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Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
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A Nanopore Phosphorylation Sensor for Single Oligonucleotides and Peptides
Yi-Lun Ying1,2, Jie Yang3, Fu-Na Meng3
1State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.
Research (Washington, D.C.)
|January 9, 2020
Summary
This study introduces a novel nanopore method for simultaneously analyzing oligonucleotide and peptide phosphorylation. This breakthrough enables precise kinase/phosphatase activity measurement for biochemical research and diagnostics.
Area of Science:
- Biochemistry
- Molecular Biology
- Nanotechnology
Background:
- Phosphorylation of oligonucleotides and peptides regulates cellular processes.
- Simultaneous characterization of these modifications is crucial for understanding regulatory pathways.
Purpose of the Study:
- To develop a single-molecule, high-throughput method for evaluating oligonucleotide and peptide phosphorylation.
- To enable label-free, general, and one-step analysis of kinase/phosphatase activity.
Main Methods:
- Utilized an aerolysin nanopore with electrochemically confined effects.
- Analyzed changes in substrate traversing speed and translocation frequency.
- Measured kinase/phosphatase activity via dose-dependent event frequency.
Main Results:
- Phosphorylation accelerated the speed of negatively charged substrates hundreds of times.
- Phosphorylation significantly enhanced the translocation frequency of positively charged substrates.
- Demonstrated nanopore evaluation of T4 oligonucleotide kinase (PNK) activity and real-time dephosphorylation monitoring.
Conclusions:
- The developed aerolysin nanopore method offers a straightforward approach for analyzing phosphorylation in both oligonucleotides and peptides.
- This technique advances nanopore enzymology for fundamental research, clinical diagnosis, and drug discovery.
