Reporter-Based BRET Sensors for Measuring Biological Functions In Vivo

  • Maitreyi Rathod
  • Arijit Mal
  • Abhijit DeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1790)


Genetic reporter systems provide a good alternative to monitor cellular functions in vitro and in vivo and are contributing immensely in experimental research. Reporters like fluorescence and bioluminescence genes, which support optical measurements, provide exquisite sensitivity to the assay systems. In recent years several activatable strategies have been developed, which can relay specialized molecular functions from inside the cells. The application of bioluminescence resonance energy transfer (BRET) is one such strategy that has been proved to be extremely valuable as an in vitro or in vivo assay to measure dynamic events such as protein-protein interactions (PPIs).

The BRET assay using RLuc-YFP was introduced in biological research in the late 1990s and demonstrated the interaction of two proteins involved in circadian rhythm. Since then, BRET has become a popular genetic reporter-based assay for PPI studies due to several inherent attributes that facilitate high-throughput assay development such as rapid and fairly sensitive ratio-metric measurement, the assessment of PPI irrespective of protein location in cellular compartment and cost effectiveness. In BRET-based screening, within a defined proximity range of 10–100 Å, the excited energy state of the luminescent molecule excites the acceptor fluorophore in the form of resonance energy transfer, causing it to emit at its characteristic emission wavelength. Based on this principle, several such donor-acceptor pairs, using Renilla luciferase or its mutants as donor and either GFP2, YFP, mOrange, TagRFP or TurboFP as acceptor, have been reported for use.

In recent years, the applicability of BRET has been greatly enhanced by the adaptation of the assay to multiple detection devices such as a luminescence plate reader, a bioluminescence microscope and a small animal optical imaging platform. Apart from quantitative measurement studies of PPIs and protein dimerization, molecular spectral imaging has expanded the scope for fast screening of pharmacological compounds that modulate PPIs by unifying in vitro, live cell and in vivo animal/plant measurement, all using one assay. Using examples from the literature, we will describe methods to perform in vitro and in vivo BRET imaging experiments and some of its applications.

Key words

Reporter gene Luciferase Fluorescent proteins Optical imaging Bioluminescence resonance energy transfer Protein-protein interactions 


  1. 1.
    Blasberg RG, Tjuvajev JG (2003) Molecular-genetic imaging: current and future perspectives. J Clin Invest 111(11):1620–1629CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ray P et al (2001) Monitoring gene therapy with reporter gene imaging. Semin Nucl Med 31(4):312–320CrossRefPubMedGoogle Scholar
  3. 3.
    Tannous BA (2009) Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nat Protoc 4(4):582–591CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Hall MP et al (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7(11):1848–1857CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Greer LF III, Szalay AA (2002) Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence 17(1):43–74CrossRefPubMedGoogle Scholar
  6. 6.
    Kelkar M, De A (2012) Bioluminescence based in vivo screening technologies. Curr Opin Pharmacol 12(5):592–600CrossRefPubMedGoogle Scholar
  7. 7.
    Mezzanotte L et al (2011) Sensitive dual color in vivo bioluminescence imaging using a new red codon optimized firefly luciferase and a green click beetle luciferase. PLoS One 6(4):e19277CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Loening AM et al (2006) Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output. Protein Eng Des Sel 19(9):391–400CrossRefPubMedGoogle Scholar
  9. 9.
    Nyati S et al (2011) Molecular imaging of TGFbeta-induced Smad2/3 phosphorylation reveals a role for receptor tyrosine kinases in modulating TGFbeta signaling. Clin Cancer Res 17(23):7424–7439CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Dogra S et al (2016) Tango assay for ligand-induced GPCR-beta-arrestin2 interaction: application in drug discovery. Methods Cell Biol 132:233–254CrossRefPubMedGoogle Scholar
  11. 11.
    Barnea G et al (2008) The genetic design of signaling cascades to record receptor activation. Proc Natl Acad Sci U S A 105(1):64–69CrossRefPubMedGoogle Scholar
  12. 12.
    Pogmore JP et al (2016) Using forster-resonance energy transfer to measure protein interactions between Bcl-2 family proteins on mitochondrial membranes. Methods Mol Biol 1419:197–212CrossRefPubMedGoogle Scholar
  13. 13.
    Ray P et al (2008) Monitoring caspase-3 activation with a multimodality imaging sensor in living subjects. Clin Cancer Res 14(18):5801–5809CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Prinz A et al (2006) Novel, isotype-specific sensors for protein kinase A subunit interaction based on bioluminescence resonance energy transfer (BRET). Cell Signal 18(10):1616–1625CrossRefPubMedGoogle Scholar
  15. 15.
    Kang JH, Chung JK (2008) Molecular-genetic imaging based on reporter gene expression. J Nucl Med 49(Suppl 2):164S–179SCrossRefPubMedGoogle Scholar
  16. 16.
    Moroz E et al (2009) Real-time imaging of HIF-1alpha stabilization and degradation. PLoS One 4(4):e5077CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Shachaf CM et al (2004) MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer. Nature 431(7012):1112–1117CrossRefPubMedGoogle Scholar
  18. 18.
    Korpal M et al (2009) Imaging transforming growth factor-beta signaling dynamics and therapeutic response in breast cancer bone metastasis. Nat Med 15(8):960–966CrossRefPubMedGoogle Scholar
  19. 19.
    Nakajima Y et al (2005) Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate. BioTechniques 38(6):891–894CrossRefPubMedGoogle Scholar
  20. 20.
    Arkin MR, Wells JA (2004) Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nat Rev Drug Discov 3(4):301–317CrossRefPubMedGoogle Scholar
  21. 21.
    Phizicky EM, Fields S (1995) Protein-protein interactions: methods for detection and analysis. Microbiol Rev 59(1):94–123PubMedPubMedCentralGoogle Scholar
  22. 22.
    Ciruela F (2008) Fluorescence-based methods in the study of protein-protein interactions in living cells. Curr Opin Biotechnol 19(4):338–343CrossRefPubMedGoogle Scholar
  23. 23.
    Gorokhovatsky AY et al (2004) Fusion of Aequorea victoria GFP and aequorin provides their Ca(2+)-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer. Biochem Biophys Res Commun 320(3):703–711CrossRefPubMedGoogle Scholar
  24. 24.
    Canals M et al (2004) Homodimerization of adenosine A2A receptors: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer. J Neurochem 88(3):726–734CrossRefPubMedGoogle Scholar
  25. 25.
    Terrillon S et al (2003) Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis. Mol Endocrinol 17(4):677–691CrossRefPubMedGoogle Scholar
  26. 26.
    Stoddart LA et al (2015) Application of BRET to monitor ligand binding to GPCRs. Nat Methods 12(7):661–663CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Siddiqui S et al (2013) BRET biosensor analysis of receptor tyrosine kinase functionality. Front Endocrinol (Lausanne) 4:46Google Scholar
  28. 28.
    Kaczor AA et al (2014) Application of BRET for studying G protein-coupled receptors. Mini Rev Med Chem 14(5):411–425CrossRefPubMedGoogle Scholar
  29. 29.
    Paulmurugan R, Umezawa Y, Gambhir SS (2002) Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies. Proc Natl Acad Sci U S A 99(24):15608–15613CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Dragulescu-Andrasi A et al (2011) Bioluminescence resonance energy transfer (BRET) imaging of protein-protein interactions within deep tissues of living subjects. Proc Natl Acad Sci U S A 108(29):12060–12065CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Ogoh K et al (2014) Bioluminescence microscopy using a short focal-length imaging lens. J Microsc 253(3):191–197CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Yamaguchi S et al (2003) Synchronization of cellular clocks in the suprachiasmatic nucleus. Science 302(5649):1408–1412CrossRefPubMedGoogle Scholar
  33. 33.
    De A et al (2009) BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals. FASEB J 23(8):2702–2709CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Coulon V et al (2008) Subcellular imaging of dynamic protein interactions by bioluminescence resonance energy transfer. Biophys J 94(3):1001–1009CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Akiyoshi R et al (2014) Bioluminescence imaging to track real-time armadillo promoter activity in live Drosophila embryos. Anal Bioanal Chem 406(23):5703–5713CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Bergkessel M, Guthrie C (2014) Colony PCR. Methods Enzymol 529:299–309CrossRefGoogle Scholar
  37. 37.
    Verveer PJ et al (2006) Imaging protein interactions by FRET microscopy: FRET measurements by acceptor photobleaching. CSH Protoc 2006(6):pii:pdb.prot4598. Scholar
  38. 38.
    Close DM et al (2011) In vivo bioluminescent imaging (BLI): noninvasive visualization and interrogation of biological processes in living animals. Sensors (Basel) 11(1):180–206CrossRefGoogle Scholar
  39. 39.
    De A, Arora R, Jasani A (2014) Engineering aspects of bioluminescence resonance energy transfer systems. In: Cai W (ed) Engineering in translational medicine. Springer, London, pp 257–300CrossRefGoogle Scholar
  40. 40.
    Mercier JF et al (2002) Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer. J Biol Chem 277(47):44925–44931CrossRefPubMedGoogle Scholar
  41. 41.
    Kroeger KM et al (2001) Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. Detection in living cells using bioluminescence resonance energy transfer. J Biol Chem 276(16):12736–12743CrossRefPubMedGoogle Scholar
  42. 42.
    Felce JH, Knox RG, Davis SJ (2014) Type-3 BRET, an improved competition-based bioluminescence resonance energy transfer assay. Biophys J 106(12):L41–L43CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Inoue Y et al (2009) Comparison of subcutaneous and intraperitoneal injection of D-luciferin for in vivo bioluminescence imaging. Eur J Nucl Med Mol Imaging 36(5):771–779CrossRefPubMedGoogle Scholar
  44. 44.
    Lee KH et al (2003) Cell uptake and tissue distribution of radioiodine labelled D-luciferin: implications for luciferase based gene imaging. Nucl Med Commun 24(9):1003–1009CrossRefPubMedGoogle Scholar
  45. 45.
    De A et al (2013) Evolution of BRET biosensors from live cell to tissue-scale in vivo imaging. Front Endocrinol (Lausanne) 4:131Google Scholar
  46. 46.
    Carriba P et al (2008) Detection of heteromerization of more than two proteins by sequential BRET-FRET. Nat Methods 5(8):727–733CrossRefPubMedGoogle Scholar
  47. 47.
    Branchini BR et al (2011) Sequential bioluminescence resonance energy transfer-fluorescence resonance energy transfer-based ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein. Anal Biochem 414(2):239–245CrossRefPubMedGoogle Scholar
  48. 48.
    Vidi PA, Watts VJ (2009) Fluorescent and bioluminescent protein-fragment complementation assays in the study of G protein-coupled receptor oligomerization and signaling. Mol Pharmacol 75(4):733–739CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Bacart J et al (2008) The BRET technology and its application to screening assays. Biotechnol J 3(3):311–324CrossRefPubMedGoogle Scholar
  50. 50.
    Machleidt T et al (2015) NanoBRET—a Novel BRET platform for the analysis of protein-protein interactions. ACS Chem Biol 10(8):1797–1804CrossRefPubMedGoogle Scholar
  51. 51.
    Kim GB, Kim YP (2012) Analysis of protease activity using quantum dots and resonance energy transfer. Theranostics 2(2):127–138CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Bakayan A et al (2011) Red fluorescent protein-aequorin fusions as improved bioluminescent Ca2+ reporters in single cells and mice. PLoS One 6(5):e19520CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.KS325, Molecular Functional Imaging LabAdvanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre(TMC)Navi MumbaiIndia

Personalised recommendations