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丁香实验推荐阅读
Purification of U Small Nuclear Ribonucleoprotein Particles

The spliceosome is the catalytic entity that removes the introns from the primary transcripts in eukaryotes. The spliceosome consists of four small nuclear ribonucleoproteins (snRNPs), named U1,U2, U4/U6, and U5 snRNP, and numerous non-snRNP proteins. Each snRNP consists of one (U1, U2, and ...

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Purification and Depletion of RNP Particles by Antisense Affinity Chromatography

Antisense oligonucleotides made of 2′-O-alkyl RNA are useful reagents for the study of the structure and function of ribonucleoprotein (RNP) particles. The nuclease resistant properties of these oligonucleotides, coupled with their ability to form specific and stable hybrids with ...

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An Immunoprecipitation-RNA:rPCR Method for the In Vivo Isolation of Ribonucleoprotein Complexes

Within the last few years, it has become well established that a given nucleic acid binding protein has the potential to interact specifically with more than one target nucleic acid sequence. Various immunoprecipitation techniques have been developed to isolate specific DNA-protein ...

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Labeling and Purification of RNA Synthesized by In Vitro Transcription

The problems of isolating sufficient quantities of rare RNAs for detailed biochemical analysis can be circumvented by synthesis of the desired RNA in vitro (1–3). Early methods of in vitro transcription included the use of eukary-otic cell extracts or Escherichia coli RNA polymerase to tra ...

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Screening Expression Libraries with Solution-Based Assay

To date, the most common approach to screening an expression library for an RNA-binding protein is one based on a procedure that was originally developed for cloning DNA-binding proteins (1,2): cDNA-encoded proteins from λ plaques are immobilized on nitrocellulose or nylon filters, which a ...

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Northwestern Screening of Expression Libraries

In the cell, all RNAs are involved in interactions with proteins that influence many aspects of their processing, localization, and expression (reviewed in refs. 1 and 2). In order to gain mechanistic insights into the role of proteins in these processes, one must (1) define the sequence elements in ...

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Identification of Specific Protein-RNA Target Sites Using Libraries of Natural Sequences

RNA-binding proteins are involved in a variety of regulatory and developmental processes such as RNA processing, transport, and translation and are integral components of ribosomes, spliceosomes, nucleoli, and other ribonucle-oprotein particles. Proteins have been shown to in ...

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In Vitro Selection of Aptamers from RNA Libraries

Methods of iterative nucleic acid selection and amplification were enabled by the invention of the polymerase chain reaction (PCR). Thus, the ability to amplify as few as a single DNA or RNA molecule made it possible to diversify sequences and to partition the desirable from the undesirable sub ...

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In Vitro Genetic Analysis of RNA-Binding Proteins Using Phage Display

RNA-binding proteins (RNA-BPs) play an essential role in key processes in all living organisms, from viruses to mammals. They are involved in RNA packaging, pre-mRNA splicing, translation, and RNA localization, to name a few. For these proteins to function properly it is imperative that they re ...

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Screening RNA-Binding Libraries Using a Bacterial Transcription Antitermination Assay

A number of genetic assays have recently been developed for detecting RNA-protein interactions, several of which have been applied to screen cDNA or combinatorial libraries for RNA-binding peptides and proteins (1–5) (see Chapters 13 and 15). These methods are useful for understanding t ...

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An Escherichia coli-Based Genetic Strategy for Characterizing RNA Binding Proteins

In recent years, interest in RNA-binding proteins (RNA-BPs) and RNA-protein interactions has grown considerably. An important reason for this interest is the increased recognition that RNA-BPs are involved in a wide variety of critical viral and cellular processes including transc ...

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Detection of Nucleic Acid Interactions Using Surface Plasmon Resonance

Several different methods can be used for detection of interactions between RNA and other molecules (e.g., proteins and other nucleic acids). Among these are: (1) enzymatic assays in which there is a measurable change in the property of the substrate such as an increase or decrease in size (mobility ...

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PACE Analysis of RNA-Peptide Interactions

The PACE assay is a relatively new addition to the arsenal of techniques used to examine quantitatively the interactions of proteins and peptides with DNA and RNA (1). Polyacrylamide coelectrophoresis (PACE) involves electrophoresis of a labeled nucleic acid through a gel medium that co ...

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Measuring Equilibrium and Kinetic Constants Using Gel Retardation Assays

When analyzing ribonucleoprotein complexes, it is important to keep in mind that any such complex in solution must coexist with some concentration of the free (uncomplexed) RNA and protein components of the RNP. This can be formalized by the equilibrium equation: where R represents the free RN ...

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Nitrocellulose Filter Binding for Determination of Dissociation Constants

Nitrocellulose filter binding has been used to measure the association of proteins to both DNA and RNA for many years. Yarus and Berg (1) first described the application of the method for characterization of aminoacyl-tRNA synthetase:tRNA association; other RNA applications include t ...

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Oligonucleotide-Targeted RNase H Protection Analysis of RNA-Protein Complexes

This chapter focuses on the analysis of RNA-protein complexes (RNPs, ribonucleoprotein particles) by oligonucleotide-targeted RNase H digestion, a powerful approach to probe the domain structure of an RNP, both in crude extracts and in purified preparations. RNase H requires DNA-RNA ...

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RNA Footprinting and Modification Interference Analysis

RNA plays a central role in a wide range of processes within the cell. In some organisms RNA replaces DNA as the genetic material. In all organisms RNA is essential at all stages in the translation of genetic information to protein (1–4). RNA-protein interactions have a key role in almost all of these biolog ...

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Identification and Sequence Analysis of RNA-Protein Contact Sites by N-Terminal Sequencing and MALDI-MS

Crosslinking techniques have been used widely to obtain meaningful structural information on RNA-protein interactions. Exact data on the contact sites of RNA-protein complexes at the molecular level are required for a detailed modeling of three-dimensional structures within t ...

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Probing RNA-Protein Interactions by Psoralen Photocrosslinking

RNA molecules can fold into extensive structures containing regions of double-stranded duplex, hairpins, internal loops, bulged bases, and pseudo-knotted structures (1). Owing to the complexity of RNA structure, the rules governing sequence-specific RNA-protein recogniti ...

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The Methylene Blue Mediated Photocrosslinking Method for Dectection of Proteins that Interact with Double-Stranded RNA

Photochemical crosslinking is an approach that is widely used to detect and analyze interactions between proteins and nucleic acids. The approach involves illuminating a mixture of protein and nucleic acid with light of a suitable wavelength to induce photochemical reactions that r ...

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