Directed evolution of a magnetic resonance imaging contrast agent for noninvasive imaging of dopamine
Abstract
The development of molecular probes that allow in vivo imaging of neural signaling processes with high temporal and spatial resolution remains challenging. Here we applied directed evolution techniques to create magnetic resonance imaging (MRI) contrast agents sensitive to the neurotransmitter dopamine. The sensors were derived from the heme domain of the bacterial cytochrome P450-BM3 (BM3h). Ligand binding to a site near BM3h's paramagnetic heme iron led to a drop in MRI signal enhancement and a shift in optical absorbance. Using an absorbance-based screen, we evolved the specificity of BM3h away from its natural ligand and toward dopamine, producing sensors with dissociation constants for dopamine of 3.3–8.9 μM. These molecules were used to image depolarization-triggered neurotransmitter release from PC12 cells and in the brains of live animals. Our results demonstrate the feasibility of molecular-level functional MRI using neural activity–dependent sensors, and our protein engineering approach can be generalized to create probes for other targets.
Additional Information
© 2010 Nature Publishing Group. Received 25 June 2009; accepted 27 January 2010; published online 28 February 2010. We thank V. Lelyveld for helpful discussions and assistance with in vitro measurements, N. Shah for help with MRI procedures and W. Schulze for help with automated analysis methods. We are grateful to C. Jennings and D. Cory for comments and suggestions about the manuscript, and to D. Vaughan for consultation regarding histology. We thank P. Caravan and again D. Cory for access to low-field relaxometers. M.G.S. thanks the Fannie and John Hertz Foundation and the Paul and Daisy Soros Fellowship for generous support. This work was funded by a Dana Foundation Brain & Immuno-Imaging grant, a Raymond & Beverley Sackler Fellowship and US National Institutes of Health (NIH) grants R01-DA28299 and DP2-OD2441 (New Innovator Award) to A.J., NIH grant R01-GM068664 and a grant from the Caltech Jacobs Institute for Molecular Medicine to F.H.A. and NIH grant R01-DE013023 to R.L. M.G.S. conceived and performed the directed evolution and in vitro assessment of dopamine sensors; G.G.W. designed and conducted the in vivo experiments; P.A.R. performed directed evolution screening for BM3h variants; J.O.S. assisted with screening and in vitro experiments; B.K. assisted with data analysis for in vivo experiments; A.S. assisted with in vivo experiments; C.R.O. worked with M.G.S. to establish BM3h screening methods; R.L. provided consultation and essential materials; F.H.A. supervised the directed evolution work; A.J. established research direction, supervised the project overall and co-wrote the paper with M.G.S. and G.G.W.Attached Files
Accepted Version - nihms280479.pdf
Supplemental Material - nbt.1609-S1.pdf
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Additional details
- PMCID
- PMC3073400
- Eprint ID
- 17801
- Resolver ID
- CaltechAUTHORS:20100326-111303498
- Fannie and John Hertz Foundation
- Paul and Daisy Soros Fellowship
- Dana Foundation
- Raymond and Beverly Sackler Foundation
- NIH
- R01-DA28299
- NIH
- DP2-OD2441
- NIH
- R01-GM068664
- Jacobs Institute for Molecular Medicine
- Created
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2010-03-30Created from EPrint's datestamp field
- Updated
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2021-11-08Created from EPrint's last_modified field