Rna Substrate Analysis
Rna Substrate Analysis - Related Videos
09:16
Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
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Cited by 1 •
2014
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 vitro.
11:53
Substrate Generation for Endonucleases of CRISPR/Cas Systems
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Cited by 4 •
2012
CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.
07:35
Computational Analysis Tutorial for Chimeric Small Noncoding RNA: Target RNA Sequencing Libraries
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Cited by 2 •
2023
Here, we present a protocol demonstrating the installation and use of a bioinformatics pipeline to analyze chimeric RNA sequencing data used in the study of in vivo RNA:RNA interactions.
07:55
An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA
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Cited by 3 •
2023
Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.
12:04
RNA Analysis of Environmental Samples Using RT-PCR
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2023
Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona Demonstrating Author: Bradley Schmitz Reverse transcription-polymerase chain reaction (RT-PCR) involves the same process as conventional PCR — cycling temperature to amplify nucleic acids. However, while conventional PCR only amplifies deoxyribonucleic acids (DNA), RT-PCR enables the amplification of ribonucleic acids (RNA) through the formation of complementary DNA (cDNA). This enables RNA-based organisms...
FAQs
Enzymes can respond to both the nucleotide sequence and the folded structure of an RNA substrate. These features influence how the enzyme binds and which molecular positions remain accessible for transformation. Comparing substrates with different sequence or structural features can therefore reveal binding determinants and explain why an enzyme acts on some RNA molecules more efficiently than others.
Binding can be evaluated by detecting enzyme association with the RNA, whereas catalysis is indicated by substrate conversion into reaction products. Tracking substrate depletion, product formation, or changes in reaction rate helps separate these outcomes. This distinction matters because an enzyme may recognize and bind a substrate without efficiently carrying out cleavage, modification, or synthesis.
Reaction rates provide quantitative evidence about how quickly an enzyme transforms an RNA substrate under defined conditions. Comparing rates can help identify catalytic activity, substrate preferences, and kinetic parameters. These measurements add information beyond observing whether a reaction occurs, allowing biochemical studies to relate RNA features or experimental conditions to the efficiency of enzyme action.
A typical workflow begins with a defined RNA substrate and an enzyme, followed by incubation under controlled conditions. The reaction is then assessed by measuring products, remaining substrate, or changes in reaction rate. The selected measurement depends on whether the investigation focuses on cleavage, modification, synthesis, binding, catalytic activity, or substrate specificity.
Product formation shows that the RNA substrate has undergone a measurable transformation, while substrate depletion indicates loss of the starting material during the reaction. Examining either outcome, together with reaction-rate changes, can establish catalytic activity and support comparisons of substrate specificity. These results also help characterize the extent and behavior of the biochemical reaction.
This approach is useful for studying RNA processing, degradation, modification, synthesis, translation, and regulation. It also supports characterization of ribozymes and RNA-dependent enzymes by connecting measurable reactions with substrate features and kinetic behavior. In applied research, the resulting information can help identify molecular determinants relevant to potential therapeutic targets without relying only on cellular observations.