A Platform of BRET-FRET Hybrid Biosensors for Optogenetics, Chemical Screening, and in Vivo Imaging

Overview

Journal Sci Rep

Specialty Science

Date 2018 Jun 14

PMID 29895862

Citations 25

Authors

Naoki Komatsu

Kenta Terai

Ayako Imanishi

Yuji Kamioka

Kenta Sumiyama

Takashi Jin

Yasushi Okada

Takeharu Nagai

Michiyuki Matsuda

Affiliations

Soon will be listed here.

Abstract

Genetically encoded biosensors based on the principle of Förster resonance energy transfer comprise two major classes: biosensors based on fluorescence resonance energy transfer (FRET) and those based on bioluminescence energy transfer (BRET). The FRET biosensors visualize signaling-molecule activity in cells or tissues with high resolution. Meanwhile, due to the low background signal, the BRET biosensors are primarily used in drug screening. Here, we report a protocol to transform intramolecular FRET biosensors to BRET-FRET hybrid biosensors called hyBRET biosensors. The hyBRET biosensors retain all properties of the prototype FRET biosensors and also work as BRET biosensors with dynamic ranges comparable to the prototype FRET biosensors. The hyBRET biosensors are compatible with optogenetics, luminescence microplate reader assays, and non-invasive whole-body imaging of xenograft and transgenic mice. This simple protocol will expand the use of FRET biosensors and enable visualization of the multiscale dynamics of cell signaling in live animals.

Citing Articles

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References

Stoddart L, Johnstone E, Wheal A, Goulding J, Robers M, Machleidt T . Application of BRET to monitor ligand binding to GPCRs. Nat Methods. 2015; 12(7):661-663. PMC: 4488387. DOI: 10.1038/nmeth.3398. View

Robers M, Dart M, Woodroofe C, Zimprich C, Kirkland T, Machleidt T . Target engagement and drug residence time can be observed in living cells with BRET. Nat Commun. 2015; 6:10091. PMC: 4686764. DOI: 10.1038/ncomms10091. View

Kotera I, Iwasaki T, Imamura H, Noji H, Nagai T . Reversible dimerization of Aequorea victoria fluorescent proteins increases the dynamic range of FRET-based indicators. ACS Chem Biol. 2010; 5(2):215-22. DOI: 10.1021/cb900263z. View

Saito K, Chang Y, Horikawa K, Hatsugai N, Higuchi Y, Hashida M . Luminescent proteins for high-speed single-cell and whole-body imaging. Nat Commun. 2012; 3:1262. PMC: 3535334. DOI: 10.1038/ncomms2248. View

Schaub F, Reza M, Flaveny C, Li W, Musicant A, Hoxha S . Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis. Cancer Res. 2015; 75(23):5023-33. PMC: 4668208. DOI: 10.1158/0008-5472.CAN-14-3538. View

Miyawaki A, Niino Y . Molecular spies for bioimaging--fluorescent protein-based probes. Mol Cell. 2015; 58(4):632-43. DOI: 10.1016/j.molcel.2015.03.002. View

Wu Y, Frey D, Lungu O, Jaehrig A, Schlichting I, Kuhlman B . A genetically encoded photoactivatable Rac controls the motility of living cells. Nature. 2009; 461(7260):104-8. PMC: 2766670. DOI: 10.1038/nature08241. View

Lam A, St-Pierre F, Gong Y, Marshall J, Cranfill P, Baird M . Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods. 2012; 9(10):1005-12. PMC: 3461113. DOI: 10.1038/nmeth.2171. View

Kennedy M, Hughes R, Peteya L, Schwartz J, Ehlers M, Tucker C . Rapid blue-light-mediated induction of protein interactions in living cells. Nat Methods. 2010; 7(12):973-5. PMC: 3059133. DOI: 10.1038/nmeth.1524. View

10.

Niwa H, Yamamura K, Miyazaki J . Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991; 108(2):193-9. DOI: 10.1016/0378-1119(91)90434-d. View

11.

Urasaki A, Morvan G, Kawakami K . Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics. 2006; 174(2):639-49. PMC: 1602067. DOI: 10.1534/genetics.106.060244. View

12.

Smith A, Mancini M, Nie S . Bioimaging: second window for in vivo imaging. Nat Nanotechnol. 2009; 4(11):710-1. PMC: 2862008. DOI: 10.1038/nnano.2009.326. View

13.

Komatsu N, Fujita Y, Matsuda M, Aoki K . mTORC1 upregulation via ERK-dependent gene expression change confers intrinsic resistance to MEK inhibitors in oncogenic KRas-mutant cancer cells. Oncogene. 2015; 34(45):5607-16. DOI: 10.1038/onc.2015.16. View

14.

Zhao H, Doyle T, Coquoz O, Kalish F, Rice B, Contag C . Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo. J Biomed Opt. 2005; 10(4):41210. DOI: 10.1117/1.2032388. View

15.

Palmer A, Qin Y, Park J, McCombs J . Design and application of genetically encoded biosensors. Trends Biotechnol. 2011; 29(3):144-52. PMC: 3433949. DOI: 10.1016/j.tibtech.2010.12.004. View

16.

Rodriguez E, Tran G, Gross L, Crisp J, Shu X, Lin J . A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat Methods. 2016; 13(9):763-9. PMC: 5007177. DOI: 10.1038/nmeth.3935. View

17.

Goedhart J, von Stetten D, Noirclerc-Savoye M, Lelimousin M, Joosen L, Hink M . Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat Commun. 2012; 3:751. PMC: 3316892. DOI: 10.1038/ncomms1738. View

18.

Aoki K, Matsuda M . Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors. Nat Protoc. 2009; 4(11):1623-31. DOI: 10.1038/nprot.2009.175. View

19.

Xu X, Soutto M, Xie Q, Servick S, Subramanian C, von Arnim A . Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues. Proc Natl Acad Sci U S A. 2007; 104(24):10264-9. PMC: 1891211. DOI: 10.1073/pnas.0701987104. View

20.

Kamioka Y, Sumiyama K, Mizuno R, Sakai Y, Hirata E, Kiyokawa E . Live imaging of protein kinase activities in transgenic mice expressing FRET biosensors. Cell Struct Funct. 2012; 37(1):65-73. DOI: 10.1247/csf.11045. View