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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...

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Autores principales: Duose, Dzifa Y., Schweller, Ryan M., Zimak, Jan, Rogers, Arthur R., Hittelman, Walter N., Diehl, Michael R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2012
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.
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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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