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Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
Hypersensitive substrate for ribonucleases
B R Kelemen1, T A Klink, M A Behlke
1Department of Biochemistry, University of Wisconsin-Madison, WI 53706, USA.
Nucleic Acids Research
|September 3, 1999
Summary
A novel, highly sensitive fluorescent substrate, 6-FAM-dArUdAdA-6-TAMRA, was developed for detecting ribonucleolytic activity. This assay enables rapid determination of enzyme inhibition constants, crucial for understanding enzyme function.
Area of Science:
- Biochemistry
- Molecular Biology
- Assay Development
Background:
- Ribonucleolytic activity is crucial in various biological processes.
- Existing assays for ribonucleolytic activity often lack sensitivity.
- Developing sensitive assays is key for studying enzyme kinetics and inhibition.
Purpose of the Study:
- To develop a novel, hypersensitive substrate for assaying ribonucleolytic activity.
- To characterize the substrate's performance with specific enzymes like Ribonuclease A and angiogenin.
- To demonstrate the substrate's utility in determining enzyme inhibition constants.
Main Methods:
- Systematic development of a fluorescence-based substrate.
- Utilizing fluorescence quenching of fluorescein by rhodamine, modulated by a ribonucleotide.
- Characterization of substrate cleavage by Bovine pancreatic ribonuclease A and human angiogenin.
Main Results:
- The optimal substrate, 6-FAM-dArUdAdA-6-TAMRA, exhibits a 180-fold fluorescence increase upon cleavage.
- Achieved high catalytic efficiency (kcat/Km) for Ribonuclease A (3.6 x 10^7 M^-1s^-1).
- Demonstrated >10-fold increased sensitivity for angiogenin cleavage compared to existing substrates.
Conclusions:
- The developed substrate is the most sensitive known for detecting ribonucleolytic activity.
- This hypersensitive assay allows for rapid determination of inhibition constants (Ki).
- The substrate is valuable for studying enzyme kinetics and screening inhibitors.
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