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Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes05:33

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

Here, a protocol for assaying nucleic acid metabolizing enzymes is presented, using examples of ligase, nuclease, and polymerase enzymes. The assay utilizes fluorescently labeled and unlabeled oligonucleotides that can be combined to form duplexes mimicking RNA and/or DNA damages or pathway intermediates, allowing for the characterization of enzyme behavior.
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Chemical Triphosphorylation of Oligonucleotides13:19

Chemical Triphosphorylation of Oligonucleotides

Oligonucleotide 5′-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5′-triphosphates, starting from oligonucleotides prepared by standard automated synthesis...
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An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes09:45

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot...
11.6K
Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution10:53

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.
9.5K
Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro09:16

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in...
13.1K
Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides16:42

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.
27.3K