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Homogeneous Dual-Parametric-Coupled Assay for Simultaneous Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring
[Image: see text] We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange and RAS/RAF-RBD interaction assays. RAS mutations are frequent drivers of multiple different human cancers, but the development of therapeutic strategies has been...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical Society
2020
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7143314/ https://www.ncbi.nlm.nih.gov/pubmed/32106676 http://dx.doi.org/10.1021/acs.analchem.9b05126 |
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author | Kopra, Kari Vuorinen, Emmiliisa Abreu-Blanco, Maria Wang, Qi Eskonen, Ville Gillette, William Pulliainen, Arto T. Holderfield, Matthew Härmä, Harri |
author_facet | Kopra, Kari Vuorinen, Emmiliisa Abreu-Blanco, Maria Wang, Qi Eskonen, Ville Gillette, William Pulliainen, Arto T. Holderfield, Matthew Härmä, Harri |
author_sort | Kopra, Kari |
collection | PubMed |
description | [Image: see text] We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange and RAS/RAF-RBD interaction assays. RAS mutations are frequent drivers of multiple different human cancers, but the development of therapeutic strategies has been challenging. Traditionally, efforts to disrupt the RAS function have focused on nucleotide exchange inhibitors, GTP-RAS interaction inhibitors, and activators increasing GTPase activity of mutant RAS proteins. As the amount of biological knowledge grows, targeted biochemical assays enabling high-throughput screening have become increasingly interesting. We have previously introduced a homogeneous quenching resonance energy transfer (QRET) assay for nucleotide binding studies with RAS and heterotrimeric G proteins. Here, we introduce a novel homogeneous signaling technique called QTR-FRET, which combine QRET technology and time-resolved Förster resonance energy transfer (TR-FRET). The dual-parametric QTR-FRET technique enables the linking of guanine nucleotide exchange factor-induced Eu(3+)-GTP association to RAS, monitored at 615 nm, and subsequent Eu(3+)-GTP-loaded RAS interaction with RAF-RBD-Alexa680 monitored at 730 nm. Both reactions were monitored in a single-well assay applicable for inhibitor screening and real-time reaction monitoring. This homogeneous assay enables separable detection of both nucleotide exchange and RAS/RAF interaction inhibitors using low nanomolar protein concentrations. To demonstrate a wider applicability as a screening and real-time reaction monitoring method, the QTR-FRET technique was also applied for G(i)α GTP-loading and pertussis toxin-catalyzed ADP-ribosylation of G(i)α, for which we synthesized a novel γ-GTP-Eu(3+) molecule. The study indicates that the QTR-FRET detection technique presented here can be readily applied to dual-parametric assays for various targets. |
format | Online Article Text |
id | pubmed-7143314 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | American
Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-71433142020-04-10 Homogeneous Dual-Parametric-Coupled Assay for Simultaneous Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring Kopra, Kari Vuorinen, Emmiliisa Abreu-Blanco, Maria Wang, Qi Eskonen, Ville Gillette, William Pulliainen, Arto T. Holderfield, Matthew Härmä, Harri Anal Chem [Image: see text] We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange and RAS/RAF-RBD interaction assays. RAS mutations are frequent drivers of multiple different human cancers, but the development of therapeutic strategies has been challenging. Traditionally, efforts to disrupt the RAS function have focused on nucleotide exchange inhibitors, GTP-RAS interaction inhibitors, and activators increasing GTPase activity of mutant RAS proteins. As the amount of biological knowledge grows, targeted biochemical assays enabling high-throughput screening have become increasingly interesting. We have previously introduced a homogeneous quenching resonance energy transfer (QRET) assay for nucleotide binding studies with RAS and heterotrimeric G proteins. Here, we introduce a novel homogeneous signaling technique called QTR-FRET, which combine QRET technology and time-resolved Förster resonance energy transfer (TR-FRET). The dual-parametric QTR-FRET technique enables the linking of guanine nucleotide exchange factor-induced Eu(3+)-GTP association to RAS, monitored at 615 nm, and subsequent Eu(3+)-GTP-loaded RAS interaction with RAF-RBD-Alexa680 monitored at 730 nm. Both reactions were monitored in a single-well assay applicable for inhibitor screening and real-time reaction monitoring. This homogeneous assay enables separable detection of both nucleotide exchange and RAS/RAF interaction inhibitors using low nanomolar protein concentrations. To demonstrate a wider applicability as a screening and real-time reaction monitoring method, the QTR-FRET technique was also applied for G(i)α GTP-loading and pertussis toxin-catalyzed ADP-ribosylation of G(i)α, for which we synthesized a novel γ-GTP-Eu(3+) molecule. The study indicates that the QTR-FRET detection technique presented here can be readily applied to dual-parametric assays for various targets. American Chemical Society 2020-02-28 2020-04-07 /pmc/articles/PMC7143314/ /pubmed/32106676 http://dx.doi.org/10.1021/acs.analchem.9b05126 Text en Copyright © 2020 American Chemical Society This is an open access article published under a Creative Commons Attribution (CC-BY) License (http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html) , which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. |
spellingShingle | Kopra, Kari Vuorinen, Emmiliisa Abreu-Blanco, Maria Wang, Qi Eskonen, Ville Gillette, William Pulliainen, Arto T. Holderfield, Matthew Härmä, Harri Homogeneous Dual-Parametric-Coupled Assay for Simultaneous Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring |
title | Homogeneous Dual-Parametric-Coupled Assay for Simultaneous
Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring |
title_full | Homogeneous Dual-Parametric-Coupled Assay for Simultaneous
Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring |
title_fullStr | Homogeneous Dual-Parametric-Coupled Assay for Simultaneous
Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring |
title_full_unstemmed | Homogeneous Dual-Parametric-Coupled Assay for Simultaneous
Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring |
title_short | Homogeneous Dual-Parametric-Coupled Assay for Simultaneous
Nucleotide Exchange and KRAS/RAF-RBD Interaction Monitoring |
title_sort | homogeneous dual-parametric-coupled assay for simultaneous
nucleotide exchange and kras/raf-rbd interaction monitoring |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7143314/ https://www.ncbi.nlm.nih.gov/pubmed/32106676 http://dx.doi.org/10.1021/acs.analchem.9b05126 |
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