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Cis-Acting Ribozymes for the Production of RNA In Vitro Transcripts with Defined 5 and 3 Ends

The use of in vitro transcribed RNA is often limited by sequence constraints at the 5′-end and the problem of transcript heterogeneity which can occur at both the 5′- and 3′-ends. This chapter describes the use of cis-acting ribozymes, 5′-end hammerhead (HH) and 3′-end hepatitis delta virus (HDV), for dir ...

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Native RNA Purification by Gel Filtration Chromatography

In vitro transcription of RNA from DNA templates by T7 RNA polymerase allows for the generation of large quantities of RNA suitable for many downstream applications. The resulting RNA can be purified by a number of methodologies. Herein, we describe the native isolation of RNA molecules by FPLC p ...

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Preparation of Short RNA by In Vitro Transcription

Short RNA oligomers are required in many biochemical and biophysical studies. In vitro transcription using T7 RNA polymerase can produce a large amount of specific RNA sufficient for structural studies in a more economical way than chemical synthesis. Here, we provided protocols optim ...

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General Protocols for Preparation of Plasmid DNA Template, RNA In Vitro Transcription, and RNA Purification by Denaturing PAGE

The development of methods for in vitro transcription of defined RNA sequences has been a key factor driving the tremendous advances in RNA biology over the last three decades. The numerous approaches available today to study RNA structure and function vary widely in their demands on the quali ...

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Preparation of Long Templates for RNA In Vitro Transcription by Recursive PCR

Preparing conventional DNA templates for in vitro RNA transcription involves PCR amplification of the DNA gene coding for the RNA of interest from plasmid or genomic DNA, subsequent amplification with primers containing a 5′ T7 promoter region, and confirmation of the amplified DNA sequ ...

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Selective RNase H Cleavage of Target RNAs from a tRNA Scaffold

In vivo overproduction of tRNA chimeras yields an RNA insert within a tRNA scaffold. For some applications, it may be necessary to discard the scaffold. Here we present a protocol for selective cleavage of the RNA of interest from the tRNA scaffold, using RNase H and two DNA oligonucleotides. After cl ...

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Purification of RNA Expressed In Vivo Inserted in a tRNA Scaffold

For structural, biochemical, or pharmacological studies, it is required to have pure RNA in large quantities. In vitro transcription or chemical synthesis are the principal methods to produce RNA. Here, we describe an alternative method allowing RNA production in bacteria and its purifi ...

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Splint Ligation of RNA with T4 DNA Ligase

Splint ligation of RNA, whereby specific RNA molecules are ligated together, can be carried out using T4 DNA ligase and a bridging DNA oligonucleotide complementary to the RNAs. This method takes advantage of the property of T4 DNA ligase to join RNA molecules when they are in an RNA:DNA hybrid. Splint ...

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Preparative Separation of Ribonucleoside Monophosphates by Ion-Pair Reverse-Phase HPLC

Structural and dynamic investigations of RNA by nuclear magnetic resonance (NMR) spectroscopy strongly benefit from isotopic-labeling strategies. Among these, nucleotide-specific and site-specific labeling methods can help tremendously in simplifying complex NMR da ...

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Biosynthetic Preparation of 13C/15N-Labeled rNTPs for High-Resolution NMR Studies of RNAs

High-resolution investigations of the structure and dynamics of RNA molecules by nuclear magnetic resonance (NMR) methodologies require the production of 13C/15N-isotopically labeled samples. A common strategy entails the preparation of 13C/15N-enriched ribonucleos ...

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Se-Derivatized RNAs for X-ray Crystallography

Selenium derivatization of RNA is an important strategy for crystal structure determination and functional studies of noncoding RNAs and protein–RNA interactions. We describe here the synthesis of nucleoside 5′-(α-P-seleno)-triphosphate analogs (Se-NTPs) and their use in vit ...

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High-Purity Enzymatic Synthesis of Site-Specifically Modified tRNA

Transfer RNA (tRNA) molecules play the key role in adapting the genetic code sequences with amino acids. The execution of this key role is highly dependent on the presence of modified nucleotides in tRNA, each of which performs a distinct function. To better understand how individual modificat ...

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End-Labeling Oligonucleotides with Chemical Tags After Synthesis

Many experimental strategies for determining nucleic acid function require labeling the nucleic acid with radioisotopes or a chemical tag. Labels enable nucleic acid detection, yield information about its state, and can serve as a handle by which the nucleic acid and associated factors ...

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In Vitro Transcription of Modified RNAs

RNAs containing a variety of terminal and internal modifications can be produced using bacteriophage polymerases often with a few simple adjustments to standard transcription protocols. RNAs containing a single phosphate or a cap structure at their 5′ ends can readily be generated eit ...

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Plasmid Template Design and In Vitro Transcription of Short RNAs Within a Structure Cassette for Structure Probing Experiments

Chemical and enzymatic RNA structure probing methods are important tools for examining RNA secondary and tertiary structures and their interactions with proteins, small molecules, and ions. The recently developed “Selective 2′-Hydroxyl Acylation Analyzed by Primer Extensi ...

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Affinity Purification of RNA Using an ARiBo Tag

The increased awareness of the importance of RNA in biology, illustrated by the recent attention given to RNA interference research and applications, has spurred structural and functional investigations of RNA. For these studies, the traditional purification method for in vitro tra ...

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Preparation of N-GST Fusion Protein for Affinity Immobilization of RNA

Affinity purification of in vitro transcribed RNA is becoming an attractive alternative to purification using standard denaturing gel electrophoresis. Affinity purification is particularly advantageous because it can be performed in a few hours under non-denaturing condi ...

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Northern Blot Analysis for Expression Profiling of mRNAs and Small RNAs

Northern analysis is a conventional but gold standard method for detection and quantification of gene expression changes. It not only detects the presence of a transcript but also indicates size and relative comparison of transcript abundance on a single membrane. In recent years, it has be ...

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Separating and Analyzing Sulfur-Containing RNAs with Organomercury Gels

Polyacrylamide gel electrophoresis is a widely used technique for RNA analysis and purification. The polyacrylamide matrix is highly versatile for chemical derivitization, enabling facile exploitation of thio-mercury chemistry without the need of tedious manipulations ...

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Detection and Quantification of Alternative Splicing Variants Using RNA-seq

Next-generation sequencing has enabled genome-wide studies of alternative pre-mRNA splicing, allowing for empirical determination, characterization, and quantification of the expressed RNAs in a sample in toto. As a result, RNA sequencing (RNA-seq) has shown tremendous power to ...

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