Characterization Of RNA Structure Research Articles

Examples of Structural Motifs in Viral Genomes and Approaches for RNA Structure Characterization.

The relationship between conserved structural motifs and their biological function in the virus replication cycle is the interest of many researchers around the world. RNA structure is closely related to RNA function. Therefore, technological progress in high-throughput approaches for RNA structure analysis and the development of new ones are very important. In this mini review, we discuss a few perspectives on the structural elements of viral genomes and some methods used for RNA structure prediction and characterization. Based on the recent literature, we describe several examples of studies concerning the viral genomes, especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV). Herein, we emphasize that a better understanding of viral genome architecture allows for the discovery of the structure-function relationship, and as a result, the discovery of new potential antiviral therapeutics.

Refining RNA solution structures with the integrative use of label-free paramagnetic relaxation enhancement NMR

NMR structure calculation is inherently integrative, and can incorporate new experimental data as restraints. As RNAs have lower proton densities and are more conformational heterogenous than proteins, the refinement of RNA structures can benefit from additional types of restraints. Paramagnetic relaxation enhancement (PRE) provides distance information between a paramagnetic probe and protein or RNA nuclei. However, covalent conjugation of a paramagnetic probe is difficult for RNAs, thus limiting the use of PRE NMR for RNA structure characterization. Here, we show that the solvent PRE can be accurately measured for RNA labile imino protons, simply with the addition of an inert paramagnetic cosolute. Demonstrated on three RNAs that have increasingly complex topologies, we show that the incorporation of the solvent PRE restraints can significantly improve the precision and accuracy of RNA structures. Importantly, the solvent PRE data can be collected for RNAs without isotope enrichment. Thus, the solvent PRE method can work integratively with other biophysical techniques for better characterization of RNA structures.

Advances in engineered trans-acting regulatory RNAs and their application in bacterial genome engineering.

Small noncoding RNAs, a large class of ancient posttranscriptional regulators, are increasingly recognized and utilized as key modulators of gene expression in a broad range of microorganisms. Owing to their small molecular size and the central role of Watson-Crick base pairing in defining their interactions, structure and function, numerous diverse types of trans-acting RNA regulators that are functional at the DNA, mRNA and protein levels have been experimentally characterized. It has become increasingly clear that most small RNAs play critical regulatory roles in many processes and are, therefore, considered to be powerful tools for genetic engineering and synthetic biology. The trans-acting regulatory RNAs accelerate this ability to establish potential framework for genetic engineering and genome-scale engineering, which allows RNA structure characterization, easier to design and model compared to DNA or protein-based systems. In this review, we summarize recent advances in engineered trans-acting regulatory RNAs that are used in bacterial genome-scale engineering and in novel cellular capabilities as well as their implementation in wide range of biotechnological, biological and medical applications.

Tracking RNA structures as RNAs transit through the cell.

RNAs perform diverse cellular functions that are mediated at least in part by their structure. However, how RNA structure changes throughout the RNA lifecycle and how these changes affect RNA function remain incompletely understood. A detailed in vivo characterization of RNA structure in various cellular subcompartments now provides insights into how RNA structural changes influence translation, RNA decay, protein binding and RNA modification.

SHAPE-Seq 2.0: systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next generation sequencing.

RNA structure is a primary determinant of its function, and methods that merge chemical probing with next generation sequencing have created breakthroughs in the throughput and scale of RNA structure characterization. However, little work has been done to examine the effects of library preparation and sequencing on the measured chemical probe reactivities that encode RNA structural information. Here, we present the first analysis and optimization of these effects for selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). We first optimize SHAPE-Seq, and show that it provides highly reproducible reactivity data over a wide range of RNA structural contexts with no apparent biases. As part of this optimization, we present SHAPE-Seq v2.0, a ‘universal’ method that can obtain reactivity information for every nucleotide of an RNA without having to use or introduce a specific reverse transcriptase priming site within the RNA. We show that SHAPE-Seq v2.0 is highly reproducible, with reactivity data that can be used as constraints in RNA folding algorithms to predict structures on par with those generated using data from other SHAPE methods. We anticipate SHAPE-Seq v2.0 to be broadly applicable to understanding the RNA sequence–structure relationship at the heart of some of life's most fundamental processes.

SHAPE–Seq: High‐Throughput RNA Structure Analysis

Knowledge of RNA structure is critical to understanding both the important functional roles of RNA in biology and the engineering of RNA to control biological systems. This article contains a protocol for selective 2'-hydroxyl acylation analyzed by primer extension and sequencing (SHAPE-Seq) that, through a combination of structure-dependent chemical probing and next-generation sequencing technologies, achieves structural characterization of hundreds of RNAs in a single experiment. This protocol is applicable in a variety of conditions, and represents an important tool for understanding RNA biology. The protocol includes methods for the design and synthesis of RNA mixtures for study, and the construction and analysis of structure-dependent sequencing libraries that reveal structural information of the RNAs in the mixtures. The methods are generally applicable to studying RNA structure and interactions in vitro in a variety of conditions, and allows for the rapid characterization of RNA structures in a high-throughput manner. Curr. Protoc. Chem. Biol. 4:275-297 © 2012 by John Wiley & Sons, Inc.

'Mutate-and-map' RNA structure characterization

'Mutate-and-map' RNA structure characterization

Function Annotation for Pseudoknot Using Structure Similarity

Many raw biological sequence data have been generated by the human genome project and related efforts. The understanding of structural information encoded by biological sequences is important to acquire knowledge of their biochemical functions but remains a fundamental challenge. Recent interest in RNA regulation has resulted in a rapid growth of deposited RNA secondary structures in varied databases. However, a functional classification and characterization of the RNA structure have only been partially addressed. This article aims to introduce a novel interval-based distance metric for structure-based RNA function assignment. The characterization of RNA structures relies on distance vectors learned from a collection of predicted structures. The distance measure considers the intersected, disjoint, and inclusion between intervals. A set of RNA pseudoknotted structures with known function are applied and the function of the query structure is determined by measuring structure similarity. This not only offers sequence distance criteria to measure the similarity of secondary structures but also aids the functional classification of RNA structures with pesudoknots.

Modeling and automation of sequencing-based characterization of RNA structure

Sequence census methods reduce molecular measurements such as transcript abundance and protein-nucleic acid interactions to counting problems via DNA sequencing. We focus on a novel assay utilizing this approach, called selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), that can be used to characterize RNA secondary and tertiary structure. We describe a fully automated data analysis pipeline for SHAPE-Seq analysis that includes read processing, mapping, and structural inference based on a model of the experiment. Our methods rely on the solution of a series of convex optimization problems for which we develop efficient and effective numerical algorithms. Our results can be easily extended to other chemical probes of RNA structure, and also generalized to modeling polymerase drop-off in other sequence census-based experiments.

Multiplexed RNA structure characterization with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPE-Seq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems.

Characterization of RNA structure by bis-pyrene-labeled 2'-O-methyloligonucleotides.

Properties of 2'-O-methyloligoribonucleotides containing two consecutive 2'-O-(1-pyrenylmethyl)uridine were investigated as a fluorescent probe to search the single strand regions of RNA. The bis-pyrene-labeled 2'-O-methyloligoribonucleotide (OMUpy2) induced the formation of pyrene dimer upon hybridization with the complementary oligoribonucleotides and showed remarkable appearance of broad structureless fluorescence at 480 nm. Contrarily, when OMUpy2 was hybridized with the complementary oligodeoxyribonucleotides, such enhancement of fluorescence was scarcely observed. When various OMUpy2 were applied to E. coli 5S-rRNA, the fluorescence intensity at 480 nm was varied in a sequence specific manner.

Rational Design and Synthesis of Ribozymes

We review our procedure for the rational design and the synthesis of oligoribonucleotides with particular reference to our recent studies of the development of a hairpin-derived twin ribozyme. Our work focuses on the design of ribozymes that are derived from naturally occurring structures but posses extended catalytic activities. Currently we are studying ribozymes that are capable of site-specific RNA double cleavage. We have designed these “twin ribozymes” by combining of two catalytic motifs in one molecule. In addition to kinetic analysis of the cleavage reaction, we are interested in the general phenomena of RNA folding and the characterization of RNA structure–function relationships. The developed twin ribozymes are particularly well suited for this purpose. They have to fold into a complex structure to achieve catalytic activity. Since there is a direct correlation between conformation and activity, the process of folding can be followed by monitoring the cleavage reaction. The twin ribozymes were synthesized by a combination of phosphoramidite chemistry and in vitro transcription techniques. We report the synthesis of a powerful reagent to replace tetrazole for activation of RNA phosphoramidites in automated solid-phase synthesis, allowing for coupling yields of >99%. Furthermore, we introduce a fast quantitative assay of ribozyme activity which is based on fluorescently labeled oligoribonucleotide substrates and DNA sequencer technology.

Epstein-Barr Virus Gene Expression in Oral Hairy Leukoplakia

A combination of Northern blotting and sequencing of clones from a cDNA library constructed using RNA isolated from oral hairy leukoplakia (OHL) has been used to study Epstein-Barr virus (EBV) gene expression in this AIDS-associated lesion, the only accessible source of in vivo-replicating EBV. Because of the limited amount of RNA available, Northern blotting was only useful for detection of very abundant EBV transcripts in the OHL biopsies. Analysis of cDNA clones containing the BdRF1, BCRF1, gp350/220, BARF0, and BKRF4 reading frames and further characterization of RNA structures spanning BCRF1 (viral IL-10) in lymphocytes infected with EBV has provided a preliminary comparison of some virus replicative gene expression in its two main host cell types. No expression of EBNA-1, EBNA-2, or EBNA-3A RNA was detected in the oral hairy leukoplakia cDNA library.