scholarly journals Chemical and Enzymatic Probing of Viral RNAs: From Infancy to Maturity and Beyond

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1894
Author(s):  
Orian Gilmer ◽  
Erwan Quignon ◽  
Anne-Caroline Jousset ◽  
Jean-Christophe Paillart ◽  
Roland Marquet ◽  
...  
Keyword(s):  
Historical Perspective ◽  
Detection Methods ◽  
Rna Structures ◽  
Viral Rnas ◽  
Rna Molecules ◽  
Rna Biology ◽  
Key Players ◽  
The 1960S ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

RNA molecules are key players in a variety of biological events, and this is particularly true for viral RNAs. To better understand the replication of those pathogens and try to block them, special attention has been paid to the structure of their RNAs. Methods to probe RNA structures have been developed since the 1960s; even if they have evolved over the years, they are still in use today and provide useful information on the folding of RNA molecules, including viral RNAs. The aim of this review is to offer a historical perspective on the structural probing methods used to decipher RNA structures before the development of the selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) methodology and to show how they have influenced the current probing techniques. Actually, these technological breakthroughs, which involved advanced detection methods, were made possible thanks to the development of next-generation sequencing (NGS) but also to the previous works accumulated in the field of structural RNA biology. Finally, we will also discuss how high-throughput SHAPE (hSHAPE) paved the way for the development of sophisticated RNA structural techniques.

scholarly journals Current and New Approaches in GMO Detection: Challenges and Solutions

2015 ◽  
Vol 2015 ◽  
pp. 1-22 ◽  
Author(s):  
Marie-Alice Fraiture ◽  
Philippe Herman ◽  
Isabel Taverniers ◽  
Marc De Loose ◽  
Dieter Deforce ◽  
...  
Keyword(s):  
Genetically Modified Organisms ◽  
Digital Pcr ◽  
Detection Methods ◽  
Gmo Detection ◽  
Freedom Of Choice ◽  
Next Generation Sequencing Ngs ◽  
Detection Strategies ◽  
Generation Sequencing ◽  
Accurate Quantification

In many countries, genetically modified organisms (GMO) legislations have been established in order to guarantee the traceability of food/feed products on the market and to protect the consumer freedom of choice. Therefore, several GMO detection strategies, mainly based on DNA, have been developed to implement these legislations. Due to its numerous advantages, the quantitative PCR (qPCR) is the method of choice for the enforcement laboratories in GMO routine analysis. However, given the increasing number and diversity of GMO developed and put on the market around the world, some technical hurdles could be encountered with the qPCR technology, mainly owing to its inherent properties. To address these challenges, alternative GMO detection methods have been developed, allowing faster detections of single GM target (e.g., loop-mediated isothermal amplification), simultaneous detections of multiple GM targets (e.g., PCR capillary gel electrophoresis, microarray, and Luminex), more accurate quantification of GM targets (e.g., digital PCR), or characterization of partially known (e.g., DNA walking and Next Generation Sequencing (NGS)) or unknown (e.g., NGS) GMO. The benefits and drawbacks of these methods are discussed in this review.

scholarly journals Structural Insights Into the 5′UG/3′GU Wobble Tandem in Complex With Ba2+ Cation

2022 ◽  
Vol 8 ◽  
Author(s):  
Agnieszka Ruszkowska ◽  
Ya Ying Zheng ◽  
Song Mao ◽  
Milosz Ruszkowski ◽  
Jia Sheng
Keyword(s):  
Electrostatic Potential ◽  
Metal Binding ◽  
Single Pair ◽  
Major Groove ◽  
Rna Structures ◽  
Rna Molecules ◽  
Rna Biology ◽  
Local Patch ◽  
Structural Insights ◽  
Unique Chemical

G•U wobble base pair frequently occurs in RNA structures. The unique chemical, thermodynamic, and structural properties of the G•U pair are widely exploited in RNA biology. In several RNA molecules, the G•U pair plays key roles in folding, ribozyme catalysis, and interactions with proteins. G•U may occur as a single pair or in tandem motifs with different geometries, electrostatics, and thermodynamics, further extending its biological functions. The metal binding affinity, which is essential for RNA folding, catalysis, and other interactions, differs with respect to the tandem motif type due to the different electrostatic potentials of the major grooves. In this work, we present the crystal structure of an RNA 8-mer duplex r[UCGUGCGA]2, providing detailed structural insights into the tandem motif I (5′UG/3′GU) complexed with Ba2+ cation. We compare the electrostatic potential of the presented motif I major groove with previously published structures of tandem motifs I, II (5′GU/3′UG), and III (5′GG/3′UU). A local patch of a strongly negative electrostatic potential in the major groove of the presented structure forms the metal binding site with the contributions of three oxygen atoms from the tandem. These results give us a better understanding of the G•U tandem motif I as a divalent metal binder, a feature essential for RNA functions.

scholarly journals Comparative analysis of viral RNA signatures on different RIG-I-like receptors

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Raul Y Sanchez David ◽  
Chantal Combredet ◽  
Odile Sismeiro ◽  
Marie-Agnès Dillies ◽  
Bernd Jagla ◽  
...  
Keyword(s):  
Rna Virus ◽  
Affinity Purification ◽  
Viral Rna ◽  
Coding Region ◽  
Chikv Infection ◽  
Rna Molecules ◽  
Positive Sense ◽  
Rna Ligands ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

The RIG-I-like receptors (RLRs) play a major role in sensing RNA virus infection to initiate and modulate antiviral immunity. They interact with particular viral RNAs, most of them being still unknown. To decipher the viral RNA signature on RLRs during viral infection, we tagged RLRs (RIG-I, MDA5, LGP2) and applied tagged protein affinity purification followed by next-generation sequencing (NGS) of associated RNA molecules. Two viruses with negative- and positive-sense RNA genome were used: measles (MV) and chikungunya (CHIKV). NGS analysis revealed that distinct regions of MV genome were specifically recognized by distinct RLRs: RIG-I recognized defective interfering genomes, whereas MDA5 and LGP2 specifically bound MV nucleoprotein-coding region. During CHIKV infection, RIG-I associated specifically to the 3’ untranslated region of viral genome. This study provides the first comparative view of the viral RNA ligands for RIG-I, MDA5 and LGP2 in the presence of infection.

RNA Sequencing of Formalin-Fixed and Paraffin-Embedded Tissue as a Complementary Method in Ophthalmopathology

2020 ◽  
Vol 237 (07) ◽  
pp. 860-866
Author(s):  
Günther Schlunck ◽  
Stefaniya Boneva ◽  
Julian Wolf ◽  
Anja Schlecht ◽  
Thomas Reinhard ◽  
...  
Keyword(s):  
Cost Effective ◽  
Messenger Rnas ◽  
Rna Molecules ◽  
Complementary Method ◽  
High Throughput Method ◽  
Next Generation Sequencing Ngs ◽  
Insight Into ◽  
Generation Sequencing ◽  
Formalin Fixed ◽  
Methodological Strategy

AbstractThe high-throughput method of “Next Generation Sequencing” (NGS) allows cost-effective decoding of the nucleotide sequences of millions of RNA molecules in a sample. This makes it possible to determine the number of distinct RNA molecules in tissues or cells and to use these data to draw conclusions. The entirety of RNAs, in particular mRNAs (messenger RNAs) as potential precursors of proteins, provides a comprehensive insight into the functional state of the cells and tissues under investigation. In addition to cell cultures or unfixed tissue, formalin-fixed and paraffin-embedded (FFPE) tissue can also be analysed for this purpose using specific methods. In this overview, the methodological strategy and its application to the field of ophthalmic histopathology are presented.

scholarly journals Mining of miRNAs using Next Generation Sequencing (NGS) data generated for Okra (Abelmoschus esculentus)

2018 ◽  
Vol 13 (2) ◽  
pp. 137-145
Author(s):  
Rekha Gupta ◽  
M Gayathri ◽  
V Radhika ◽  
M Pichaimuthu ◽  
K V Ravishankar
Keyword(s):  
Gene Expression ◽  
Mirna Family ◽  
Abelmoschus Esculentus ◽  
Stem Loop ◽  
Rna Molecules ◽  
Non Coding Rna ◽  
Precursor Rna ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Generation Sequencing

MicroRNAs (miRNAs) are small, highly conserved non-coding RNA molecules involved in theregulation of gene expression in eukaryotes. Gene expression involves post-transcriptionalgene regulation by miRNAs. miRNAs are formed from precursor RNA molecules that fold intoa stem loop secondary structure. The mature miRNA is one end of the precursor miRNA,defined by the cut from ‘Drosha’ on either the 5’ or 3’ arm. In this study, we have used abioinformatics approach to identify miRNAs in 3,361 contigs obtained from partial genomesequence data of Abelmoschus esculentus (okra) sequenced by NGS technology. Using C-miiand psRNA Target tools, we identified two miRNAs and their target RNAs for which a regulatorymiRNA binding has been verified. Their targets consisted of transcription factors involved ingrowth and development, gene regulation and metabolism. Phylogenetic analysis of the newlyidentified miRNA family has been done to compare their level of conservation with respect tothe other members of the plant kingdom.

Comparison of detection methods for vaginal lactobacilli

2015 ◽  
Vol 6 (5) ◽  
pp. 747-751 ◽  
Author(s):  
I. Smidt ◽  
R. Kiiker ◽  
H. Oopkaup ◽  
E. Lapp ◽  
T. Rööp ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Detection Methods ◽  
Lactobacillus Gasseri ◽  
Vaginal Lactobacilli ◽  
Lactobacillus Jensenii ◽  
Child Bearing ◽  
Good Concordance ◽  
Tract Infections ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Vaginal lactobacilli offer protection against microbiota imbalance and genitourinary tract infections. We compared vaginal lactobacilli in 50 Estonian women of child-bearing age applying culture-based methods, quantitative PCR and next-generation sequencing (NGS). The culture-based methods found three different lactobacilli: Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus gasseri. Using NGS revealed the presence of L. crispatus in 76%, Lactobacillus iners in 52%, L. jensenii in 47% and L. gasseri in 33% of the samples. According to qPCR, L. iners was present in 67% and L. crispatus in 64% of the samples. The proportions of L. crispatus revealed by qPCR and NGS were in good correlation (R=0.79, P<0.001), while that of L. iners correlated poorly (R=0.13, P>0.05). Good concordance for L. crispatus was also found between the results of the culture-based method and qPCR. Finally, good overlap between the results of the culture-based method and NGS was revealed: in case of a positive NGS result for L. crispatus, the same species was isolated in 95% of samples. The corresponding percentages were 82% for L. jensenii and 86% for L. gasseri. Our data indicate fairly general concordance of the three methods for detecting vaginal lactobacilli, except for L. iners. This points out the importance of standardisation of techniques, and the respective studies should involve cultures applying a medium suitable for the fastidious L. iners.

scholarly journals CNNdel: Calling Structural Variations on Low Coverage Data Based on Convolutional Neural Networks

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Jing Wang ◽  
Cheng Ling ◽  
Jingyang Gao
Keyword(s):  
Neural Networks ◽  
Convolutional Neural Networks ◽  
Sequence Data ◽  
High Sensitivity ◽  
False Positives ◽  
Detection Methods ◽  
Structural Variations ◽  
Next Generation Sequencing Ngs ◽  
Low Coverage ◽  
Generation Sequencing

Many structural variations (SVs) detection methods have been proposed due to the popularization of next-generation sequencing (NGS). These SV calling methods use different SV-property-dependent features; however, they all suffer from poor accuracy when running on low coverage sequences. The union of results from these tools achieves fairly high sensitivity but still produces low accuracy on low coverage sequence data. That is, these methods contain many false positives. In this paper, we present CNNdel, an approach for calling deletions from paired-end reads. CNNdel gathers SV candidates reported by multiple tools and then extracts features from aligned BAM files at the positions of candidates. With labeled feature-expressed candidates as a training set, CNNdel trains convolutional neural networks (CNNs) to distinguish true unlabeled candidates from false ones. Results show that CNNdel works well with NGS reads from 26 low coverage genomes of the 1000 Genomes Project. The paper demonstrates that convolutional neural networks can automatically assign the priority of SV features and reduce the false positives efficaciously.

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