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Fluorescence resonance energy transfer (FRET)-based biosensors: visualizing cellular dynamics and bioenergetics

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Abstract

Förster (or fluorescence) resonance energy transfer (FRET) is a process involving the radiation-less transfer of energy from a “donor” fluorophore to an “acceptor” fluorophore. FRET technology enables the quantitative analysis of molecular dynamics in biophysics and in molecular biology, such as the monitoring of protein–protein interactions, protein–DNA interactions, and protein conformational changes. FRET-based biosensors have been utilized to monitor cellular dynamics not only in heterogeneous cellular populations, but also at the single-cell level in real time. Lately, applications of FRET-based biosensors range from basic biological to biomedical disciplines. Despite the diverse applications of FRET, FRET-based sensors still face many challenges. There is an increasing need for higher fluorescence resolution and improved specificity of FRET biosensors. Additionally, as more FRET-based technologies extend to medical diagnostics, the affordability of FRET reagents becomes a significant concern. Here, we will review current advances and limitations of FRET-based biosensor technology and discuss future FRET applications.

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References

  • Ai H, Hazelwood K, Davidson M, Campbell R (2008) Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors. Nat Methods 5:401–403. doi:10.1038/nmeth.1207

    Article  CAS  Google Scholar 

  • Ansbacher T, Srivastava HK, Stein T, Baer R, Merkx M, Shurki A (2012) Calculation of transition dipole moment in fluorescent proteins—towards efficient energy transfer. Phys Chem Chem Phys 14(12):4109–4117

    Article  CAS  Google Scholar 

  • Bailey VJ, Easwaran H, Zhang Y, Griffiths E, Belinsky SA, Herman JG, Baylin SB, Carraway HE, Wang TH (2009) MS-qFRET: a quantum dot-based method for analysis of DNA methylation. Genome Res 19(8):1455–1461

    Article  CAS  Google Scholar 

  • Bellau-Pujol S, Vabret A, Legrand L, Dina J, Gouarin S, Petitjean-Lecherbonnier J, Pozzetto B, Ginevra C, Freymuth FJ (2005) Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses. Virol Methods 126(1–2):53–63

    Article  CAS  Google Scholar 

  • Bujalowski WM, Jezewska MJ (2012) Using structure–function constraints in FRET studies of large macromolecular complexes. Methods Mol Biol 875:135–164

    Article  CAS  Google Scholar 

  • Cardullo RA, Agrawal S, Flores C, Zamecnik PC, Wolf DE (1988) Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc Natl Acad Sci U S A 85(23):8790–8794

    Article  CAS  Google Scholar 

  • Crivat G, Da Silva SM, Reyes DR, Locascio LE, Gaitan M, Rosenzweig N, Rosenzweig Z (2010) Quantum dot FRET-based probes in thin films grown in microfluidic channels. J Am Chem Soc 132(5):1460–1461

    Article  CAS  Google Scholar 

  • Davydov DR, Davydova NY, Halpert JR (2008) Allosteric transitions in cytochrome P450eryF explored with pressure-perturbation spectroscopy, lifetime FRET, and a novel fluorescent substrate, Fluorol-7GA. Biochemistry 47(43):11348–11359

    Article  CAS  Google Scholar 

  • Day RN, Periasamy A, Schaufele F (2001) Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus. Methods 25(1):4–18

    Article  CAS  Google Scholar 

  • Deuschle K, Chaudhuri B, Okumoto S, Lager I, Lalonde S, Frommer WB (2006) Rapid metabolism of glucose detected with FRET glucose nanosensors in epidermal cells and intact roots of Arabidopsis RNA-silencing mutants[W][OA]. Plant Cell 18(9):2314–2325. doi:10.1105/tpc.106.044073

    Article  CAS  Google Scholar 

  • Dietrich A, Buschmann V, Müller C, Sauer M (2002) Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data. J Biotechnol 82(3):211–231

    CAS  Google Scholar 

  • Ellingson JLE, Koziczkowski JJ, Anderson JL, Carlson SA, Sharma VK (2005) Rapid PCR detection of enterohemorrhagic Escherichia coli (EHEC) in bovine food products and feces. Mol Cell Probes 213–217

  • Fernando H, Halpert JR, Davydov DR (2006) Resolution of multiple substrate binding sites in cytochrome P450 3A4: the stoichiometry of the enzyme-substrate complexes probed by FRET and Job’s titration. Biochemistry 45:4199–4209

    Article  CAS  Google Scholar 

  • Gill R, Willner I, Shweky I, Banin U (2005) Fluorescence resonance energy transfer in CdSe/ZnS-DNA conjugates: probing hybridization and DNA cleavage. J Phys Chem B 109(49):23715–23719

    Article  CAS  Google Scholar 

  • Ha T, Enderle T, Ogletree DF, Chemla DS, Selvin PR, Weiss S (1996) Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc Natl Acad Sci U S A 93:6264–6268

    Article  CAS  Google Scholar 

  • Halivni S, Sitt A, Hadar I, Banin U (2012) Effect of nanoparticle dimensionality on fluorescence resonance energy transfer in nanoparticle-dye conjugated systems. ACS Nano 6(3):2758–2765

    Article  CAS  Google Scholar 

  • Hangauer MJ, Bertozzi CR (2008) A FRET-based fluorogenic phosphine for live cell imaging with the Staudinger ligation. Angew Chem Int Ed Engl 47(13):2394–2397. doi:10.1002/anie.200704847

    Article  CAS  Google Scholar 

  • Hoppe A, Christensen K, Swanson JA (2002) Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys J 83:3652–3664

    Article  CAS  Google Scholar 

  • Howell WM (2006) Detection of DNA hybridization using induced fluorescence resonance energy transfer. Methods Mol Biol 335:33–41

    CAS  Google Scholar 

  • Jares-Erijman EA, Jovin TM (2003) FRET imaging. Nat Biotechnol 21:1387–1395. doi:10.1038/nbt896

    Article  CAS  Google Scholar 

  • Johansson MK (2006) Choosing reporter-quencher pairs for efficient quenching through formation of intramolecular dimers. Methods Mol Biol 335:17–29

    CAS  Google Scholar 

  • Joo C, Ha T, Selvin P (2007) Single-molecule FRET with total internal reflection microscopy. Single Mol Tech Lab Man 3–36

  • Kajihara D, Abe R, Iijima I, Komiyama C, Sisido M, Hohsaka T (2006) FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids. Nat Methods 3(11):923–929

    Article  CAS  Google Scholar 

  • Kapanidis AN, Weiss S (2002) Fluorescent probes and bioconjugation chemistries for single-molecule fluorescence analysis of biomolecules. J Chem Phys 117:10953–10964

    Article  CAS  Google Scholar 

  • Kehl SC, Kumar S (2009) Utilization of nucleic acid amplification assays for the detection of respiratory viruses. Clin Lab Med 29(4):661–671

    Article  Google Scholar 

  • Kim K, Barhoumi R, Burghardt R, Safe S (2005) Analysis of estrogen receptor alpha-Sp1 interactions in breast cancer cells by fluorescence resonance energy transfer. Mol Endocrinol 19(4):843–854

    Article  CAS  Google Scholar 

  • Kohl T, Heinze KG, Kuhlemann R, Koltermann A, Schwille P (2002) A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins. Proc Natl Acad Sci U S A 17; 99(19): 12161–12166. doi:10.1073/pnas.192433499

    Google Scholar 

  • Komatsua N, Aokia K, Yamadac M, Yukinagac H, Fujitac Y, Kamiokac Y, Matsudaa M (2011) Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol Biol Cell 22(23)

  • Kwak CK, Kim DM, Lee CS, Lee M, Lee TS (2010) Aldehyde-functionalized, water-soluble poly(para-phenylene): synthesis and streptavidin assay using FRET. J Nanosci Nanotechnol 10(10):6920–6924

    Article  CAS  Google Scholar 

  • Lassaunière R, Kresfelder T, Venter M (2010) A novel multiplex real-time RT-PCR assay with FRET hybridization probes for the detection and quantitation of 13 respiratory viruses. J Virol Methods 165(2):254–260

    Article  Google Scholar 

  • Lee AJ, Ensign AA, Krauss TD, Bren KL (2010) Zinc porphyrin as a donor for FRET in Zn(II)cytochrome c. J Am Chem Soc 132(6):1752–1753

    Article  CAS  Google Scholar 

  • Leriche G, Budin G, Darwich Z, Weltin D, Mély Y, Klymchenko AS, Wagner A (2012) A FRET-based probe with a chemically deactivatable quencher. Chem Commun 48:3224–3226

    Article  CAS  Google Scholar 

  • Li H, Luo Y, Sun X (2011) Fluorescence resonance energy transfer dye-labeled probe for fluorescence-enhanced DNA detection: an effective strategy to greatly improve discrimination ability toward single-base mismatch. Biosens Bioelectron 27(1):167–171

    Article  Google Scholar 

  • Linder KE, Metcalfe E, Nanjappan P, Arunachalam T, Ramos K, Skedzielewski TM, Marinelli ER, Tweedle MF, Nunn AD, Swenson RE (2011) Synthesis, in vitro evaluation, and in vivo metabolism of fluor/quencher compounds containing IRDye 800CW and Black Hole Quencher-3 (BHQ-3). Bioconjug Chem 22(7):1287–1297

    Article  CAS  Google Scholar 

  • Liu H, Liang G, Abdel-Halim ES, Zhu J (2011) A sensitive and selective quantum dots-based FRET biosensor for the detection of cancer marker type IV collagenase. Anal Methods 3:1797–1801

    Article  CAS  Google Scholar 

  • Long F, Gu C, Gu§ AZ, Shi H (2012) Quantum dot/carrier–protein/haptens conjugate as a detection nanobioprobe for FRET-based immunoassay of small analytes with all-fiber microfluidic biosensing platform. Anal Chem 84(8):3646–3653

    Google Scholar 

  • Lovell JF, Chen J, Jarvi MT, Cao WG, Allen AD, Liu Y, Tidwell TT, Wilson BC, Zheng G (2009) FRET quenching of photosensitizer singlet oxygen generation. J Phys Chem B 113(10):3203–3211

    Article  CAS  Google Scholar 

  • Marras SAE, Kramer FR, Tyagi S (2002) Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res 30(21):e122

    Article  Google Scholar 

  • Means JA, Hines JV, Merchant KA, Best RB, Louis JM, Gopich IV, Eaton WA (2005) Fluorescence resonance energy transfer studies of aminoglycoside binding to a T box antiterminator RNA. Bioorg Med Chem Lett 15(8):2169–2172

    Article  CAS  Google Scholar 

  • Merchant KA, Best RB, Louis JM, Gopich IV, Eaton WA (2007) Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations. Proc Natl Acad Sci U S A 104:1528–1533

    Article  CAS  Google Scholar 

  • Meyer T, Teruel MN (2003) Fluorescence imaging of signaling networks. Trends Cell Biol 13:101–106

    Article  CAS  Google Scholar 

  • Moerner WE, Fromm DP (2003) Methods of single-molecule fluorescence spectroscopy and microscopy. Rev Sci Instrum 74:3597–3619

    Article  CAS  Google Scholar 

  • Ogawa M, Kosaka N, Longmire M, Urano Y, Choyke PL, Kobayashi H (2009) Fluorophore-quencher based activatable targeted optical probes for detecting in vivo cancer metastases. Mol Pharm 6(2):386–395. doi:10.1021/mp800115t

    Article  CAS  Google Scholar 

  • Okamura Y, Watanabe Y (2006) Detecting RNA/DNA hybridization using double-labeled donor probes with enhanced fluorescence resonance energy transfer signals. Methods Mol Biol 335:43–56

    CAS  Google Scholar 

  • Paulsson JF, Schultz SW, Köhler M, Leibiger I, Berggren P, Westermark GT (2008) Real-time monitoring of apoptosis by caspase-3-like protease induced FRET reduction triggered by amyloid aggregation. Exp Diabetes Res Vol 2008. doi:10.1155/2008/865850

  • Periasamy A (2001) Fluorescence resonance energy transfer microscopy: a mini review. J Biomed Opt 6(3):287–291

    Article  CAS  Google Scholar 

  • Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5:507–516. doi:10.1038/nmeth.1208

    Article  CAS  Google Scholar 

  • Sturmey RG, O’Toole PJ, Leese HJ (2006) Fluorescence resonance energy transfer analysis of mitochondrial:lipid association in the porcine oocyte. Reproduction 132(6):829–837

    Article  CAS  Google Scholar 

  • Tian H, Ip L, Luo H, Chang DC, Lu KQ (2007) A high throughput drug screen based on fluorescence resonance energy transfer (FRET) for anticancer activity of compounds from herbal medicine. Br J Pharmacol 150(3):321–334. doi:10.1038/sj.bjp.0706988

    Article  CAS  Google Scholar 

  • Tsuji A, Sato Y, Hirano M, Suga T, Koshimoto H, Taguchi T, Ohsuka S (2001) Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer. Biophys J 81(1):501–515

    Article  CAS  Google Scholar 

  • Wang Z, Elbaz J, Remacle F, Levine RD, Willner I (2010) All-DNA finite-state automata with finite memory. Proc Natl Acad Sci U S A 107(51):21996–22001. doi:10.1073/pnas.1015858107

    Article  CAS  Google Scholar 

  • Waterhouse BR, Gijsen M, Barber PR, Tullis ID, Vojnovic B, Kong A (2011) Assessment of EGFR/HER2 dimerization by FRET-FLIM utilizing Alexa-conjugated secondary antibodies in relation to targeted therapies in cancers. Oncotarget 2(9):728–736

    Google Scholar 

  • Wiedenmann J, Oswald F, Nienhaus GU (2009) Fluorescent proteins for live cell imaging: opportunities, limitations, and challenges. IUBMB Life 61(11):1029–1042

    Article  CAS  Google Scholar 

  • Wigelsworth DJ, Krantz BA, Christensen KA, Lacy DB, Juris SJ, Collier RJ (2004) Binding stoichiometry and kinetics of the interaction of a human anthrax toxin receptor, CMG2, with protective antigen. J Biol Chem 279:23349–23356

    Article  CAS  Google Scholar 

  • Willemsel M, Janssen E, de Lange F, Wieringa B, Fransen J (2007) ATP and FRET—a cautionary note. Nat Biotechnol 25:170–172. doi:10.1038/nbt0207-170

    Article  Google Scholar 

  • Yang J, Zhang Z, Lin J, Lu J, Liu BF, Zeng S, Luo Q (2007) Detection of MMP activity in living cells by a genetically encoded surface-displayed FRET sensor. Biochem Biophys Acta 1773(3):400–407

    Article  CAS  Google Scholar 

  • Zadran S, Qin Q, Bi X, Zadran H, Kim Y, Foy MR, Thompson R, Baudry M (2009) 17-Beta-estradiol increases neuronal excitability through MAP kinase-induced calpain activation. Proc Natl Acad Sci U S A 106(51):21936–21941

    Article  Google Scholar 

  • Zhu X, Fu A, Luo KQ (2012) A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents. Biochem Biophys Res Commun 418(4):641–646

    Article  CAS  Google Scholar 

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Acknowledgments

We are thankful guidance and suggestions provided by Dr. Raphael Levine for this review. We would also like to thank the UCLA Department of Pathology and Laboratory Medicine and the David Geffen School of Medicine for providing Early Career Award and support to SZ. This mini-review is dedicated to the late Professor Paola S. Timiras.

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Authors declare no competing financial interests.

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Correspondence to Sohila Zadran.

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Zadran, S., Standley, S., Wong, K. et al. Fluorescence resonance energy transfer (FRET)-based biosensors: visualizing cellular dynamics and bioenergetics. Appl Microbiol Biotechnol 96, 895–902 (2012). https://doi.org/10.1007/s00253-012-4449-6

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  • DOI: https://doi.org/10.1007/s00253-012-4449-6

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