Photocrosslinking of RNA and PhotoMet‐Containing Amphiphilic α‐Helical Peptides

Discovering RNA–protein interactions : A library of photoMet‐containing peptides was synthesized by using an Arg‐ and Leu‐rich α‐helical amphiphilic peptide. Irradiation of mixtures of these peptides and Rev‐responsive element (RRE) hairpin RNA promoted formation of covalent adducts. Analysis of one adduct showed that U26 in the bulged stem is responsible for covalent bond formation with the carbene intermediate. This strategy can provide important structural information about RNA–peptide interactions.

     

影响氨解法RNA提取率因素的探讨与研究

啤酒废酵母是啤酒生产的重要副产物,其营养成分十分丰富,具有很高的利用价值,其中核酸(DNA和RNA)含量尤其是RNA的含量较高.据报道,从啤酒废酵母提取RNA的方法有浓盐法、混合盐法、表面活性剂法、稀碱法等,常用的有浓盐法和稀碱法两种.采用氨解法,通过正交实验和响应面法,探讨和研究了影响氨解法RNA提取率的主要因素,确定氨解法从啤酒废酵母中提取RNA的最佳工艺条件:酵母浓度10.0%,氨水浓度为1.0%,破壁温度60℃,破壁时间55min.     

Exploration of long noncoding RNA in bovine milk exosomes and their stability during digestion in vitro

Previous studies have demonstrated that bovine milk contains mRNA and microRNA that are largely encapsulated in milk-derived exosomes. However, little information is available about long noncoding RNAs (lncRNA) in bovine milk. Increasing evidence suggests that lncRNA are of particular interest given their key role in gene expression and development. We performed a comprehensive analysis of lncRNA in bovine milk exosomes by RNA sequencing. We used a validated human in vitro digestion model to investigate the stability of lncRNA encapsulated in bovine milk exosomes during the digestion process. We identified 3,475 novel lncRNA and 6 annotated lncRNA. The lncRNA shared characteristics with those of other mammals in terms of length, exon number, and open reading frames. However, lncRNA showed higher expression than mRNAs. We selected 12 lncRNA of high-expression abundance and identified them by PCR. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that lncRNA regulate immune function, osteoblastogenesis, neurodevelopment, reproduction, cell proliferation, and cell–cell communication. We also investigated the 12 lncRNA using quantitative real-time PCR to reveal their expression profiles in milk exosomes during different stages of lactation (colostrum 2 d, 30 d, 150 d, and 270 d); their resulting expression levels in milk exosomes showed variations across the stages. A digestion experiment showed that bovine milk exosome lncRNA was resistant to in vitro digestion with different digestive juices, including saliva, gastric juice, pancreatic juice, and bile juice. Taken together, these results show for the first time that cow milk contains lncRNA, and that their abundance varied at different stages of lactation. As expected, bovine milk exosomal lncRNA were stable during in vitro digestion. These findings provide a basis for further understanding of the physiological role of milk lncRNA.     

Near‐field microscopy and fluorescence spectroscopy: application to chromosomes labelled with different fluorophores

We have coupled a spectrophotometer with a scanning near‐field optical microscope to obtain, with a single scan, simultaneously scanning near‐field optical microscope fluorescence images at different wavelengths as well as topography and transmission images. Extraction of the fluorescence spectra enabled us to decompose the different wavelengths of the fluorescence signals which normally overlap. We thus obtained images of the different fluorescence emissions of acridine orange bound to single or double stranded nucleic acids in human metaphase chromosomes before and after DNAse I or RNAse A treatment. The analysis of these images allowed us to visualize some specific chromatin areas where RNA is associated with DNA showing that such a technique could be used to identify multiple components within a cell.     

Method for rapid detection of viable Escherichia coli in water using real-time NASBA

A rapid real-time NASBA method was developed for detection of Escherichia coli in water samples. In this method, a fragment of the clpB-mRNA is amplified and a specific molecular beacon probe is used to detect the amplified mRNA fragment during the NASBA reaction. The method was shown to be specific and sensitive (1 viable E. coli in 100 ml) and can be performed within 3-4 h. Different inactivation processes (starvation, heat, UV-irradiation and chlorine) were employed to study the relationship between culturability and the ability to detect E. coli using NASBA. Detection of clpB-mRNA correlated with culturability after starvation or chlorine treatment. After UV-irradiation or heat-inactivation, detection of the increase in production of clpB-mRNA in viable E. coli cells after heat-shock induction correlated with culturability. Application of the NASBA method on tap water, treated sewage and surface water samples showed that culture and NASBA yielded comparable results in these different matrices. This study demonstrates that the NASBA method has high potential as a rapid test for microbiological water quality monitoring.     

Biodegradable Dextran Nanogels for RNA Interference: Focusing on Endosomal Escape and Intracellular siRNA Delivery

The successful therapeutic application of small interfering RNA (siRNA) largely relies on the development of safe and effective delivery systems that are able to guide the siRNA therapeutics to the cytoplasm of the target cell. In this report, biodegradable cationic dextran nanogels are engineered by inverse emulsion photopolymerization and their potential as siRNA carriers is evaluated. The nanogels are able to entrap siRNA with a high loading capacity, based on electrostatic interaction. Confocal microscopy and flow cytometry analysis reveal that large amounts of siRNA‐loaded nanogels can be internalized by HuH‐7 human hepatoma cells without significant cytotoxicity. Following their cellular uptake, it is found that the nanogels are mainly trafficked towards the endolysosomes. The influence of two different strategies to enhance endosomal escape on the extent of gene silencing is investigated. It is found that both the application of photochemical internalization (PCI) and the use of an influenza‐derived fusogenic peptide (diINF‐7) can significantly improve the silencing efficiency of siRNA‐loaded nanogels. Furthermore, it is shown that an efficient gene silencing requires the degradation of the nanogels. As the degradation kinetics of the nanogels can easily be tailored, these particles show potential for intracellular controlled release of short interfering RNA.     

Complex Conformational Dynamics of the Heart Failure-Associated Pre-miRNA-377 Hairpin Revealed by Single-Molecule Optical Tweezers

Pre-miRNA-377 is a hairpin-shaped regulatory RNA associated with heart failure. Here, we use single-molecule optical tweezers to unzip pre-miRNA-377 and study its stability and dynamics. We show that magnesium ions have a strong stabilizing effect, and that sodium ions stabilize the hairpin more than potassium ions. The hairpin unfolds in a single step, regardless of buffer composition. Interestingly, hairpin folding occurs either in a single step (type 1) or through the formation of intermediates, in multiple steps (type 2) or gradually (type 3). Type 3 occurs only in the presence of both sodium and magnesium, while type 1 and 2 take place in all buffers, with type 1 being the most prevalent. By reducing the size of the native hairpin loop from fourteen to four nucleotides, we demonstrate that the folding heterogeneity originates from the large size of the hairpin loop. Further, while efficient pre-miRNA-377 binders are lacking, we demonstrate that the recently developed C2 ligand displays bimodal activity: it enhances the mechanical stability of the pre-miRNA-377 hairpin and perturbs its folding. The knowledge regarding pre-miRNA stability and dynamics that we provide is important in understanding its regulatory function and how it can be modulated to achieve a therapeutic effect, e.g., in heart failure treatment.     

Modelling the dynamics of viral suppressors of RNA silencing

Virus infection in plants is limited by RNA silencing. In turn, viruses can counter RNA silencing with silencing suppressors. Viral suppressors of RNA silencing have been shown to play a role in symptom development in plants. We here study four different strategies employed by silencing suppressors: small interfering RNA (siRNA) binding, double-strand RNA (dsRNA) binding and degrading or inactivating Argonaute. We study the effect of the suppressors on viral accumulation within the cell as well as its spread on a tissue with mathematical and computational models. We find that suppressors which target Argonaute are very effective in a single cell, but that targeting dsRNA or siRNA is much more effective at the tissue level. Although targeting Argonaute can be beneficial for viral spread, it can also cause hindrance in some cases owing to raised levels of siRNAs that can spread to other cells.