Springer protocols feed by Genetics/Genomics 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Modification Interference Analysis of the Ribosome
RNAs are versatile molecules involved in myriad functions in the cell. To understand how a RNA molecule functions in the cell it is important to identify the nucleotides in the RNA molecule that are important for its structure and function. There are several biochemical methods such as footprinting, cross-linking, and modification interference analysis that can be used to study RNA–RNA and RNA–protein interactions. Ribosome is a classical example of a RNA–protein complex that has been extensively studied using these methods. Here, we describe a modification interference method that was used to identify ba...
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
Single Molecule Studies of RNA–RNA Interactions
Non-coding RNAs including microRNAs, siRNAs, and snoRNAs interact with their targets directly through RNA–RNA interactions by base-paring (van Himbergen et al., Nucleic Acids Res 21(8):1713–1717, 1993). RNA–RNA interactions play important roles in gene transcription and protein translation, which can be investigated with several experimental techniques including single molecule methods. Here, we describe how single molecule Förster resonance energy transfer (FRET) can be used to study RNA–RNA interactions in vitro by either surface immobilization or vesicle encapsulation. (Source: Springer prot...
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
Native Purification and Labeling of RNA for Single Molecule Fluorescence Studies
The recent discovery that non-coding RNAs are considerably more abundant and serve a much wider range of critical cellular functions than recognized over previous decades of research into molecular biology has sparked a renewed interest in the study of structure–function relationships of RNA. To perform their functions in the cell, RNAs must dominantly adopt their native conformations, avoiding deep, non-productive kinetic traps that may exist along a frustrated (rugged) folding free energy landscape. Intracellularly, RNAs are synthesized by RNA polymerase and fold co-transcriptionally starting from the 5′ end,...
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
Transactivation of Large Ribozymes
The domains of large ribozymes are composed of secondary structural elements that fold independently in solution. Interactions between these structures contribute to the overall structure of the ribozyme. These elements can also be deleted, reducing catalytic activity, and their structure–function relationships determined by assaying the ability of variant elements to activate the deleted ribozyme in trans. This is illustrated for an unusual loop structure from Bacillus subtilis Ribonuclease P RNA. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
Identification of Antisense RNA Stem-Loops That Inhibit RNA–Protein Interactions Using a Bacterial Reporter System
RNA–protein interactions play important roles in gene regulation, functional RNA–protein complexes such as the ribosome, and in viral replication. Molecules that regulate specific RNA–protein interactions may be used to dissect biological processes, and to establish the validity of targeting an RNA–protein interaction. There are many examples of biological regulation by antisense RNA stem-loops that form loop-loop and loop-linear RNA–RNA interactions. Here, a bacterial reporter system for the identification of RNA stem-loops that inhibit the formation of RNA–protein complexes through RNA...
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
RNA–RNA SELEX
Systematic evolution of ligands by exponential enrichment (SELEX) protocol is a valuable technique to identify RNA aptamers interacting with RNA structural motifs. RNA aptamers are mainly resolved with affinity column chromatography and electrophoretic mobility shift assay (EMSA). Here, we describe the separation of the RNA aptamers binding to an RNA stem-loop target with affinity chromatography using the column attached the target RNA and nondenaturing polyacrylamide gel electrophoresis to obtain a single predominant RNA aptamer family. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
RNA-Directed Recombination of RNA In Vitro
Construction of long RNAs can be achieved in vitro by using ribozymes to recombine shorter RNAs. This can be a useful technique to prepare RNAs when the final product is either very long or contains chemical modifications that are difficult to incorporate using standard in vitro transcription techniques. Here, we describe the use of the Azoarcus group I intron ribozyme to recombine shorter RNAs into longer ones. This ribozyme is a generalized RNA recombinase ribozyme that operates rapidly and with high efficiency. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
Identifying RNA Recombination Events and Non-covalent RNA–RNA Interactions with the Molecular Colony Technique
Molecular colonies (also known under names nanocolonies, polonies, RNA or DNA colonies, PCR colonies) form when nucleic acids are amplified in a porous solid or semi-solid medium, such as a gel, which contains a system for the exponential multiplication of RNA or DNA. As an individual colony comprises many copies of a single molecule (a molecular clone), the method can be used for the detection, enumeration, and analysis of individual DNA or RNA molecules, including the products of such rare events as RNA recombinations. Here we describe protocols for the detection of RNA molecules by growing colonies of RNA (in a gel cont...
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
Fusion RNAs in Crystallographic Studies of Double-Stranded RNA from Trypanosome RNA Editing
Head-to-head fusions of two identical double-stranded fragments of RNA can be designed to self-assemble from a single RNA species and form a double-stranded helix with a twofold rotation axis relating the two strands. These symmetrical RNA molecules are more likely to crystallize without end-on-end statistical packing disorder because the two halves of the molecule are identical. This approach can be used to study many fragments of double-stranded RNA or many isolated helical domains from large single-stranded RNAs that may not yet be amenable to high-resolution studies by crystallography or NMR. We used fusion RNAs to stu...
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
Structural Studies of a Double-Stranded RNA from Trypanosome RNA Editing by Small-Angle X-Ray Scattering
We used small-angle X-ray scattering (SAXS) to evaluate the solution structure of a double-stranded RNA with 32 base pairs. We wanted to compare the solution structure to the crystal structure to assess the impact of the crystal lattice on the overall conformation of the RNA. The RNA was designed to self-anneal and form a head-to-head fusion of two identical mRNA/oligo(U) tail domains (the U-helix) from a trypanosome RNA editing substrate formed by the annealing of a guide RNA to a pre-edited mRNA. This substrate is from the U insertion/deletion RNA editing system of trypanosomes. Each strand in the fusion RNA had 16 purin...
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
Electrophoretic Mobility Shift Assay of RNA–RNA Complexes
A simple, rapid, and sensitive electrophoretic mobility shift assay (EMSA) can be successfully used to analyze RNA–RNA interactions. The EMSA of RNA–RNA complexes can be further used to evaluate the specificity of interactions using competitor RNAs in combination with their mutated versions or nonspecific RNAs, such as yeast tRNA. RNA is simply prepared by in vitro transcription from PCR product templates. Detailed experimental descriptions for EMSA-based analysis of specific RNA–RNA interactions between Sib RNAs and ibs mRNAs as a representative example are presented. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
Fluorescence Anisotropy: Analysis of tRNA Binding to the T Box Riboswitch Antiterminator RNA
Fluorescence anisotropy can be utilized in drug discovery screening assays to identify compounds that disrupt medicinally important RNA–macromolecular complexes. Here we describe the application of this technique to monitor tRNA binding to T box riboswitch antiterminator RNA. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
Electrophoretic Mobility Shift Assays: Analysis of tRNA Binding to the T Box Riboswitch Antiterminator RNA
Changes in electrophoretic mobility upon complex formation with RNA can be used to probe structure–function relationships that are critical for complex formation. Here, we describe the application of this technique to monitor tRNA binding to the T box riboswitch antiterminator RNA. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - October 30, 2014 Category: Genetics & Stem Cells Source Type: news
Structure Modeling of Toll-Like Receptors
Toll-like receptors (TLRs) recognize invasion of microbial pathogens and initiate innate immune responses that are essential for inhibiting pathogen dissemination and for the development of acquired immunity. To understand how these receptors work, it is crucial to investigate them from a structural perspective. High-throughput genome sequencing projects have led to the identification of more than 3,000 TLR sequences. However, only several structures of TLRs have been determined because structure determination by X-ray diffraction or nuclear magnetic resonance spectroscopy experiments remains difficult and time-consuming. ...
Source: Springer protocols feed by Genetics/Genomics - June 24, 2014 Category: Genetics & Stem Cells Source Type: news
Isolation of RIG-I-Associated RNAs from Virus-Infected Cells
When a novel innate pattern recognition receptor (PRR) is identified, a question comes up immediately: Which molecular pattern(s) can it recognize? One approach that can be taken to answer this question for nucleic acid-binding receptors is the detailed analysis of synthetic ligands (DNA, RNA, or hybrids) to narrow in on the minimal patterns that activate a given receptor. However, this may not always lead to a satisfying answer. A complementary albeit technically more demanding way to tackle this question is to examine which nucleic acids are actually bound by the receptor in a setting of cellular infection. Here, we desc...
Source: Springer protocols feed by Genetics/Genomics - June 24, 2014 Category: Genetics & Stem Cells Source Type: news
