcellular fractionation was performed to assess BC200 distribution and efficient knock-down of BC200 was established using both locked nucleic acid (LNA) GapmeRs and conventional siRNAs. Cell viability following BC200 knockdown and overexpression was assessed by MTT assay and induction of apoptosis was monitored by Annexin V/PI staining and flow cytometry. Cell cycle arrest and synchronization were performed using serum withdrawal as well as the specific inhibitors Lovastatin, Thymidine, RO3306 and Nocodazole. Synchronization was monitored by fluorescent analysis of cellular DNA content by flow cytometryResultsBC200 expression was substantially upregulated in brain and elevated expression was also observed in testes, small intestine and ovary. Expression in cultured tumour cells was dramatically higher than corresponding normal tissue; however, expression in cultured primary cells was similar to that in immortalized and cancer cell lines. BC200 knockdown resulted in a dramatic loss of viability through growth arrest and induction of apoptosis that could be partially rescued by overexpression of wild-type BC200 but not an siRNA-resistant sequence mutant. A substantial decrease in BC200 expression was observed upon cell confluence or serum deprivation, as well as drug induced cell cycle arrest in G1 or G2 but not S- or M-phases. Upon release from cell cycle arrest, BC200 expression was recovered as cells entered S-phase, but did not follow a periodic expression pattern during synchronized progression through the cell cycle. This elevated expression was critical for the survival of proliferating cancerous and non-cancerous cells, but is dispensable upon senescence or cell cycle arrest.ConclusionsBC200 expression is elevated in proliferating cultured cells regardless of origin. In primary cells, expression is dramatically reduced upon cell cycle arrest by confluence, serum deprivation or chemical inhibition. The lethality of BC200 knockdown is restricted to actively proliferating cells, making it a promising therapeutic target for a broad spectrum of cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-017-0679-7) contains supplementary material, which is available to authorized users.

The identification of four-stranded G-quadruplexes (G4s) has highlighted the fact that DNA has additional spatial organisations at its disposal other than double-stranded helices. Recently, it became clear that the formation of G4s is not limited to the traditional G3+NL1G3+NL2G3+NL3G3+ sequence motif. Instead, the G3 triplets can be interrupted by deoxythymidylate (DNA) or uridylate (RNA) where the base forms a bulge that loops out from the G-quadruplex core. Here, we report the first high-resolution X-ray structure of a unique unimolecular DNA G4 with a cytosine bulge. The G4 forms a dimer that is stacked via its 5′-tetrads. Analytical ultracentrifugation, static light scattering and small angle X-ray scattering confirmed that the G4 adapts a predominantly dimeric structure in solution. We provide a comprehensive comparison of previously published G4 structures containing bulges and report a special γ torsion angle range preferentially populated by the G4 core guanylates adjacent to bulges. Since the penalty for introducing bulges appears to be negligible, it should be possible to functionalize G4s by introducing artificial or modified nucleotides at such positions. The presence of the bulge alters the surface of the DNA, providing an opportunity to develop drugs that can specifically target individual G4s.

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