Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy
Abstract The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of the...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-12-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-45413-8 |
| _version_ | 1797397965354041344 |
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| author | Guillaume A. Castillon Sebastien Phan Junru Hu Daniela Boassa Stephen R. Adams Mark H. Ellisman |
| author_facet | Guillaume A. Castillon Sebastien Phan Junru Hu Daniela Boassa Stephen R. Adams Mark H. Ellisman |
| author_sort | Guillaume A. Castillon |
| collection | DOAJ |
| description | Abstract The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques. |
| first_indexed | 2024-03-09T01:18:51Z |
| format | Article |
| id | doaj.art-b07668336e4f4e469bef9ea0ee763d1a |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-09T01:18:51Z |
| publishDate | 2023-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-b07668336e4f4e469bef9ea0ee763d1a2023-12-10T12:19:14ZengNature PortfolioScientific Reports2045-23222023-12-0113111410.1038/s41598-023-45413-8Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopyGuillaume A. Castillon0Sebastien Phan1Junru Hu2Daniela Boassa3Stephen R. Adams4Mark H. Ellisman5Department of Neurosciences, University of California San DiegoDepartment of Neurosciences, University of California San DiegoDepartment of Neurosciences, University of California San DiegoDepartment of Neurosciences, University of California San DiegoDepartment of Pharmacology, University of California San DiegoDepartment of Neurosciences, University of California San DiegoAbstract The binding and interaction of proteins with nucleic acids such as DNA and RNA constitutes a fundamental biochemical and biophysical process in all living organisms. Identifying and visualizing such temporal interactions in cells is key to understanding their function. To image sites of these events in cells across scales, we developed a method, named PROMPT for PROximal Molecular Probe Transfer, which is applicable to both light and correlative electron microscopy. This method relies on the transfer of a bound photosensitizer from a protein known to associate with specific nucleic acid sequence, allowing the marking of the binding site on DNA or RNA in fixed cells. The method produces a fluorescent mark at the site of their interaction, that can be made electron dense and reimaged at high resolution in the electron microscope. As proof of principle, we labeled in situ the interaction sites between the histone H2B and nuclear DNA. As an example of application for specific RNA localizations we labeled different nuclear and nucleolar fractions of the protein Fibrillarin to mark and locate where it associates with RNAs, also using electron tomography. While the current PROMPT method is designed for microscopy, with minimal variations, it can be potentially expanded to analytical techniques.https://doi.org/10.1038/s41598-023-45413-8 |
| spellingShingle | Guillaume A. Castillon Sebastien Phan Junru Hu Daniela Boassa Stephen R. Adams Mark H. Ellisman Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy Scientific Reports |
| title | Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy |
| title_full | Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy |
| title_fullStr | Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy |
| title_full_unstemmed | Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy |
| title_short | Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy |
| title_sort | proximal molecular probe transfer prompt a new approach for identifying sites of protein nucleic acid interaction in cells by correlated light and electron microscopy |
| url | https://doi.org/10.1038/s41598-023-45413-8 |
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