Cellular encoding of Cy dyes for single-molecule imaging
A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and C...
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2016-12-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/19088 |
| _version_ | 1811253352038662144 |
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| author | Lilia Leisle Rahul Chadda John D Lueck Daniel T Infield Jason D Galpin Venkatramanan Krishnamani Janice L Robertson Christopher A Ahern |
| author_facet | Lilia Leisle Rahul Chadda John D Lueck Daniel T Infield Jason D Galpin Venkatramanan Krishnamani Janice L Robertson Christopher A Ahern |
| author_sort | Lilia Leisle |
| collection | DOAJ |
| description | A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single-molecule resolution in a cellular environment. |
| first_indexed | 2024-04-12T16:48:41Z |
| format | Article |
| id | doaj.art-217eeb09083548d5bba5fc63e7377a1b |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T16:48:41Z |
| publishDate | 2016-12-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-217eeb09083548d5bba5fc63e7377a1b2022-12-22T03:24:27ZengeLife Sciences Publications LtdeLife2050-084X2016-12-01510.7554/eLife.19088Cellular encoding of Cy dyes for single-molecule imagingLilia Leisle0Rahul Chadda1John D Lueck2Daniel T Infield3Jason D Galpin4Venkatramanan Krishnamani5Janice L Robertson6https://orcid.org/0000-0002-5499-9943Christopher A Ahern7https://orcid.org/0000-0002-7975-2744Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United StatesDepartment of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United StatesDepartment of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United StatesDepartment of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United StatesDepartment of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United StatesDepartment of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United StatesDepartment of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United StatesDepartment of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, United StatesA general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single-molecule resolution in a cellular environment.https://elifesciences.org/articles/19088single molecule imaginggenetic code expansionTIRFmembrane proteinscell-free protein synthesisorthogonal tRNA |
| spellingShingle | Lilia Leisle Rahul Chadda John D Lueck Daniel T Infield Jason D Galpin Venkatramanan Krishnamani Janice L Robertson Christopher A Ahern Cellular encoding of Cy dyes for single-molecule imaging eLife single molecule imaging genetic code expansion TIRF membrane proteins cell-free protein synthesis orthogonal tRNA |
| title | Cellular encoding of Cy dyes for single-molecule imaging |
| title_full | Cellular encoding of Cy dyes for single-molecule imaging |
| title_fullStr | Cellular encoding of Cy dyes for single-molecule imaging |
| title_full_unstemmed | Cellular encoding of Cy dyes for single-molecule imaging |
| title_short | Cellular encoding of Cy dyes for single-molecule imaging |
| title_sort | cellular encoding of cy dyes for single molecule imaging |
| topic | single molecule imaging genetic code expansion TIRF membrane proteins cell-free protein synthesis orthogonal tRNA |
| url | https://elifesciences.org/articles/19088 |
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