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Configuring robust DNA strand displacement reactions for in situ molecular analyses
The number of distinct biomolecules that can be visualized within individual cells and tissue sections via fluorescence microscopy is limited by the spectral overlap of the fluorescent dye molecules that are coupled permanently to their targets. This issue prohibits characterization of important fun...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326323/ https://www.ncbi.nlm.nih.gov/pubmed/22156404 http://dx.doi.org/10.1093/nar/gkr1209 |
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author | Duose, Dzifa Y. Schweller, Ryan M. Zimak, Jan Rogers, Arthur R. Hittelman, Walter N. Diehl, Michael R. |
author_facet | Duose, Dzifa Y. Schweller, Ryan M. Zimak, Jan Rogers, Arthur R. Hittelman, Walter N. Diehl, Michael R. |
author_sort | Duose, Dzifa Y. |
collection | PubMed |
description | The number of distinct biomolecules that can be visualized within individual cells and tissue sections via fluorescence microscopy is limited by the spectral overlap of the fluorescent dye molecules that are coupled permanently to their targets. This issue prohibits characterization of important functional relationships between different molecular pathway components in cells. Yet, recent improved understandings of DNA strand displacement reactions now provides opportunities to create programmable labeling and detection approaches that operate through controlled transient interactions between different dynamic DNA complexes. We examined whether erasable molecular imaging probes could be created that harness this mechanism to couple and then remove fluorophore-bearing oligonucleotides to and from DNA-tagged protein markers within fixed cell samples. We show that the efficiency of marker erasing via strand displacement can be limited by non-toehold mediated stand exchange processes that lower the rates that fluorophore-bearing strands diffuse out of cells. Two probe constructions are described that avoid this problem and allow efficient fluorophore removal from their targets. With these modifications, we show one can at least double the number of proteins that can be visualized on the same cells via reiterative in situ labeling and erasing of markers on cells. |
format | Online Article Text |
id | pubmed-3326323 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-33263232012-04-16 Configuring robust DNA strand displacement reactions for in situ molecular analyses Duose, Dzifa Y. Schweller, Ryan M. Zimak, Jan Rogers, Arthur R. Hittelman, Walter N. Diehl, Michael R. Nucleic Acids Res Synthetic Biology and Chemistry The number of distinct biomolecules that can be visualized within individual cells and tissue sections via fluorescence microscopy is limited by the spectral overlap of the fluorescent dye molecules that are coupled permanently to their targets. This issue prohibits characterization of important functional relationships between different molecular pathway components in cells. Yet, recent improved understandings of DNA strand displacement reactions now provides opportunities to create programmable labeling and detection approaches that operate through controlled transient interactions between different dynamic DNA complexes. We examined whether erasable molecular imaging probes could be created that harness this mechanism to couple and then remove fluorophore-bearing oligonucleotides to and from DNA-tagged protein markers within fixed cell samples. We show that the efficiency of marker erasing via strand displacement can be limited by non-toehold mediated stand exchange processes that lower the rates that fluorophore-bearing strands diffuse out of cells. Two probe constructions are described that avoid this problem and allow efficient fluorophore removal from their targets. With these modifications, we show one can at least double the number of proteins that can be visualized on the same cells via reiterative in situ labeling and erasing of markers on cells. Oxford University Press 2012-04 2011-12-10 /pmc/articles/PMC3326323/ /pubmed/22156404 http://dx.doi.org/10.1093/nar/gkr1209 Text en © The Author(s) 2011. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Synthetic Biology and Chemistry Duose, Dzifa Y. Schweller, Ryan M. Zimak, Jan Rogers, Arthur R. Hittelman, Walter N. Diehl, Michael R. Configuring robust DNA strand displacement reactions for in situ molecular analyses |
title | Configuring robust DNA strand displacement reactions for in situ molecular analyses |
title_full | Configuring robust DNA strand displacement reactions for in situ molecular analyses |
title_fullStr | Configuring robust DNA strand displacement reactions for in situ molecular analyses |
title_full_unstemmed | Configuring robust DNA strand displacement reactions for in situ molecular analyses |
title_short | Configuring robust DNA strand displacement reactions for in situ molecular analyses |
title_sort | configuring robust dna strand displacement reactions for in situ molecular analyses |
topic | Synthetic Biology and Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326323/ https://www.ncbi.nlm.nih.gov/pubmed/22156404 http://dx.doi.org/10.1093/nar/gkr1209 |
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