COMRADES determines in vivo RNA structures and interactions.

The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs.This work was supported by Cancer Research UK (C13474/A18583, C6946/A14492) and the Wellcome Trust (104640/Z/14/Z, 092096/Z/10/Z) to E.A.M. O.Z. was supported by the Human Frontier Science Program (HFSP, LT000558/2015), the European Molecular Biology Organization (EMBO, ALTF1622-2014), and the Blavatnik Family Foundation postdoctoral fellowship. G.K. and M.G. were supported by Wellcome Trust grant 207507 and UK Medical Research Council. A.T.L.L. and J.C.M. were supported by core funding from Cancer Research UK (award no. 17197 to JCM). J.C.M was also supported by core funding from EMBL. I.G. and L.W.M. were supported by the Wellcome Trust Senior Fellowship in Basic Biomedical Science to I.G. (207498/Z/17/Z). I.J.M., L.F.G. and J.S.-G. were supported by grants R01GM104475 and R01GM115649 from NIGMS. C.K.K was supported by City University of Hong Kong Projects 9610363 and 7200520, Croucher Foundation Project 9500030 and Hong Kong RGC Projects 9048103 and 9054020. C.-F.Q. was supported by the NSFC Excellent Young Scientist Fund 81522025 and the Newton Advanced Fellowship from the Academy of Medical Sciences, UK

Spiegelzymes® mirror-image hammerhead ribozymes and mirror-image DNAzymes, an alternative to siRNAs and microRNAs to cleave mRNAs in vivo?

With the discovery of small non-coding RNA (ncRNA) molecules as regulators for cellular processes, it became intriguing to develop technologies by which these regulators can be applied in molecular biology and molecular medicine. The application of ncRNAs has significantly increased our knowledge about the regulation and functions of a number of proteins in the cell. It is surprising that similar successes in applying these small ncRNAs in biotechnology and molecular medicine have so far been very limited. The reasons for these observations may lie in the high complexity in which these RNA regulators function in the cells and problems with their delivery, stability and specificity. Recently, we have described mirror-image hammerhead ribozymes and DNAzymes (Spiegelzymes®) which can sequence-specifically hydrolyse mirror-image nucleic acids, such as our mirror-image aptamers (Spiegelmers) discovered earlier. In this paper, we show for the first time that Spiegelzymes are capable of recognising complementary enantiomeric substrates (D-nucleic acids), and that they efficiently hydrolyse them at submillimolar magnesium concentrations and at physiologically relevant conditions. The Spiegelzymes are very stable in human sera, and do not require any protein factors for their function. They have the additional advantages of being non-toxic and non-immunogenic. The Spiegelzymes can be used for RNA silencing and also as therapeutic and diagnostic tools in medicine. We performed extensive three-dimensional molecular modelling experiments with mirror-image hammerhead ribozymes and DNAzymes interacting with D-RNA targets. We propose a model in which L/D-double helix structures can be formed by natural Watson-Crick base pairs, but where the nucleosides of one of the two strands will occur in an anticlinal conformation. Interestingly enough, the duplexes (L-RNA/D-RNA and L-DNA/D-RNA) in these models can show either right- or left-handedness. This is a very new observation, suggesting that molecular symmetry of enantiomeric nucleic acids is broken down

Can spectral power predict subjective sleep quality in healthy individuals?

The aim of this study was to assess the relationship between electroencephalogram (EEG) power spectral density and subjective sleep quality in healthy individuals. The sample was selected from the archival database of the Sleep Center at the Department for Psychiatry and Psychotherapy, Medical Center - University of Freiburg, and consisted of 206 healthy adults aged 19-73 years (85 male, 121 female) who underwent a polysomnographic examination for two consecutive nights. A multivariate analysis of variance (MANOVA) with spectral power variables of different frequency bands as dependent variables and subjective sleep quality, night number, age and gender as independent variables was statistically significant for subjective sleep quality, age and gender, but not for night number. In subsequent separate ANOVAs, higher subjective sleep quality was significantly related to decreased non-rapid eye movement (NREM) stage 2 sigma 2 and rapid eye movement (REM) delta 1; however, the relation between REM delta 1 and sleep quality did not remain significant when REM duration was accounted for. The effect sizes of the correlations between sleep quality and spectral power were small (r = -0.1). In contrast to common assumptions, the amount of variance in subjective sleep quality that can be explained through EEG power spectral density variables is small. This finding indicates that subjective and objective sleep are different constructs, the interrelations of which are not yet well understood

The activity of D-DNAzyme and L-DNAzyme in HeLa cells.

<p><b>(A)</b> HeLa cells were transfected with 1 ug EGFP-plasmid and 25, 50 and 100 nM of D-DNAzyme or L-DNAzyme. Fluorescence measurements were carried out 48 h after the transfection. <b>(B)</b> Quantification by real-time PCR of the L- and D-DNAzyme hydrolytic activities of GFP mRNAs in HeLa cells. After 48 h, total RNA was isolated and a reverse transcription cDNA was done. PCR reactions were performed to assess GFP expression relative to Hprt and Actb genes in a thermocycler Stratagene Mx3005P instrument. All experiments were repeated at least three times. <b>(C)</b> Western blot analysis of GFP gene expression in HeLa cells after transfection with 25, 50 and 100 nM of L-DNAzyme and D-DNAzyme. After 48 h, incubation cells were sonicated in 10 mM Tris HCl, pH 7.5. Proteins were separated and Western blot analysis was carried out.</p

Western Blot Table with Details for Actb, Hprt and Gfp Proteins analysed.

<p>Western Blot Table with Details for Actb, Hprt and Gfp Proteins analysed.</p

Incubation of D- and L-RNA targets with D-and L-DNAzymes.

<p>Lanes 1 and 2: D- and L-RNA targets (0.2 µM) incubated in 50 mM Tris-HCl buffer, pH 7.5, and 10 mM MgCl₂. Lanes 3 and 4 incubation of D- RNA targets (0.2 µM) with D-and L-DNAzyme (2 µM) in the presence of 10 mM MgCl₂, lanes 5 and 6 incubation of L-RNA target (0.2 µM) with D-and L-DNAzyme (2 µM) in the presence of 10 mM MgCl₂. All incubations were carried out for 5 h at 37°C. The arrow shows products of hydrolysis as expected when the RNA target is hydrolyzed by the Spiegelzymes as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086673#pone-0086673-g008" target="_blank">Figure 8</a>. Separation of reaction products was accomplished by 20% polyacrylamide gel electrophoresis and 7 M urea.</p