Modulation of RNA Splicing by Oligonucleotides: Mechanisms of Action and Therapeutic Implications.

Dysregulation of RNA splicing causes many diseases and disorders. Several therapeutic approaches have been developed to correct aberrant alternative splicing events for the treatment of cancers and hereditary diseases, including gene therapy and redirecting splicing, using small molecules or splice switching oligonucleotides (SSO). Significant advances in the chemistry and pharmacology of nucleic acid have led to the development of clinically approved SSO drugs for the treatment of spinal muscular dystrophy and Duchenne muscular dystrophy (DMD). In this review, we discuss the mechanisms of SSO action with emphasis on "less common" approaches to modulate alternative splicing, including bipartite and bifunctional SSO, oligonucleotide decoys for splice factors and SSO-mediated mRNA degradation via AS-NMD and NGD pathways. We briefly discuss the current progress and future perspectives of SSO therapy for rare and ultrarare diseases.

Mesyl Phosphoramidate Oligonucleotides as Potential Splice-Switching Agents: Impact of Backbone Structure on Activity and Intracellular Localization.

A series of 2'-deoxy and novel 2'-O-methyl and 2'-O-(2-methoxyethyl) (2'-MOE) oligonucleotides with internucleotide methanesulfonyl (mesyl, µ) or 1-butanesulfonyl (busyl, ß) phosphoramidate groups has been synthesized for evaluation as potential splice-switching oligonucleotides. Evaluation of their splice-switching activity in spinal muscular atrophy patient-derived fibroblasts revealed no significant difference in splice-switching efficacy between 2'-MOE mesyl oligonucleotide and the corresponding phosphorothioate (nusinersen). Yet, a survival study with model neonatal mice has shown the antisense 2'-MOE mesyl oligonucleotide to be inferior to nusinersen at the highest dose of 40 mg/kg. A reason for their lower activity in vivo as ascertained by cellular uptake study by fluorescent confocal microscopy in HEK293 cell line could possibly be ascribed to compromised endosomal release and/or nuclear uptake of the 2'-OMe or 2'-MOE µ- and ß-oligonucleotides compared to their phosphorothioate analog.

Differences between male and female university students in sleepiness, weekday sleep loss, and weekend sleep duration.

INTRODUCTION: Women and men experience sleep differently and the difference in intrinsic desire for sleep might underlie some of the observed male-female differences. The objective of this cross-sectional questionnaire study of university students was to determine male-female differences in self-reported sleepiness and sleep-wake patterns. METHODS: Five questionnaires were completed by 1650 students at four Russian universities. RESULTS: Compared to male students, female students reported a lower subjective sleep quality score, had a higher morning sleepability score and lower nighttime and daytime wakeability scores. They more often reported excessive daytime sleepiness and expected to be sleepier at any time of the day with the largest male-female difference around the times of sleep onset and offset. On free days, they reported a longer sleep duration and an earlier sleep onset. Free-weekday difference was larger for sleep duration and smaller for sleep onset. Such male-female differences showed similarity to the differences observed in university and high school students from different countries around the globe. There was no significant male-female difference in weekly averaged sleep duration, weekday sleep duration, hours slept, midpoint of sleep on free days, free-weekday difference in sleep offset, social jetlag, and morningness-eveningness score. Therefore, when studies rely on these self-reports, the most salient male-female differences might not be immediately evident. CONCLUSIONS: It seems that the intrinsic desire for longer sleep duration might contribute to a higher susceptibility of female students to weekday sleep loss. Among these students, negative effects of reduced sleep duration might be more common and more detrimental.

Multifunctional nanostructured drug delivery carriers for cancer therapy: Multimodal imaging and ultrasound-induced drug release.

Development of multimodal systems for therapy and diagnosis of neoplastic diseases is an unmet need in oncology. The possibility of simultaneous diagnostics, monitoring, and therapy of various diseases allows expanding the applicability of modern systems for drug delivery. We have developed hybrid particles based on biocompatible polymers containing magnetic nanoparticles (MNPs), photoacoustic (MNPs), fluorescent (Cy5 or Cy7 dyes), and therapeutic components (doxorubicin). To achieve high loading efficiency of MNP and Dox to nanostructured carriers, we utilized a novel freezing-induced loading technique. To reduce the systemic toxicity of antitumor drugs and increase their therapeutic efficacy, we can use targeted delivery followed by the remote control of drug release using high intensity-focused ultrasound (HIFU). Loading of MNPs allowed performing magnetic targeting of the carriers and enhanced optoacoustic signal after controlled destruction of the shell and release of therapeutics as well as MRI imaging. The raster scanning optoacoustic mesoscopy (PA, RSOM), MRI, and fluorescent tomography (FT) confirmed the ultrasound-induced release of doxorubicin from capsules: in vitro (in tubes and pieces of meat) and in vivo (after delivery to the liver). Disruption of capsules results in a significant increase of doxorubicin and Cy7 fluorescence initially quenched by magnetite nanoparticles that can be used for real-time monitoring of drug release in vivo. In addition, we explicitly studied cytotoxicity, intracellular localization, and biodistribution of these particles. Elaborated drug delivery carriers have a good perspective for simultaneous imaging and focal therapy of different cancer types, including liver cancer.

Multispectral sensing of biological liquids with hollow-core microstructured optical fibres.

The state of the art in optical biosensing is focused on reaching high sensitivity at a single wavelength by using any type of optical resonance. This common strategy, however, disregards the promising possibility of simultaneous measurements of a bioanalyte's refractive index over a broadband spectral domain. Here, we address this issue by introducing the approach of in-fibre multispectral optical sensing (IMOS). The operating principle relies on detecting changes in the transmission of a hollow-core microstructured optical fibre when a bioanalyte is streamed through it via liquid cells. IMOS offers a unique opportunity to measure the refractive index at 42 wavelengths, with a sensitivity up to ~3000 nm per refractive index unit (RIU) and a figure of merit reaching 99 RIU-1 in the visible and near-infra-red spectral ranges. We apply this technique to determine the concentration and refractive index dispersion for bovine serum albumin and show that the accuracy meets clinical needs.

Excimer-FRET Cascade in Dual DNA Probes: Open Access to Large Stokes Shift, Enhanced Acceptor Light up, and Robust RNA Sensing.

The efficacy of fluorescent hybridization assays is often limited by the low signal-to-background ratio of the probes that can be partially overcome by sophisticated signal amplification methods. Deep understanding of the mechanisms of fluorescence quenching and energy transfer in complex DNA probes and the choice of optimal donor/acceptor pairs along with rational design can significantly enhance the performance of DNA probes. Here, we proposed and studied novel Förster resonance energy transfer (FRET) dual DNA probes with the excimer-forming pyrene pair as a donor and sulfo-Cy3 dye as an acceptor, which demonstrated remarkable 75-fold enhancement of sulfo-Cy3 fluorescence upon target capturing. Stokes shift up to 220 nm minimizes fluorescence crosstalk. Time-correlated single-photon counting revealed two excited states of pyrene excimer wherein only one is directly involved in the resonance energy transfer to sulfo-Cy3. Optimized DNA probes demonstrated high sensitivity with excellent signal-to-background ratio, which were applied for visualization of 18S rRNA by fluorescent in situ hybridization in HEK-293T cells.

eIF4G2 balances its own mRNA translation via a PCBP2-based feedback loop.

Poly(rC)-binding protein 2 (PCBP2, hnRNP E2) is one of the most abundant RNA-binding proteins in mammalian cells. In humans, it exists in seven isoforms, which are assumed to play similar roles in cells. The protein is shown to bind 3'-untranslated regions (3'-UTRs) of many mRNAs and regulate their translation and/or stability, but nothing is known about the functional consequences of PCBP2 binding to 5'-UTRs. Here we show that the PCBP2 isoform f interacts with the 5'-UTRs of mRNAs encoding eIF4G2 (a translation initiation factor with a yet unknown mechanism of action, also known as DAP5) and Cyclin I, and inhibits their translation in vitro and in cultured cells, while the PCBP2 isoform e only affects Cyclin I translation. Furthermore, eIF4G2 participates in a cap-dependent translation of the PCBP2 mRNA. Thus, PCBP2 and eIF4G2 seem to regulate one another's expression via a novel type of feedback loop formed by the translation initiation factor and the RNA-binding protein.

NMR assignments of the WBSCR27 protein related to Williams-Beuren syndrome.

Williams-Beuren syndrome is a genetic disorder characterized by physiological and mental abnormalities, and is caused by hemizygous deletion of several genes in chromosome 7. One of the removed genes encodes the WBSCR27 protein. Bioinformatic analysis of the sequence of WBSCR27 indicates that it belongs to the family of SAM-dependent methyltransferases. However, exact cellular functions of this protein or phenotypic consequences of its deficiency are still unknown. Here we report nearly complete 1H, 15N, and 13C chemical shifts assignments of the 26 kDa WBSCR27 protein from Mus musculus in complex with the cofactor S-adenosyl-L-methionine (SAM). Analysis of the assigned chemical shifts allowed us to characterize the protein's secondary structure and backbone dynamics. The topology of the protein's fold confirms the assumption that the WBSCR27 protein belongs to the family of class I methyltransferases.

Neurophysiological markers of high anxiety level in man during the process of preparing for a visual recognition.

By means of EEG analysis the functional state of subjects with high and low levels of anxiety was studied in different periods preceding a cognitive task - a visual expression recognition. Several conditions were investigated: background/eyes closed; background/eyes opened; listening the instruction for the cognitive task; operative rest (time lapse between listening the instruction and the beginning of the task), as well as short intervals immediately preceding the exposition of target stimuli (stage of preparation) - pairs of faces pictures with identical or different emotional expressions. At all these pre-task stages high-anxiety subjects exhibited much lower amplitude values in alpha and theta bands (as compared with low-anxiety subjects). The most prominent differences were revealed in the phases of instruction listening and operative rest. These data could provide more precise electrophysiological markers of anxiety level in conditions preceeding cognitive task performance.

Synthesis and biological evaluation of novel mono- and bivalent ASGP-R-targeted drug-conjugates.

Asialoglycoprotein receptor (ASGP-R) is a promising biological target for drug delivery into hepatoma cells. Nevertheless, there are only few examples of small-molecule conjugates of ASGP-R selective ligand equipped by a therapeutic agent for the treatment of hepatocellular carcinoma (HCC). In the present work, we describe a convenient and versatile synthetic approach to novel mono- and multivalent drug-conjugates containing N-acetyl-2-deoxy-2-aminogalactopyranose and anticancer drug - paclitaxel (PTX). Several molecules have demonstrated high affinity towards ASGP-R and good stability under physiological conditions, significant in vitro anticancer activity comparable to PTX, as well as good internalization via ASGP-R-mediated endocytosis. Therefore, the conjugates with the highest potency can be regarded as a promising therapeutic option against HCC.

N6-Methylated Adenosine in RNA: From Bacteria to Humans.

N6-methyladenosine (m(6)A) is ubiquitously present in the RNA of living organisms from Escherichia coli to humans. Methyltransferases that catalyze adenosine methylation are drastically different in specificity from modification of single residues in bacterial ribosomal or transfer RNA to modification of thousands of residues spread among eukaryotic mRNA. Interactions that are formed by m(6)A residues range from RNA-RNA tertiary contacts to RNA-protein recognition. Consequences of the modification loss might vary from nearly negligible to complete reprogramming of regulatory pathways and lethality. In this review, we summarized current knowledge on enzymes that introduce m(6)A modification, ways to detect m(6)A presence in RNA and the functional role of this modification everywhere it is present, from bacteria to humans.

Impact of methylations of m2G966/m5C967 in 16S rRNA on bacterial fitness and translation initiation.

The functional centers of the ribosome in all organisms contain ribosomal RNA (rRNA) modifications, which are introduced by specialized enzymes and come at an energy cost for the cell. Surprisingly, none of the modifications tested so far was essential for growth and hence the functional role of modifications is largely unknown. Here, we show that the methyl groups of nucleosides m(2)G966 and m(5)C967 of 16S rRNA in Escherichia coli are important for bacterial fitness. In vitro analysis of all phases of translation suggests that the m(2)G966/m(5)C967 modifications are dispensable for elongation, termination and ribosome recycling. Rather, the modifications modulate the early stages of initiation by stabilizing the binding of fMet-tRNA(fMet) to the 30S pre-initiation complex prior to start-codon recognition. We propose that the m(2)G966 and m(5)C967 modifications help shaping the bacterial proteome, most likely by fine-tuning the rates that determine the fate of a given messenger RNA (mRNA) at early checkpoints of mRNA selection.

Properties of small rRNA methyltransferase RsmD: mutational and kinetic study.

Ribosomal RNA modification is accomplished by a variety of enzymes acting on all stages of ribosome assembly. Among rRNA methyltransferases of Escherichia coli, RsmD deserves special attention. Despite its minimalistic domain architecture, it is able to recognize a single target nucleotide G966 of the 16S rRNA. RsmD acts late in the assembly process and is able to modify a completely assembled 30S subunit. Here, we show that it possesses superior binding properties toward the unmodified 30S subunit but is unable to bind a 30S subunit modified at G966. RsmD is unusual in its ability to withstand multiple amino acid substitutions of the active site. Such efficiency of RsmD may be useful to complete the modification of a 30S subunit ahead of the 30S subunit's involvement in translation.

How much can we learn about the function of bacterial rRNA modification by mining large-scale experimental datasets?

Modification of ribosomal RNA is ubiquitous among living organisms. Its functional role is well established for only a limited number of modified nucleotides. There are examples of rRNA modification involvement in the gene expression regulation in the cell. There is a need for large data set analysis in the search for potential functional partners for rRNA modification. In this study, we extracted phylogenetic profile, genome neighbourhood, co-expression and phenotype profile and co-purification data regarding Escherichia coli rRNA modification enzymes from public databases. Results were visualized as graphs using Cytoscape and analysed. Majority linked genes/proteins belong to translation apparatus. Among co-purification partners of rRNA modification enzymes are several candidates for experimental validation. Phylogenetic profiling revealed links of pseudouridine synthetases with RF2, RsmH with translation factors IF2, RF1 and LepA and RlmM with RdgC. Genome neighbourhood connections revealed several putative functionally linked genes, e.g. rlmH with genes coding for cell wall biosynthetic proteins and others. Comparative analysis of expression profiles (Gene Expression Omnibus) revealed two main associations, a group of genes expressed during fast growth and association of rrmJ with heat shock genes. This study might be used as a roadmap for further experimental verification of predicted functional interactions.