Application of fluorescence resonance energy transfer in the clinical laboratory: Routine and...

Review Article

Application of Fluorescence Resonance Energy Transferin the Clinical Laboratory: Routine and Research

Janos Szollo9 si,* Sandor Damjanovich, and Laszlo MatyusDepartment of Biophysics and Cell Biology, University Medical School of Debrecen, Debrecen, Hungary

Fluorescence resonance energy transfer (FRET) phenomenon has been applied to a variety of scientificchallenges in the past. The potential utility of this biophysical tool will be revisited in the 21st century. Therapid digital signal processing in conjunction with personal computers and the wide use of multicolor lasertechnology in clinical flow cytometry opened an opportunity for multiplexed assay systems. The concept isvery simple. Color-coded microspheres are used as solid-phase matrix for the detection of fluorescent labeledmolecules. It is the homogeneous assay methodology in which solid-phase particles behave similarly to thedynamics of a liquid environment. This approach offers a rapid cost-effective technology that harnesses awide variety of fluorochromes and lasers. With this microsphere technology, the potential applications forclinical flow cytometry in the future are enormous. This new approach of well-established clinically provenmethods sets the stage to briefly review the theoretical and practical aspects of FRET technology. The reviewshows various applications of FRET in research and clinical laboratories. Combination of FRET withmonoclonal antibodies resulted in a boom of structural analysis of proteins in solutions and also in biologicalmembranes. Cell surface mapping of cluster of differentiation molecules on immunocompetent cells hasgained more and more interest in the last decade. Several examples for biological applications are discussedin detail. FRET can also be used to improve the spectral characteristics of fluorescent dyes and dyecombinations, such as the tandem dyes in flow and image cytometry and the FRET primers in DNA sequencingand polymerase chain reactions. The advantages and disadvantages of donor-acceptor dye combinations areevaluated. In addition, the sensitivity of FRET provides the basis for establishing fast, robust, and accurateenzyme assays and immunoassays. Benefits and limitations of FRET-based assays are thoroughly scrutinized. At theend of the paper we review the future of FRET methodology. Cytometry (Comm. Clin. Cytometry) 34:159–179,1998. r 1998 Wiley-Liss, Inc.

Key terms: fluorescence resonance energy transfer; tandem dyes; enzyme assay; immunoassays; DNAsequencing; cell surface mapping

Although the phenomenon of fluorescence resonanceenergy transfer (FRET) was observed by Perrin at thebeginning of the century, it was Theodor Forster whoproposed a theory describing long-range dipole-dipoleinteractions between fluorescent molecules approxi-mately 50 years ago (29,30). He derived an equation thatrelates the transfer rate to the interchromophore and thespectroscopic properties of the chromophores. The inge-nious discovery that a fluorescence dipole-dipole interac-tion, besides orientational and other spectroscopic param-eters, which can be kept under control, depends on thenegative sixth power of their distance provided one of themost sensitive methods to measure molecular and atomicdistance relations at the nanometer level. The utilization ofthis method in chemistry and biochemistry reached apinnacle in the 1970s. Cell biological applications alsostarted in the 1970s, but widespread application beganonly a decade later and is still flourishing.

FRET is widely utilized for a variety of applications. Inone series of studies, FRET is used as a tool for ensuringhigh sensitivity. FRET technology can be incorporated intochromatographic assays, electrophoresis, microscopy, andflow cytometry. FRET also can be used for improvingspectral characteristics of fluorescent dyes. In anothergroup of studies, FRET is used to obtain structural informa-tion that is otherwise difficult to obtain. The majoradvantage of applying FRET for structural studies is that

Contract grant sponsor: Hungarian Academy of Sciences; Contractgrant numbers: OTKA T019372, T023835, and 6221; Contract grantsponsor: Ministry of Public Health; Contract grant numbers: ETT 344/96and 359/96; Contract grant sponsor: Ministry of Education; Contractgrant number: FKFP 1015/1997.

*Correspondence to: Janos Szollo9si, Department of Biophysics and CellBiology, University Medical School of Debrecen, P.O. Box 39, Nagyerdeikrt. 98, H-4012 Debrecen, Hungary.

E-mail: [email protected] 9 March 1988; Accepted 29 May 1988

Cytometry (Communications in Clinical Cytometry) 34:159–179 (1998)

r 1998 Wiley-Liss, Inc.

owing to the specific labeling the experimental object canbe investigated in situ and/or in vivo with little or nointerference regardless of the complexity and heterogene-ity of the system.

Although numerous reviews are available on fluores-cence resonance energy transfer (17,18,21,28,69–71,90,94,98,100,106–108,127), there is paucity of information re-garding the clinical applications of the technology. In thisreview, an attempt has been made to summarize anddescribe recent applications of the FRET in routine andresearch clinical laboratories. The topics and publicationsdiscussed in this review undoubtedly reflect the interest ofthe authors, but we did our best to review relevantliterature. First, we describe briefly the theory behindFRET, then the measuring techniques are introduced.Next, papers dealing with the structure and cell surfacedistribution of cluster of differentiation (CD) molecules aresummarized and the analytical applications of FRET aredescribed. At the end of the paper, the future prospects ofFRET applications are discussed. The papers reviewedwere selected because they had introduced new or im-proved methods for FRET measurements and analysis orled to a better understanding of important biologicalstructures.

Three directions with enormous clinical potential influ-enced the selection of reviewed topics in the future. Thecurrent interest in quantitative fluorescence determinationwith simultaneous multicolor immunophenotyping as itapplies to clinical immunology. Secondly, the rapidlyincreasing interest in flow cytometer-based multiplexedimmunoassays. This microsphere-based technology revis-its the well-characterized solid-phase immunoassays andbioassays that were developed in the 1970s. In the case ofthe multiplexed solid-phase technology, the epitopes aresuspended uniformly in a liquid environment on solidphase (31,73). This paradox-like situation permits harness-ing the benefit of both liquid and solid-phase technologies.The novel application is based on rapid identifications ofvarious microsphere populations with subtle differencesattributed to spectral emission profiles related to variousdistinct shades of dyes embedded in their surfaces. Thepermutations of discrete microsphere types rendered byfour-color clinical flow cytometry is staggering. Eachcolor-coded population of microsphere set carries reac-tants for a distinct bioassay. The solid-phase compartment-based technology opens new doors in unrelated areas,such as pharmacokinetics for drug discovery studies,nucleic acid-based tissue typing, and competitive DNAhybridization, that were not readily available for rapid flowcytometric-based applications in the past. Finally, in thefuture, the miniaturization of flow cytometric instrumenta-tion will force a new approach to evaluate the relationshipbetween solid phase and liquid phase in the context ofrapid immunochemistry. There is a need to revisit the roleof transfer rate equation that relates to the interchromo-phore and the spectroscopic properties of the chromo-phores. The ultimate exploitation of FRET will come at theend of this century by focusing on the interface betweenmolecular pharmacology and medical chemistry for drug

development. Will flow cytometry have a role in thisfascinating scientific challenge?

THEORY OF FRET

The theory of FRET was first described by Forster in thelate forties, its application to measure distances betweendonor and acceptor molecules came decades later(20,21,28–30,58,69–71,94,100,106–108). FRET is a radia-tionless process in which energy is transferred from anexcited donor molecule to an acceptor molecule underfavorable conditions. One of the most important factors isthe distance between the donor and acceptor molecules.Because the rate of energy transfer is inversely propor-tional to the sixth power of the distance between thedonor and acceptor, the energy transfer efficiency isextremely sensitive to distance changes. Energy transferoccurs in the 1- to 10-nm distance range with measurableefficiency, and these distances correlate well with macro-molecular dimensions.

Consider a system with two different fluorophores inwhich the molecule with higher energy absorption isdefined as the donor (D) and the one with lower energyabsorption is defined as acceptor (A). If the donor is in theexcited state, it will lose energy by internal conversionuntil it reaches the ground vibrational level of the firstexcited state. If the donor emission energies overlap withthe acceptor absorption energies, through weak coupling,the following resonance can occur:

D* 1 Al D 1 A* (1)

where D and A denote the donor and the acceptormolecules in ground state, and D* and A* denote the firstexcited states of the fluorophores. The rate of the forwardprocess is kT and the rate of the inverse process is k2T.Because vibrational relaxation converts the excited accep-tor into the ground vibrational level, the inverse process ishighly unlikely to occur. As a result, the donor moleculesbecome quenched, while the acceptor molecules becomeexcited and, under favorable conditions, can emit fluores-cent light. This latter process is called sensitized emission(Fig. 1).

According to the theory of Forster, the rate (kT) andefficiency (E) of energy transfer can be written as:

kT 5 const Jn24R26k2 (2)

E 5kT

kT 1 kF 1 kD

(3)

where kF is the rate constant of fluorescence emission ofthe donor and kD is the sum of the rate constants of allother deexcitation processes of the donor. R is theseparation distance between the donor and acceptormolecules, and k2 is an orientation factor that is a functionof the relative orientation of the donor’s emission dipoleand the acceptor’s absorption dipole. Other parametersare n, the refractive index of the medium, and J, the

160 SZOLLO9 SI ET AL.

spectral overlap integral, which is proportional to theoverlap in the emission spectrum of the donor and theabsorption spectrum of the acceptor:

J 5e FD(l)eA(l)l24 dl

e FD(l) d(l)(4)

where FD(l) is the fluorescence intensity of the donor atwavelength l, eA(l) is the molar extinction coefficient ofthe acceptor.

For dipole-dipole energy transfer it can be shown that:

k2 5 (cos a 2 3 cos b cos g)2 (5)

where a is the angle of the transition moments of thedonor and the acceptor, and b and g are the anglesbetween the line joining the centers of the fluorophoresand their transition moments (Fig. 2). Uncertainties in thevalue of k2 cause the greatest error in distance determina-tion by energy transfer. (Fortunately R depends on (k2)1/6).The direct measurement of its value is impossible. Fromtheoretical considerations k2 is in the range between 0 and4. Assuming random orientation of the donor and theacceptor k2 becomes 2/3. In the case of cell surfacecomponents this assumption is reasonable (19). It can beshown that:

E 5R26

R26 1 R026

(6)

From Equations 2 and 3 it follows that:

kT 51

t 1R0

R 26

(7)

where t is the donor’s lifetime in the absence of theacceptor, and R0 is the characteristic distance between thedonor and the acceptor when the transfer efficiency is50%.

R0 5 const( JK2QDn24)1/6 (8)

In this equation, QD is the quantum efficiency of the donorin the absence of the acceptor.

The energy transfer efficiency, as follows from the aboveformulas, can be determined in a number of differentways. Since energy is transferred from the excited donorto the acceptor, the lifetime (t), quantum efficiency (Q),and fluorescence intensity (F) of the donor decrease, if theacceptor is present. As a consequence, the fluorescenceintensity of the acceptor increases if the donor is present.

1 2 E 5tD

A

tD

(9)

FIG. 1. Energy balance of FRET. The top part of the figure shows theJablonski energy level diagram. The donor fluorophore is excited andrapidly drops to the lowest vibrational level of the excited state, where itcan decay radiatively (fluorescence) or by internal conversion (heat) to theground state, or transfer energy to the acceptor. Only those levels of thedonor and acceptor with similar energies contribute significantly to thetransfer rate. Once the acceptor is excited, rapid vibrational relaxationprevents back transfer. The acceptor then decays to the ground state viafluorescence or heat. The bottom part of the figure shows the spectralcharacteristics and changes of the donor and acceptor undergoing FRET.The donor intensity decreases and the acceptor increases (i.e., issensitized) with energy transfer. The spectral overlap that makes FRETpossible is shown in gray. The absorbance and emission intensities arenormalized for display purposes.

FIG. 2. Orientation of the transition moments of donor and acceptormolecules. a is the angle between the transition moments, b is the anglebetween the transition moment of the donor and the line joining thefluorophores, and g is the angle between the transition moment of theacceptor and the line joining the fluorophores.

161FRET IN CLINICAL LABORATORY

1 2 E 5F D

A

FD

5QD

A

QD

(10)

FAD

FA

5 1 1 1eDCD

eACA2 E (11)

In the previous formulas the lower indexes refer to thedonor (D) or acceptor (A), whereas the upper indexesindicate the presence of the donor (D) or the acceptor (A)in the system, while CD and CA are the molar concentra-tions, eD and eA are the molar absorption coefficients of thedonor and the acceptor. Another possibility for determin-ing the energy transfer efficiency is based on the moredepolarized emission of the acceptor. Detailed evaluationof the energy transfer measurements can be found inseveral recent reviews (69–71,100,106,108).

Calculation of distance relationships from energy trans-fer efficiencies is easy in the case of a single donor singleacceptor system if the localization and relative orientationof the fluorophores is known. However, if cell membranecomponents are investigated, a two dimensional restric-tion applies for the labeled molecules. Analytical solutionsfor randomly distributed donor and acceptor moleculesand numerical solutions for nonrandom distribution havebeen elaborated by different groups (24,26,27,35,36,92,124). In order to differentiate between random andnonrandom distributions, energy transfer efficiencies haveto be determined at different acceptor concentrations.

MEASURING TECHNIQUESSpectrofluorimetry

For the case of spectrofluorometric measurements,Equations 10 and 11 are used to determine the energytransfer efficiency. A complete set of samples for transferefficiency determination should contain at least one unla-beled, two single-labeled (one labeled with donor only andone labeled with acceptor only) and a double-labeled(labeled with donor and acceptor) sample. The measuredfluorescence intensities have to be corrected intensities,i.e. for autofluorescence and light scattering. This can beachieved using the unlabeled sample. At the same time thefluorescence intensities should be normalized to the samedonor (Equation 10) or acceptor (Equation 11) concentra-tion. For both corrections very accurate sample prepara-tion is required. The dye concentration should be carefullycontrolled.

In the case of cell suspension another possible errorsource is the contribution from unbound fluorophores andcell debris to the specific fluorescence. These are verydifficult, if not impossible to control, especially if thefluorescent label has a low binding constant. Multiplewashing decreases the contribution of free fluorophoresto the fluorescence intensity, but unavoidably increasesthe amount of cell debris. Fluorescence microscopy over-comes most of the problems one faces using spectrofluo-rometry. The distortion caused by dead cells or cell debriscan be avoided, and uncertainties in cell concentration donot cause a problem either. Measurement of energy

transfer in a microscope has the advantage of the spatialresolution, thus providing structural information at thesame time. The only disadvantage is statistical accuracy,because only a relatively small number of cells can beinvestigated.

Flow Cytometry

Flow cytometry offers a good compromise for bothmeasuring energy transfer on cell surfaces, combiningsome of the advantages of the spectrofluorometric andmicroscopic methods. In the case of flow cytometry, theeffect of light scattering on fluorescence intensities ispractically negligible. Because the receptor densities havegreat variation in a cell population, normalization offluorescence intensities on a cell-by-cell basis can not bedone using the spectrofluorometric approach. Real single-cell determination of energy transfer requires the measure-ment of all parameters on the same cell. In flow cytometricmeasurements there are three unknown parameters: theunquenched donor fluorescence intensity, the nonen-hanced acceptor intensity, and the efficiency of the energytransfer. In order to determine these parameters one has tomeasure three independent signals from the same cell.Two of these parameters are the emission intensitiesdetected from different spectral bands. The third emissionintensity is the one resulting from a second exciting laserbeam. To this end, a conventional flow cytometer may bemodified by the introduction of a second excitation laserbeam. The technical details of such a system were de-scribed elsewhere (22,100,102,103,109). Briefly, in such asystem the 488-nm and the 514-nm lines of an argon ionlaser are used for excitation. The laser beams are displacedby about 0.5 mm at the so-called intersection point, wherethe laser beams illuminate the cells. Spectral ranges offluorescence are detected around the emission maximumof fluorescein (535 nm) and usually above 590 nm (emis-sion of rhodamine). Data collection is done in list mode,meaning that the corresponding light scatter and fluores-cence intensities from each cell are stored separately. Thecalculation of energy transfer efficiency is done by acomputer using Equations 15 and 16. In Equations 12–15,the measured intensities are I1, I2, and I3 and the excitationand emission wavelengths are given in parentheses. IF andIR stand for the theoretical (unquenched and nonen-hanced) intensities of the donor (excited at 488 nm,emission detected at 535 nm) and of the acceptor (excitedat 514 nm, detected at .590 nm), respectively. Becausethe emission spectra of the fluorescein and rhodamineoverlap, and both molecules can be excited by the use ofboth laser beams, correction factors have to be intro-duced. These factors are S1, S2, and S3. The definitions ofthese parameters are as follows:

S1 5 I2/I1

(determined using only donor labeled cells);

S2 5 I2/I3


(determined using only acceptor labeled cells); and

S3 5 I3/I1

(determined using only donor labeled cells).The three detected intensities can be expressed as

I1(488 = 535) 5 IF(1 2 E) (12)

I2(488 = .590) 5 IF(1 2 E)S1 1 IRS2 1 IFEa (13)

I3(514 = .590) 5 IF(1 2 E)S3 1 IR 1S3

S1

IFEa (14)

I1 is smaller than IF because the energy transfer causesdonor quenching. I2 consists of three additive terms: 1) theoverlapping fraction of the quenched fluorescein inten-sity, 2) the direct contribution of rhodamine, and 3)sensitized emission due to energy transfer. The proportion-ality factor a is the ratio of I2 for a given number ofrhodamine molecules and I1 for the same number offluorescein molecules. a is constant for each experimentalsetup, and has to be determined for every defined case. I3

is a sum of: 1) a fraction of the quenched fluoresceinintensity, 2) the rhodamine intensity, and 3) the sensitizedemission of rhodamine due to energy transfer correctedfor the lower molar extinction coefficient of fluorescein at514 nm than at 488 nm.

From Equations 12–14, the following equation can bederived:

E

1 2 E5

1

a 3(I2 2 S2I3)

11 2S3S2

S12 I1

2 S14 (15)

All the parameters in Equation 15 can be determinedexperimentally. If we substitute B in the right side ofEquation 15, E can be expressed as follows:

E 5B

1 1 B(16)

Because fluorescein is used as a donor and rhodamine asan acceptor in most flow cytometric energy transferexperiments, the above considerations apply to them. Theequations are valid for other donor acceptor pairs, but thedifferent spectral characteristics must be considered.

In Equations 12–15, it is assumed that the contributionof cellular autofluorescence to the specific fluorescencesignals is negligible. If the autofluorescence is substantial,corrections should be done. In this case, however, thecorrection for autofluorescence can be done using theaverage autofluorescence intensities of the entire cellpopulation. It should be noted that since in most cell typesthere is a good correlation between the autofluorescence

detected at different regions of the spectrum, a moreelaborate correction method is also possible. Here anotherindependent parameter should be detected, a fourthfluorescence intensity, and this way the autofluorescencecan be calculated on a cell-by-cell basis. Naturally highautofluorescence may decrease the precision of the mea-surements.

IMAGE CYTOMETRYPhotobleaching FRET Digital Imaging Microscopy

Jovin and Arndt-Jovin introduced another approach todetermine transfer efficiencies in a microscope (47,48).The energy transfer is calculated from the photobleachingkinetics of the donor in the presence and in the absence ofthe acceptor. Their method is based on the fact that theintegrated fluorescence intensity during complete photo-bleaching is independent from the quantum efficiency ofthe fluorophore and therefore it is proportional to thedonor concentration (41). It is assumed that the donor’sphotobleaching occurs from the excited singlet state. Itcan be derived that the energy transfer efficiency can becalculated as follows:

E 5 1 2tble

tbleA

5 1 2(I0

A/IintA )

(I0/Iint)(17)

where tble is the time constant of photobleaching, I0 is theinitial fluorescence intensity, and Iint is the integratedfluorescence intensity upon complete photobleaching ofthe donor. The A upper indexes indicate the presence ofan adequate acceptor. The detailed derivation of the aboveformula is described in references (47,48). In the case ofenergy transfer, because there is extra possibility forde-excitation, the availability of excited donors for photo-bleaching will decrease. As a consequence, the rate ofphotobleaching will be slower, starting from a quenchedinitial fluorescence intensity, but the integrated fluores-cence intensity remains unchanged.

This method offers a greater sensitivity with an internalcontrol for real donor concentration. The only drawbackof the method is the limited number of cells that can bemeasured. This may cause that inhomogeneities in thesample are not revealed. However, if the same experimentis performed on a flow cytometer this disadvantage can beovercome. Since the introduction of this approach severalsuccessful adaptations for different systems have beenelaborated (2,95–97).

Intensity-Based Microscopy

The first semiquantitative intensity-based microscopicmethod for measuring FRET was introduced approxi-mately 10 years ago (112,113). Uster and Pagano installedan additional filter combination for detecting the ‘‘transfersignal’’ by using the excitation wavelength of the donorand detecting the sensitized emission of the acceptor. Thisapproach is suitable for proving the existence of energytransfer, but the accurate determination of the transfer


efficiency is not possible. More recently, others (83,132)used a similar approach with slight modifications.

In the family of the intensity-based microscopic meth-ods, two relatively new versions emerged. The first (79)uses a set of equations similar to those described by Tron(109), whereas the other calculates corrected ratio imagestaken from the donor and the acceptor as well (54). Bothversions are suitable for determining the transfer efficien-cies on pixel basis.

APPLICATION OF FRETResearch

Cell surface distribution of hematopoietic clusterof differentiation (CD) molecules. A nonrandomizedcodistribution of membrane-bound proteins play an impor-tant role in signal transduction across the cell membrane.The primary target for external stimuli is the plasmamembrane. In addition to the well-known biochemicalmechanisms of ligand-receptor interaction there are numer-ous physical events that induce alterations of the cellsurface in the vicinity of the receptor. Signal transductionis often accompanied by the dynamic rearrangement ofthe two dimensional patterns of the macromolecularconstituents at the cell surface.

The most commonly used methods for determiningmolecular associations in membranes include cocapping,co-immunoprecipitations, chemical cross-linking, modi-fied fluorescence recovery after photobleaching, electronmicroscopy, atomic force microscopy, and FRET measure-ments. Application of one method alone usually does notgive a conclusive picture. Results of two or more methods,however, reinforce each other and lead to a consistentpicture of the molecular interactions in the plasma mem-brane. We will focus mostly on results gained by FRETtechnique, some of the supporting data obtained by otherbiochemical and biophysical methods will also be men-tioned. There have been several general reviews onmapping cell surface elements using FRET technique(17,18,21,70,71,98,108,127); this chapter will focus mainlyon the latest results concerning this field.

Although the major histocompatibility complex (MHC)class I and class II molecules are structurally and function-ally distinct, there is evidence that MHC class I and class IImolecules can be more intimately related than previouslythought. It has been reported that MHC class I-specificantibodies can cocap MHC class II antigens on B lympho-cytes (77,78). These observations prompted us to performstudies in which a more direct approach, FRET technique,was applied for the investigation of the possible associa-tion between HLA class I and class II molecules on PGF andJY B lymophoblastoid cells (99). A panel of monoclonalantibodies specific for various class I and class II antigenswas labeled with either FITC (donor) or TRITC (acceptor).Flow cytometric energy transfer measurements were madeon cells labeled with fluoresceinated and rhodaminatedantibodies simultaneously. FRET efficiency was calculatedon a cell-by-cell basis and the results were displayed asenergy transfer distribution histograms. Mean values ofsuch distribution histograms were used to draw conclu-

sions about the proximity relationship of cell surfaceproteins under investigation. This type of FRET studyrevealed that HLA class I and class II molecules areexpressed mostly in a monomeric state on the cell surface.Class II antigens may form heteroassociations amongthemselves and there is association between class I andclass II molecules. There is an equilibrium between freenonassociated HLA class I and class II molecules and theassociated class I-class II antigen complexes. The numberof class I-class II molecule associations formed dependsupon the available class I and class II molecules expressedin the plasma membrane. These results are also in agree-ment with cocapping experiments demonstrating classI-class II interaction. Our data, however, demonstratedthat these complexes are physically associated beforecocapping (99). While there was no homoassociationbetween HLA class I molecules on PGF cells, we coulddetect homoassociation between them on JY cells. Thedegree of homoassociation of class I antigens highlydepends on the culturing conditions, whether the cellswere in log phase or in plateau phase. HLA class Iclustering correlated with the expression level of b2-microglobulin-free HLA class I heavy chains, and theaddition of exogenous b2-microglobulin greatly reducedthe HLA class I homoassociation (9). Moreover, modula-tion of the composition of plasma membrane also influ-enced the HLA class I clustering. Addition of cholesteroldecreased the membrane fluidity and also the degree ofhomoassociation of HLA class I molecules (9).

We have extended these types of flow cytometricenergy transfer measurements and molecular associationshave been detected between intercellular adhesion mol-ecule 1 (ICAM-1; CD54), HLA class I heavy chain, b2-microglobulin, and HLA-DR on the cell surface of JY Blymphoma cells (6). Similar heteroassociations were foundin the plasma membrane of HUT-102B2 T lymphoma cells,but the significantly different quantitative data suggested asomewhat different cell surface distribution pattern of themolecules. In this case, interleukin-2 (IL-2) receptor asubunit (IL-2Ra, CD25), was also included in the proximitystudies. FRET data suggested that ICAM-1 and/or IL-2Raare closer to the b2-microglobulin than to heavy chain ofthe HLA class I complex. In addition, a high degree ofhomoassociation of ICAM-1 molecules was observed. Het-eroassociations involving ICAM-1 molecules may play animportant role in antigen presentation, T-cell recognition,cytotoxicity, site-directed lymphokine secretion, and otherimmunological processes (6).

Flow cytometric energy transfer measurements havealso been used to study the topological distribution oftransferrin receptor (TfR; CD71) relative to the heavy andlight chains of the HLA class I molecules, class II mol-ecules, interleukin receptor a-chain (CD25), and ICAM-1(CD54) molecules on HUT-102B2 T and JY B cell lines(72). TfR showed high degree of homoassociation, and itscell surface distribution depended upon the growingcondition of cells. TfR was in close vicinity to HLA class Imolecules on the surface of JY cells in both logarithmicand plateau phase, whereas it was not associated with HLA


class I on the surface JY cells (Figs. 3 and 4). HLA class IImolecules heteroassociated with TfR on HUT-102B2 cells,whereas only a modest association was found on JY cells,and only in the logarithmic phase (6,72).

Flow cytometric energy transfer measurements havebeen used to confirm whether coprecipitation of tetraspanmolecules (CD37, CD53, CD81, CD82) with HLA class II(DR) antigens is an experimental artefact or reflects realmolecular associations in the plasma membrane of livecells. Results of energy transfer experiments carried outwith fluorescently labeled monoclonal antibodies (wholeantibodies and Fab fragments) demonstrated that thetetraspan molecules are in a single complex with DRmolecules rather than each of them is separately associ-ated with different DR molecules (101). The coclusteringof these molecules may have functional significance; this iscurrently under investigation.

Combination of atomic force, electron microscopy, andphotobleaching energy transfer made the discovery and

description of the clustering of MHC class I molecules at 2to 10 nm and also at µm levels (23). Using flow cytometricand photobleaching energy transfer measurements a non-random distribution pattern of HLA class I molecules wasobserved in the 2 to 10 nm range. A second, nonrandom,and larger-scale topological organization of the HLA class Imolecules was detected by transmission electron micros-copy and atomic force microscopy using immunogoldlabeling. These data suggested that HLA class I antigensexhibit a hierarchical arrangement consisting of specificpatterns of localizations but with a degree of randomnessin the distribution. The possible function of the higherorder clusterization is that by increasing the local densityof adhesion molecules and thereby the multiplicity ofintercellular interactions, clusters may serve to stabilizeweak contacts between cells (23). A similar hierarchicaldistribution pattern was observed for HLA class II mol-ecules. Electron microscopy also revealed that a fraction ofthe HLA class II molecules was heteroclustered with HLAclass I molecules at the same hierarchical level (46).

CD7 is a 40-kDa glycoprotein that is expressed on amajor subset of human peripheral blood T cells. Cross-linking of CD7 monoclonal antibody (mAb) is mitogenicand signals delivered via CD7 molecule stimulated integrin-mediated adhesion (59). Co-immunoprecipitation datasuggested that CD7 associate with CD3 and CD45. Toconfirm this observation, flow cytometric energy transfermeasurements were performed using FITC- and TRITC-labeled mAbs as donor acceptor pairs. There was signifi-cant increase in the sensitized emission when FITC-CD7and TRITC-CD45, or FITC-CD7 and TRITC-CD3 interactionwas investigated, indicating molecular associations be-tween these entities. These data supported the hypothesisthat CD7 exists in an oligomeric complex with CD3/T cellreceptor (TCR), CD45, and tyrosine kinase, thereby provid-ing physical basis for the accessory role of the CD7molecule in T cell activation (59).

Flow cytometric energy transfer measurements wereapplied to monitor the aggregation of IL-1 type I receptor(CD121a) on transfected C127 mouse mammary carci-noma cells or on Chinese hamster ovary (CHO) cells.Noncompetitive anti-CD121a mAb, M5, was conjugatedseparately with either FITC (donor) or Cy3 (acceptor) andthe cells were labeled simultaneously with a mixture ofFITC-M5 and Cy3-M5 antibodies. The ratio of acceptor todonor emission was monitored to detect sensitized emis-sion and donor quenching that occurred upon addition ofIL-1a. FRET results indicated that IL-1 binding led totime-dependent aggregation of IL-1 type I receptor, andthat this aggregation is likely to play an important role inIL-1-dependent signal transduction (34).

Vignali and coworkers used also flow cytometry todetect FRET between various domains of CD4 and TCR(114). They used FITC- and TRITC-conjugated mAbs tolabel specific epitopes on these molecules. CD4 is inti-mately involved in colocalizing TCR with its specificpeptide ligand bound to MHC class II molecules. Theywanted to determine which portion and function of CD4was responsible for the fidelity of the interaction between

FIG. 3. Schematic representation of the lateral distribution of transfer-rin receptor (TfR), HLA class I (HLA I), HLA class II (HLA II) molecules onthe surface of JY lymphoblastoid B cells. Note that the transferrinreceptor shows high degree of homoassociation and associates with HLAclass II molecules but not with HLA class I molecules. The complexesshown are only examples of possible clusters.

FIG. 4. Schematic representation of the lateral organization of transfer-rin receptor (TfR), IL-2 receptor a chain (Tac), intercellular adhesionmolecule-1 (ICAM-1), HLA class I (HLA I), and HLA class II (HLA II)molecules on the surface of HUT-102B2 lymphoblastoid T cells. b2-microglobulin is the light chain of HLA class I molecule. Note that thetransferrin receptor shows high degree of homoassociation and associateswith HLA class I, HLA class II, ICAM-1, and Tac. The complexes shownare only examples of possible clusters.


TCR and peptide-loaded MHC class II molecules, the distalD1/D2 domains that bind to MHC class II molecules or themembrane proximal D3/D4 domain. FRET data indicatedthat the D3/D4 domains of CD4 might interact directly orindirectly with the TCR-CD3 complex and influence thesignal transduction processes (114).

In contrast to the above-mentioned examples in whichmostly flow cytometric energy transfer measurementswere used for mapping cell surface distribution of mem-brane proteins, the application of microscopic energytransfer measurements will be discussed in the followingexamples. In one article, the photobleaching fluorescenceresonance energy transfer (pbFRET) method was applied,in the other paper, intensity-based FRET studies wereperformed using microscope.

pbFRET technique was used to study the role of CD4 insignal transduction via TCR/CD3 complex. Szabo et al.wanted to reveal whether the inhibition of T cell activationvia TCR/CD3 by anti-CD4 antibodies is attributed by thetopological separation of CD4-p56lck from CD3, or byimproper apposition (97). The epitopes were ligandedwith FITC-conjugated antibodies (donor) and PE-conju-gated antibodies (acceptor) simultaneously. Results ofpbFRET experiments showed that CD4 stayed in themolecular vicinity of CD3, whereas anti-CD3 stimulationwas suppressed by anti-CD4 antibodies. Negative signalingvia CD4 may be interpreted in terms of functional uncou-pling rather than physical separation of CD4 from theTCR/CD3 complex (97).

Jurgens and coworkers also used the pbFRET approachto reveal proximity relationships between the type Ireceptor for Fce (FceRI) and the mast cell function-associated antigen (MAFA) (53). Monoclonal antibodiesagainst FceRI and MAFA were conjugated with FITC(donor) and TRITC (acceptor) whereas the IgE, the ligandfor FceRI, was conjugated with bis(sulfate)-indocarbocya-nine Cy3.18-OSu (Cy3) (donor) and bis(sulfate)-indocarbo-cyanine Cy5.18-OSu (Cy5) (acceptor) and FRET analysiswas performed using donor photobleaching digital imag-ing microscopy. Results of pbFRET analysis suggested thatMAFA was in close proximity to at least some of the FceRI,and clustering of FceRI leads to no significant change in theproximity of the two molecular species. The association ofFceRI and MAFA establishes a molecular base for MAFA-mediated inhibition of mast cell activation (53).

The best examples for intensity-based FRET measure-ments were provided by Petty and coworkers. Theysystematically investigated the interreceptor interactionsbetween FcgRIIB (CD16b), a GPI-linked protein, and theleukocyte integrin CR3 (aMb2; CD11b/CD18) (84). Theyused 3T3 cell lines transfected with either FcgRIIB or CR3or both proteins. Monoclonal antibodies were labeledwith FITC and TRITC, and the energy transfer measure-ments were performed in a microscope. Using appropriatefilters and background subtraction FRET signal was de-tected with a photon counting apparatus. Their resultsindicated that these two membrane proteins can exist inclose physical proximity in membranes and that thisassociation can be affected by an exogenous compoundsuch as N-acetyl-D-glucosamine (84). Using the same

resonance energy transfer microscopy approach the groupstudied the interaction between complement receptortype 3 (CR3) and FcgRIIA (CD32). Normal and a tail-minusmutant of FcgRIIA were transfected into fibroblasts thatdid or did not express CR3. Physical proximity wasdetected between CR3 and FcgRIIA on the cell surfaces.Cells expressing only the tail-minus mutant of FcgRIIAwere unable to internalize opsonized erythrocytes butshowed significant binding ability. In contrast, cells express-ing both mutant FcgRIIA and C3 internalized opsonizederythrocytes. Results showed that CR3 could complementthe phagocytic function of defective FcgRII (126). FRETmicroscopy was used to image the spatial distribution ofenergy transfer efficiency and to follow the kinetics of theassociation and dissociation of CR3 and the urokinase-typeplasminogen activator receptor (uPAR; CD87), a GPI-linked protein. Initial level of FRET dramatically fell duringcell polarization, but did not change on cells fixed withparaformaldehyde. This means that interreceptor associa-tions correlate well with cell activity: CR3 and uPAR arecoclustered on stationary cells and uncoupled on polar-ized cells initiating locomotion (56). CD14, another GPI-linked membrane protein, also affected the cell surfacedistribution of CR3. When cells were treated with endo-toxin LPS, which binds CD14, formation of CD14-CR3complex was initiated. Kinetic studies showed that CD14-CR3 complexes dissociate as neutrophils attach to sub-strate (129).

Conformation of membrane proteins. In additionto the cell surface distribution of membrane proteins theconformation of these molecules can also be investigatedby FRET technique. In one approach reactive groups of aprotein under investigation are labeled with fluorescentprobes, i.e., with donor acceptor pairs, and the variation inFRET efficiency is used to characterize altered conforma-tion of the protein molecule. In the other approachvarious epitopes of a membrane protein can be labeledwith appropriately conjugated monoclonal antibodies orFab fragments and the change in the intramolecular FRETefficiency is monitored during various treatments.

Zheng et al. have chosen the first approach in order tomonitor conformations of IgE bound to its receptor FceRIand in solution (131). They prepared a mutant IgE (e/Cg3*)that has a cysteine replacing a serine near the C-terminalend of the heavy chain. This sulfhydryl group was selec-tively labeled with fluorescein-5-maleimide serving as do-nor, and the 5-(dimethylamino)naphthalene-1-sulfonyl(DNS) bound to the antigen binding site of the IgE servedas acceptor. The resonance energy transfer between thesegroups was monitored by spectrofluorometry and theaverage end-to-end distances for IgE in solution and onmembranes after forming receptor-IgE complex werecompared. These distance measurements clearly demon-strated a bent confirmation for IgE bound to its receptoron the cell membrane and also provided evidence for asurprisingly similar conformation for IgE in solution (131).

In contrast to the antibodies, T cells do not recognizeantigens in their native conformation, but only after partialproteolysis within antigen processing and presenting cells


into constituent peptides that are then bound to MHCmolecules. The bimolecular complex of antigen peptideand MHC molecule is then displayed on the surface ofantigen presenting cells and recognized by T cell clonesbearing antigen receptor specific for it. MHC class Imolecules primarily specialized to present peptide derivedfrom endogenous proteins whereas class II moleculespresent peptides derived from endocytosed antigens.Tampe and coworkers wanted to address the question ofwhether MHC class II molecules can bind one or twopeptides (105). They compared the peptide binding capac-ity of the ‘‘floppy’’ and ‘‘compact’’ forms of MHC class IImolecules. The floppy confirmation is an intermediate inthe dissociation of compact into separate a and b chains. A170 amino acid peptide from chicken ovalbumin waslabeled with fluorescein at the amino terminus or withTexas Red (TR) near the carboxyl terminus. Systematic,spectrofluorometric FRET measurements between fluores-cein- and TR-labeled peptides revealed that two full-lengthpeptides can bind to the floppy ab heterodimer, and twotruncated peptides can bind to the compact ab het-erodimer and also to floppy ab heterodimer. No simulta-neous binding of the two full-length peptides to thecompact ab conformation was detected. The biologicalsignificance of the above-mentioned findings may be thatthey suggest a mechanism or peptide exchange in class IIantigen (105).

Catipovic and coworkers compared the conformation ofempty and peptide-loaded MHC class I molecules (11)using flow cytometric FRET measurements. Fluorescein-ated Fab fragments of monoclonal antibody specific for a2domain of the heavy chain served as donor, and TR-conjugated Fab fragment of monoclonal antibody specificto b2-microglobulin as acceptor. No FRET was foundbetween empty MHC class I complex, but FRET wasdetected when the complex was loaded with peptides.These data indicated that empty MHC class I moleculeshave a flexible and extended conformation that is acces-sible by peptides. Upon peptide binding, MHC class Imolecules adopt a more compact conformation. Becausethe amount of FRET depends on the sequence of thebound peptide, it appears that this compact MHC confor-mation is influenced by the peptide (11).

Fluoresceinated and rhodaminated, noncompeting mAbs,which bind to the same H-2Kk antigen but to differentepitopes, were applied to label the surface of HK 22murine T lymphoma cells. Using flow cytometric tech-nique, significant FRET efficiency was detected betweenFITC-labeled 30/6 and TRITC-labeled 27/55 antibody be-cause it would be expected for intramolecular transferefficiency. Addition of specific peptide to the double-labeled cells increased the FRET efficiency significantly. Atthe same time addition of the specific peptide had noeffect on the monomeric state of H-2Kk molecules. Be-cause a large portion of the MHC class I antigens expressedon the cell surface is already occupied by endogenouspeptides the increase in FRET efficiency upon addition ofexogenous peptide can be interpreted in the followingways. Addition of peptide induces a huge conformationalchange in formerly free MHC class I molecules, which can

be 10% to 30% of the total class I molecules. Anotherpossibility is that exogenously added peptides replacealmost all the endogenous peptides, and due to its differ-ent structure induces a slight conformational change on allclass I molecules (98).

Flow cytometric FRET measurements were performedto detect reversible conformational changes in the MHCclass I complex in the plasma membrane of JY cells upondepolarization of the transmembrane potential (7). Theheavy chain of the MHC class I molecules was labeled withfluoresceinated mAb, while the b2-microglobulin withrhodaminated mAb. Reduction of transmembrane poten-tial increase the intramolecular FRET efficiency betweenthese antibodies (Fig. 5). Repolarization of the depolarizedsamples restored the energy transfer efficiency to theoriginal values measured before depolarization. Depolariza-tion caused similar relative changes in FRET efficiencywhen Fab fragment was used for labeling MHC class Icomplex, suggesting that the observed phenomenon is notrestricted to whole antibodies (7). The magnitude of theconformational change was similar to that observed inMHC class I complex upon specific peptide binding (98).This finding suggested that peptides presenting MHC classI molecules might alter their conformation upon transmem-brane potential changes to such an extent that antigenpresentation and, thus, cell-mediated cytotoxicity may beinfluenced. Toward this end, the same group studied theeffect of membrane potential changes of target cells on thefunction of cytotoxic T lymphocytes. Alterations of theresting potential of target cells in both directions resultedin enhanced cytotoxic activity (3). These observationssuggested the alteration of membrane potential coulddirectly influence the conformation of proteins critical forimmune recognition.

Analytical Applications

Tandem fluorescence dyes in immunophenotyp-ing. The detection of multiple fluorescent biomarkers ona single cell by flow cytometry provides a powerful tool forcell analysis. Therefore, there is always a need for morefluorophores that can be used simultaneously in immuno-fluorescence applications. Because most clinical flowcytometers utilize single-laser excitation, conventionallow molecular weight fluorescent dyes having small Stokesshift can not be used for the detection of more than twofluorescent parameters on a single cell. The introductionof the phycobiliprotein-based tandem dyes has signifi-cantly enhanced the capabilities of these single-laser flowcytometers for performing multiparametric analysis(5,60,86,115). R-PE, a protein of molecular weight 240kDa containing 34 bilin fluorophores (80) can be appliedsimultaneously with fluorescein because both can beexcited at 488 nm and the PE emission at 575 nm andfluorescein emission at 525 nm can readily be discrimi-nated with optical band-pass interference filters. In addi-tion, PE can be used as energy donor due to its high molarabsorption coefficient between 450 and 550 nm, with arange of potential acceptor molecules possessing favor-able spectral overlap, including TR, Allophycocyanine


(APC), and cyanine dyes (Cy5 or Cy7). The Texas Red-PEtandem conjugates have been commercially available un-der the following trademarks: Duochrome (Becton Dickin-son Immunocytometry Systems, Belgium), ECD (BeckmanCoulter) and Red 613 (Life Technologies, Rockville, MD).The large Stokes shift associated with the fluorescenceresonance energy transfer of these tandems producesemission that can easily be resolved from direct PE orfluorescein emissions. The PE-TR tandem emits at 613 nm,the APC-PE and Cy5-PE at 660–670 nm, and the Cy7-PEat 780 nm, respectively (60,86,115). The largest Stokesshift of 300 nm is provided by the Cy7-PE conjugate thatcan be efficiently excited at 488 nm and emits at 780 nm.The Cy5-PE and Cy7-PE tandems are very bright fluores-cent reagents and can be used with fluorescein and PEwith excitation from a single laser light (488 nm) provid-ing a useful fluorophore set for four-color immunofluores-cence (86).

Development of tandem reagents is more difficult thandevelopment of simple fluorophores primarily because ofthe added variable: the acceptor-to-donor molar ratio(A/D). In general, a high A/D ratio yields the best transferefficiency, however, at such ratios the acceptor oftenself-quenches (producing less fluorescence) and may causethe tandem to become ‘‘sticky.’’ In such an event, themonoclonal antibody-tandem conjugate looses its specific-ity. Therefore, compromise between optimal transferefficiency (i.e., low or no direct emission of the PE shouldbe present) and optimal acceptor fluorescence is neces-sary. In the case of Cy5-PE tandem less Cy5 is needed toquench the direct PE fluorescence than in the case ofCy7-PE conjugate because the spectral overlap betweenPE and Cy5 is larger than between PE and Cy7 (60,86).Self-quenching due to acceptor dimerization starts above 5Cy5/PE ratio for Cy5-PE conjugate and above 3 Cy7/PEratio for Cy7-PE conjugate. Interestingly, the stickiness ismuch less of a problem for Cy7-PE than for Cy5-PE, whichhas nonspecific staining when applied to peripheral bloodmononuclear cells. It has been suggested that humanmonocytes might have a ‘‘Cy5’’ receptor resulting in highbackground binding of Cy5-PE. Cy7-PE conjugated nonrel-evant antibodies do not show nonspecific binding onmonocytes (86). Although these tandem dyes are veryuseful in single-laser flow cytometry, not all of them can beutilized for dual-laser application, because the second lasermight directly excite the acceptor molecule of the energytransfer pair. For example, in a flow cytometer in whichthe second laser emits at 633 nm, the application of Cy5-PEtandem dye as a third color on the first laser beam (488nm) may not be effective. Cy5-PE is efficiently excited withthe second laser and, unfortunately, it emits energy in thesame region as APC. The exploitation of the second laserbeam has been greatly enhanced with the introduction oftandem dyes that work in the red and far-red region (5,86).The other motivation in the development of the redexcitable tandem dyes was the challenge to avoid fluores-cence near or at cellular autofluorescence, which is often alimiting factor in signal detection. Competition with auto-fluorescence is much less of a problem when the dyeselected is in the red and far-red region. Cy7 dye with its780-nm emission is a good candidate as a fluorescentprobe for flow cytometry, however, the 744-nm excitationis usually unavailable on standard dual-laser flow cytom-eters. Conjugation of Cy7 to APC resulted in a new tandemconstruct, the Cy7-APC or ALLO-7 (5,86). The sensitivity ofphotomultipliers tubes in the 700 to 800 nm region shouldbe increased in order to obtain wider applications of thisred tandem dye. ALLO-7 has the potential to increase anddiversify the combinations of fluorochromes used withconventional flow cytometers.

The tandem dyes discussed above are bright fluoro-phores and can easily be coupled to mAbs. These reagentsare well suited for a variety of fluorescence applications,including multilaser clinical flow cytometry and micros-copy.

Enzyme assays. Usually this type of FRET measure-ment involving synthesized oligomers is not of concern to

FIG. 5. Schematic representation of HLA class I complex labeled withnoncompeting fluorescently tagged antibodies bound to the same com-plex but to different epitopes. Monoclonal antibody W6/32 binds to theheavy (a) chain of the HLA I complex, while L368 to the light chain(b2-microglobulin) of the complex. Depolarization of the membraneresults in conformational change of the HLA class I molecule bringing thetagged epitopes closer to each other.


clinical immunologists. However, the current interest inmultiplexed flow cytometric analysis provides an opportu-nity to revisit some of the classical assay systems involvingfluorophores and solid-phase binding. The distance be-tween the locations of the donor and acceptor moleculesis of great concern in this type of flow cytometric assaybecause many analytes form complexes in close proximityto each other. Utilizing microspheres suspended in a liquidmedium attached to oligomers with and without fluores-cent label sets the stage for a new approach to homoge-neous assay systems.

The general scheme of a FRET-based enzyme assay is thefollowing: Oligomer substrates are synthesized that aredouble labeled with donor and acceptor fluorophores.Usually the distance between the locations of the donorand acceptor molecules is less than R0, so the donorfluorescence is significantly quenched in this doubleconjugate. This intramolecular quenching is relieved bythe action of an enzyme that cleaves the oligomer sub-strate, thereby releasing the donor and acceptor moietiesseparately into the solution, resulting in tremendousincrease in donor fluorescence (Fig. 6). These fluorometricassays are widely used because they provide high sensitiv-ity and the assay can easily be automated.

Proteases are among the most often assayed enzymes.Matayoshi and coworkers have developed a new fluoro-genic substrate for assaying retroviral proteases using theFRET principle (66). The assayed enzyme, the 11-kDaprotease (PR) encoded with human immunodeficiencyvirus (HIV) 1, is essential for the correct processing of viralpolyproteins. The maturation of infectious virus is there-fore a target for the design of selective HIV diseasetherapeutics. The assay used the quenched fluorogenicsubstrate 4-[[48-(dimethylamino)-phenyl]azo]benzoic acid(DABCYL)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-5-[(28-aminoeth-yl)amino]naphthalene-1-sulfonic acid (EDANS), whose pep-tide sequence was derived from a natural processing site

for HIV-1 PR. The fluorogenic peptide is cleaved at theTyr-Pro bond resulting in a 40-fold increase in the fluores-cence quantum yield. Application of this assay facilitatesthe identification of novel inhibitors of HIV-1 PR, andpermits detailed studies on the activity and inhibition ofthis enzyme. Because of its simplicity, speed, sensitivity,and precision in kinetic analyses, this method is superiorto the more commonly used high-performance liquidchromatography or electrophoresis-based assays for pep-tide substrate hydrolysis by retroviral PR (66).

The serine protease involved in spreading of an otherinfectious virus, hepatitis C, is contained within theN-terminal region of nonstructural protein 3 (NS3 prote-ase) and is among the possible targets for therapeuticintervention. Taliani and coworkers also used the above-mentioned DABCYL-EDANS pair to design a fluorogenicsubstrate for NS3 protease, however, in vitro characteriza-tion of synthetic substrates based on all of the naturalcleavage sites has consistently revealed poor kinetic param-eters, making them unsuitable for sensitive high-through-put screening (104). Instead, they have developed adepsipeptide substrate incorporating an ester bond withinthe molecule containing the two fluorophores. With thehelp of this new substrate a continuous assay for NS3protease activity was developed, and the detection limitfor NS3 was estimated between 1 nM and 250 pM (104).

Wang and coworkers used the same DABCYL-EDANSdonor-acceptor pair to design a fluorogenic substrate forcontinuous assay of renin activity (116). Human renin, anaspartic protease, is one of the most specific proteases andplays an important role in the regulation of blood pressureand in electrolyte homeostasis. The DABCYL-gaba-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS substrate incorpo-rates the renin cleavage site that occurs in the N-terminalpeptide of human angiotensinogen. The cleavage of thesubstrate occurs specifically at the Leu-Val bond, whichcorresponds to the renin cleavage site of angiotensinogen,and leads to a time-dependent increase in fluorescenceintensity. It was estimated that with extended incubationtime (2 to 3 h) the assay can detect renin at 0.5 ng/mlconcentration. The automated, high-throughput fluoromet-ric renin assay version for the 96-well microtiter-platefluorescence reader is useful for studying enzyme inhibi-tors and enzyme stability (116).

The DABCYL-EDANS pair proved to be also very usefulin developing fluorogenic substrate for interleukin-1b(IL-1b)-converting enzyme (ICE) (82). ICE is a heterodi-meric cystein protease that catalyzes the conversion of theinactive 33-kDa or 31-kDa IL-1b precursor to the 17.5-kDamature biologically active molecule. The unique cleavagesite appears to be conserved in all known IL-1b moleculesand occurs between Asp 116 and Ala 117. Upon cleavageof the newly developed DABCYL-Tyr-Val-Ala-Asp-Ala-Pro-Val-EDANS substrate, an increase in fluorescence intensityis observed, permitting continuous assay of ICE. Besidescharacterizing the enzyme activity of ICE, this assay isuseful in screening inhibitory compounds (82).

Another protease, the stromelysin is a member of thematrix metalloproteinase family of enzymes and has been

FIG. 6. Enzyme assays based on FRET principle. Fluorogenic sub-strates are synthesized in which donor and acceptor molecules areattached to monomers (e.g., amino acids, saccharides, nucleotides) andlocated within the FRET distance. Upon cleavage, the FRET efficiencydrops to zero, the intensity of donor increases and the intensity ofacceptor decreases.


implicated in the pathogenesis of tumor metastasis andinflammatory diseases such as rheumatoid arthritis. Toscreen prospective inhibitors of this protease, Bickett andcoworkers developed a fluorogenic substrate with excita-tion and emission spectra compatible with commerciallyavailable 96-well plate readers (8). The substrate is basedon conjugation of 6[N-(7-nitrobenz-2-oxa-1,3-diazol-4-ylamino] hexanoic acid (NBD) (excitation: 467 nm; emis-sion: 534 nm) and 7-dimethylaminocoumarin-4-acetate(DMC) (excitation: 368 nm; emission: 465 nm) to apeptide substrate for stromelysin. The new substrateNBD-Arg-Pro-Lys-Pro-Leu-Ala-Nva-TRP-Lys-(DMC)-NH2 is95% quenched and the fluorescent product Nva-TRP-Lys-(DMC)-NH2 is easily detected at 368 nm excitation and 465nm emission. Because of the action of the enzyme, a20-fold increase in the fluorescence quantum yield can beobserved. These characteristics make this compound anexcellent substrate for routine determination of in vitroactivities of stromelysin inhibitors (8).

Goudreau and coworkers synthesized a novel fluoro-genic substrate, dansyl-Gly-(p- NO2)Phe-bAla, as a selectivesubstrate for neutral endopeptidase 24.11, found on thesurface of various cells and involved in processes asdiverse as hypertension and the control of vasoactivepeptides (33). The neutral endopeptidase also behaves as alymphocyte marker and is identical to the common acutelymphocytic leukemia antigen (CALLA; CD10). Cleavageof the substrate Gly-(p-NO2)Phe amide bond leads to anincrease in fluorescence related to the disappearance ofthe intramolecular energy transfer between the dansyl andthe nitrophenyl residues. The substrate has advantagesover the commercially available dansyl-D-Ala-Gly-(p-NO2)Phe-Gly, because the Gly residue in the fourth posi-tion has been replaced by b-alanine. This increases theselectivity of the substrate for neutral endopeptidase viaeliminating a residual sensitivity of the peptide towardangiotensin converting enzyme. In addition, deletion ofthe D-Ala residue in the second position resulted in anincrease in the quenching efficiency, thus raising thesensitivity of the assay (33).

In another set of fluorogenic substrates, only oneextrinsic fluorophore is added to the polypeptide chain,because the Trp is used as intrinsic fluorophore in creatingthe donor/acceptor pair for FRET-based analysis. Stack andGray designed a fluorogenic substrate for vertebrate colla-genase and gelatinase, dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (93). Tryptophan fluorescence was effi-ciently quenched by the NH2-terminal dinitrophenyl group.Increased fluorescence accompanied hydrolysis of thepeptide by collagenase or gelatinase. Amino acid analysisof the two-product peptides showed that collagenase andgelatinase cleaved the substrate at the Gly-Leu bond. Thespecificity of the substrate was also proven by the fact thatsoluble type I collagen was a competitive inhibitor ofpeptide hydrolysis by collagenase (93). In the othersubstrate, designed by Bouvier et al. (10), the indolefluorescence of the tryptophan residue was quenched byan N-terminal dansyl group located five amino acid resi-dues away. The heptopeptide substrate, dansyl-Ala-Tyr-Leu-

Lys-Lys-Trp-Val-NH2, is a specific substrate for the promati-gote surface protease (PSP) of Leishmania. The fluorescentoligopeptide substrate was cleaved by the PSP betweenthe tyrosine and leucine residues and the hydrolysisresulted in a time-dependent increase in fluorescenceintensity of 3- to 7-fold. Wang and Liang described a newapproach for synthesizing fluorogenic substrates contain-ing only a-amino acids for renin, by incorporating trypto-phan and p-nitrophenylalanine into peptides (117). In thismanner, the substrates can be prepared on a peptidesynthesizer, and both ends of these peptides are free;other residues can be attached to increase their solubilitiesor to label them with affinity ligands. They tested theapplicability of this new approach by developing a continu-ous assay for renin. Hydrolysis of the peptide specific torenin resulted in a 4.5-fold increase in tryptophan fluores-cence, suggesting that p-nitrophenylalanine quenches about78% of the fluorescence of tryptophan in this peptide(117).

Not only proteases but also other enzymes have beenassayed using FRET-based fluorogenic substrates. For ex-ample, introduction of bifluorescent-labeled substrates forendo-type carbohydrases provided a continuous, homoge-neous assay for glycoamidases and ceramide glycanases.Earlier, the activity of these enzymes was measured inassays involving the separation of the products. Lee andcoworkers prepared a doubly fluorescence-labeled bianten-nary glycopeptide for glycoamidases and an alkyl lactosidefor ceramide glycanases (61). For the glycopeptide sub-strate, dansyl group (donor) is attached to the terminalgalactose and naphthyl group (acceptor) is placed on theN-terminal amino acid. For the alkyl lactoside substratedansyl group is attached to the omega-amino group of thealkyl aglycon and naphthyl group is attached to the68-hydroxyl of lactose. The fluorescence emission of thenaphthyl increased as glycoamidase or ceramide glycanasehydrolyzed their respective substrates. Using these sub-strates, sensitive and convenient assays of these enzymeswere established (61). Armand and coworkers synthesizeda bifunctionalized tetrasaccharide a substrate to studycellulases, which are usually classified as endoglucanasesand cellobiohydrolases (1). The substrate, which carries a5-(2-aminoethylamino)-1-naphthalenesulfonate group onthe nonreducing end and an indolethyl group on thereducing end, could be of general use to measure thekinetic constants of cellulases able to act on oligomers ofdegree of polymerization less than 5. Their data alsoproved that cellobiohydrolases I and II are able to degradean oligosaccharide substrate carrying noncarbohydratesubstituents at both ends (1).

Retroviruses require viral DNA to be synthesized byreverse transcription in the cytoplasm followed by integra-tion of the resulting viral DNA into the host chromosomein the nucleus. Reverse transcription and integration,essential steps in the life cycle of retroviruses, are possibletargets in the development of antiviral reagents. Oneattractive target is the integrase protein, a product of theretroviral pol gene, which is solely responsible for theretroviral integration process through cutting and joining


reactions. For screening of large numbers of antiviralagents a rapid and precise assay, based on the FRETprinciple, has been developed (62). Oligonucleotide con-taining the terminal sequence of HIV-1 DNA and FITC(donor) and EITC (acceptor) were synthesized and an-nealed to form a fluorogenic substrate for HIV integrase.The authors were able to overcome the problem of earlierattempts in which the fluorophores interacted with theDNA causing quenched fluorescence even after cleavage,via applying a nucleotide analog with a 12-carbon linkedarm, 5 amino (12)-28deoxyuridine b-cyanoethyl phosphora-madite. The fluorogenic substrate-based assay was standard-ized with radioactive DNA cleavage reaction. The advan-tages of the fluorescent assay over the other assays includeits speed, continuity of reaction monitoring, sensitivity,specificity, and capacity for automation through a 96-wellfluorescence microplate reader (62).

Ghosh and coworkers developed a FRET-based assay formonitoring the kinetics of PaeR7 endonuclease enzymeactivity (32). The authors synthesized a series of duplexsubstrates with an internal CTCGAG PaeR7 recognitionsite and donor (fluorescein) and acceptor (rhodamine)dyes conjugated to the opposing 58 termini applying a 6mer carbon spacer. Restriction cleavage of the fluorogenicsubstrate resulted in time-dependent increase in donorfluorescence. The steady state kinetic parameters for thesesubstrates were in agreement with the rate constantsobtained from a gel electrophoresis-based fixed time pointassay using radiolabeled substrates. The FRET-based methodprovides a rapid continuous assay as well as high sensitiv-ity and reproducibility (32).

Ribonucleases (Rnases) have also been assayed usingfluorogenic substrate. Zelenko and coworkers synthesizedDUPAAA, a novel fluorogenic substrate for pancreaticRnases (130). It consists of the dinucleotide uridylyl-38,58-deoxyadenosine to which a fluorophore, o-aminobenzoicacid, and a quencher, 2,4-dinitroaniline, have been at-tached by means of phosphodiester linkages. Cleavage ofthe phosphodiester bond at the 38-side of the uridylylresidue by RNase caused a 60-fold increase in fluores-cence. The substrate was hydrolyzed efficiently by pancre-atic RNases, but no cleavage was observed with themicrobial RNase T1 (130).

Luminescence from lanthanides has also been used inFRET-based enzyme assay (16). The lanthanides binds to amutant Ca21 binding protein, oncomodulin, in whichsalicylic acid is conjugated to a cysteine residue. Lumines-cence of Tb31 resulting from energy transfer form thesalicylic group continuously decreased as it was monitoredusing time resolved luminescence in the presence ofproteolytic enzymes such as subtilisin, chymotrypsin,cathepsin B, and HIV-1 protease. The simplicity of theassay coupled with its high level of sensitivity makes ituseful for the detection of proteases at very low concentra-tions (16).

All the above fluorogenic substrates can be applied invitro. Mitra and coworkers introduced a novel fluorogenicsubstrate that can be applied for monitoring proteaseactivity within a cell, based on genetic engineering of the

green fluorescent protein (GFP) (74). They fused the Cterminus of a red-shifted variant of GFP (RSGFP4) to aflexible polypeptide linker containing a factor Xa proteasecleavage site. The C terminus of this linker was fused tothe N terminus of a blue variant of GFP (BFP5). TheRSGFP4-BFP5 concatamer was cleaved with factor Xaresulting in marked decrease in energy transfer, i.e.,increase in donor fluorescence. Although this substratewas tested only in vitro, the authors suggested that thisconcatamer might have advantages over other methods ofmonitoring protease activity or screening for proteaseinhibitors because the assay could be carried out in livingcells and in real time. Towards this end cells should becotransfected with the gene for the protease of interestand the gene for the BFP:RSGFRP concatmer. An intracel-lular assay may be particularly useful for finding proteaseinhibitors because factors such as the cytotoxicity of apotential inhibitor and its ability to enter into the cell areautomatically determined when screening with this assay.

Immunoassays. Interest in measuring the binding in-teractions between antibodies and their specific antigenshas resulted in the application of a broad array of technolo-gies to the development of immunodiagnostics. Fluores-cence immunoassays have received considerable attentionrecently due to their high sensitivity (25,75). Such immuno-assays are divided into two categories, heterogeneousassays, which involve physical separation of the assaymixture before detection, and homogeneous assay, inwhich no separation steps are required. The majority ofexisting fluoroimmunoassays are heterogeneous. Further-more, they are often competitive assays in which afluorescently labeled antigen (or antibody) competes forbinding with an unlabeled antigen (or antibody). In suchan assay, the fluorescently labeled species are referred toas the tracer, and the unlabeled species as the analyte.Depending on the assay being performed, the analyte canbe either an antigen (Ag) or an antibody (Ab). Afterremoval of unbound analyte and tracer, the signal intensityfrom the bound tracer is found to be inversely propor-tional to the analyte concentration in the original solu-tions. The separation step always complicates the designof immunoassays and can degrade their overall perfor-mance.

Consequently, homogeneous immunoassays are advanta-geous because they are conducted entirely in the originalsample mixture, requiring fewer manipulations, renderingthe assay for easy automation. In general this results infewer sources of imprecision, shorter times to results anddecreased hazards due to sample handling. Homogeneousimmunofluorescence assays can be based on several typesof physicochemical interactions. They modulate the labelemission within the immunological complex (39), includ-ing spectral changes of a fluorescent labeled antibodyupon binding of unlabeled Ag (63). Changes may arise influorescence polarization and fluorescence lifetime duringassay reaction (38,40), and in FRET processes broughtabout during immune complex formation (110).

Ullman et al. (111) in 1976 developed the first immuno-assay based on FRET. They applied fluorescein labeled


antigen as donor and rhodamine labeled antibody asacceptor. The size of the antigen and antibody complexwas compatible with the FRET distance, thus the complexled to fluorescence quenching. Inclusion of unlabeledantigen reduced the available binding sites by competitivebinding thus reducing the amount of quenching (Fig. 7).

In the second approach, separate portions of antibodywere labeled with fluorescein and rhodamine, respec-tively. Provided that multivalent antigen is used, admixtureof the differently labeled antibody fractions with unlabeledantigen should reduce the fluorescence intensity by bring-ing the donor and acceptor within close proximity (Fig. 8).The first approach was applied successfully for detectionof fluorescein-labeled morphine conjugates in the 100 pMrange and above. In the second approach morphine-albumin conjugate was used as multivalent antigen and upto 20 anti-morphine antibody was able to bind to morphine-albumin conjugate (111).

Thorough analysis of the optimal conditions of thisFRET-based immunoassay system revealed that althoughfluorescein and rhodamine as the donor and acceptorlabels are perfectly practicable, these fluorophores are byno means ideal for use in such an assay (64,110). Particulardisadvantages include the poor stability of rhodamine-labeled antibodies, and the overlap of fluorescein andrhodamine absorption spectra. In addition, the Stokes shiftfor fluorescein is relatively small, so interference fromscattered light may limit the sensitivity of the assay inbiological samples. Usually the intensity of rhodamineemission at 580 nm is too feeble to permit its routine use inan assay based on fluorescence sensitization (64,110).

To overcome some of the above-mentioned problems,48,58-dimethoxy-6-carboxyfluorescein was introduced as anew acceptor in FRET-based immunoassay (55). Thiscompound is nonfluorescent and participates in ‘‘dark’’transfer process, giving no contribution to the backgroundfluorescence of the assay mixture. In addition, due to the

better spectral overlap with the fluorescein emissionspectra, R0 value of 6.2 nm was determined as comparedto that of 5.4 nm for the fluorescein rhodamine pair. Thehigher R0 value made it possible to achieve efficientquenching of the fluorescent antigen without overlabelingthe antibodies, increasing the stability and bench lifetimeof the acceptor labeled antibody. The usefulness of thisnew probe was demonstrated in assaying morphine (55).

The other approach to improve FRET-based immunoas-say took advantage of excellent spectral properties ofphycobilliprotein fluorescent dyes (57). Two dye combina-tions were used to demonstrate the basic principlesinvolved. In the first, fluorescein-conjugated human immu-noglobulin G (IgG) was quenched by a conjugate of PE and

FIG. 7. FRET-based homogeneous,competitive immunoassay. Antibodies andantigens are labeled with donor and ac-ceptor molecules respectively. The ana-lyte is the unlabeled antigen in thiscompetitive assay. The extent of quench-ing (FRET) decreases in the presence ofthe unlabeled antigen.

FIG. 8. FRET-based homogeneous immunoassay for polyhaptenic anti-gen. Two monoclonal antibodies raised against the antigen and labeledrespectively with donor and acceptor fluorophore. FRET is observed onlyin the presence of the antigen.


goat anti-human IgG. In the second, the fluorescence of PEconjugate to human IgG was quenched by Texas Red-labeled goat anti-human IgG. The reason PE can be used aseither donor or acceptor is that the absorption spectrumof PE overlaps the emission spectrum of fluorescein andthe absorption spectrum of Texas Red overlaps the emis-sion spectrum of PE. Both approaches produced sensitiveand reliable assays for human IgG. In the latter combina-tion, a potential advantage of the system is operation in thered end of this spectrum, where biological interferencesshould be much less. This is most important in homoge-neous assays in which the assay mixture is not separatedfrom the tested serum. In addition, the phycobiliproteinsgive more intense signals and less nonspecific binding.Immunity from random collision quenching with ions ofthe serum should also be significantly decreased becausechromophores in phycobiliproteins are protected by theproteins to which they are attached (57).

FRET immunoassays based upon acceptor fluorescenceemission are plagued with background fluorescence result-ing from absorption of the excitation light by the acceptordye. To overcome this problem a long-lifetime donorfluorophore (pyrenebutyrate) and a short-lifetime accep-tor fluorophore (PE) were applied in combination withpulsed-laser excitation and electronic gating of detectorsignals. This permitted the separation of the component ofacceptor emission due to energy transfer from the compo-nent arising from the absorption of the excitation light(75). Human IgG was measured in a competitive assayusing pyrene-conjugated F(ab8)2 fragments against humanIgG and PE-conjugated human IgG as a tracer. Acceptoremission was measured with 0- and 20-ns integrationdelays and the ratios of the FRET components to thelaser-excited component increased by 9- to 15-fold whenthe 20-ns delay was used in the immunoassay and thesensitivity was in the order of 10 nM. Increasing, thesensitivity required lowering the concentration of labeledantigen and antibody reagents. Although the fluorometercan detect the acceptor fluorescence at a concentrationthousand-fold lower than those used by the assay de-scribed above, the formation of appreciable amounts ofantibody-antigen complexes at lower reagent concentra-tions required the use of antibodies with affinity constantsconsiderably higher than those applied by Morrison (75).

Energy transfer process between PE-conjugated thyrox-ine and Cy5-conjugated anti-thyroxine antibody was usedto measure thyroxine concentration using competitiveassay format (81). In this assay format, however, thefluorescence intensity change was not monitored but thefluorescence lifetime was determined by multifrequencyphase-modulation fluorescence. Dose-response curves ofphase angle versus thyroxine concentration were compa-rable to steady-state fluorescence intensity curves. Becausephase modulation lifetime measurements are largely inde-pendent of total signal intensity, sources of optical interfer-ence are minimized (81). This approach was furtherimproved when long-lifetime luminescent metal-ligandcomplexes were used in a FRET-based immunoassay forhuman serum albumin (128). Human serum albumin

(HSA) was labeled with [Ru(bpy)2(phen-ITC)]21, and theantibody to HSA with a nonfluorescent absorber, ReactiveBlue 4. The concentration of HSA was determined in acompetitive assay in which the presence of analyte pre-vented the decrease of fluorescence lifetime by immunecomplex formation. The advantages of the ruthenium-ligand fluorophore include its long wavelength absorptionand emission, long fluorescence lifetime, and high photo-stability. Long wavelengths minimize problems of autofluo-rescence from biological samples, and long lifetimes allowoff-gating of the prompt autofluorescence (128).

Mathis also used rare earth (Eu) cryptates to developFRET immunoassay allowing double discrimination of theemitted light through spectral and temporal selectivity(67,68). Eu(III) trisbipyridine cryptate [TBP(Eu(III))]–conjugated antibody was used as donor, and APC-labeledantibody was used as acceptor. Both antibodies bindprolactin and in the presence of prolactin an immunecomplex is formed and energy transfer can take place.Because sensitized emission of APC is detected, themethod is called as amplified homogeneous assay. Time-resolved detection eliminates the direct fluorescence ofunbound APC-antibody, spectral selection eliminates theluminescence of TBP(Eu(III)). The author introduceddouble wavelength detection, which fully shields the assayfrom perturbations of the media. This feature of the assayis based on the following phenomenon: because of theamount of the analyte and the bound donor-labeledantibody is always small compared to the overall concentra-tion of the donor-labeled antibody, the free donor-labeledantibody concentration can be taken as a constant. Theemission yield of free donor becomes an internal lumines-cence standard that takes into account quenching artifactsdue to absorptions by interfering species in the sample tobe analyzed. These features have allowed the developmentof a rapid homogeneous immunoassay that can detect aslittle as 0.3 µg/l of prolactin. A plate reader has also beendeveloped to permit simultaneous dual-wavelength time-resolved luminescence detection for this assay (25,67,68).

In FRET immunoassays, random labeling of whole anti-body with a fluorescent donor could result in some donormoieties that are so distant from the binding site that theyare unable of undergoing energy transfer to bound accep-tor-conjugated antigen. This circumstance is unsatisfactorybecause there would be no change in intensity and lifetimefor that donor population. Thus the extent of immunecomplex formation would be poorly determined and thedynamic range of the assay would be reduced. In contrast,site-specific labeling results in greater homogeneity ofFRET distances. To achieve this goal, Chang and cowork-ers applied photoaffinity labeling in which fluorescein wasconjugated to the biotin hapten and tetramethylrhoda-mine was attached to anti-biotin antibodies (13). Anenergy transfer efficiency of almost 50% was observedupon binding of fluoresceinated biotin, which indicatedthat the photoaffinity labeling technique attached tetra-methylrhodamine groups at positions within energy trans-fer distance (5.2 nm) from the antigen-combining site.


A novel concept was described for directly couplingfluorescence enhancement to protein-ligand binding (121).Wei and coworkers used the phenomenon that somefluorescent dyes (e.g., fluorescein and rhodamine mol-ecules) form dimers in aqueous solution when they are athigh concentrations or within close proximity to eachother, resulting in significant quenching of the fluoro-phores. This sometimes adverse effect can be used toadvantage to quench the fluorescence of free tracers andto enhance the fluorescence of bound tracers if thedimer t monomer equilibrium can be modulated byantibody antigen reactions. Toward this end, a 13-residuepeptide, recognized by a monoclonal antibody againsthuman chorionic gonadotrophin (hCG) was labeled withfluorescein and tetramethylrhodamine at its N- and C-termini. Due to intramolecular dimerization, the fluores-cence of fluorescein and rhodamine was quenched by 98%and 90%, respectively. Binding of anti-hCG antibody to thelabeled peptide resulted in a 7.8-fold increase in rhoda-mine fluorescence due to dissociation of intramoleculardimers brought about conformational changes of theconjugate upon binding. Fluorescein fluorescence was stillquenched because of the FRET to rhodamine. In the assaymixture the presence of the analyte (hCG) will inhibit theenhancement in rhodamine fluorescence. The double-labeled peptide technique is a promising assay, especiallyof large molecular analytes, given that fluorescence homo-geneous assays are simple, reproducible, and can readilybe adapted to 96-well fluorescence plate readers (121).

DNA analysis. DNA analysis is becoming increasinglyimportant in the diagnosis of hereditary diseases, detec-tion of infections agents, tissue typing for histocompatibil-ity, identification of individuals in forensic and paternitytesting, and monitoring the genetic composition of plantsand animals in agricultural breeding programs. DNA analy-sis usually involves DNA sequencing, polymerase chainreaction (PCR), and DNA hybridization or combinations ofthese techniques. Application of FRET-based fluorescentprobes fertilized and revolutionized all of these fields: thefluorescence-based DNA sequencing, quantitative monitor-ing of PCR reactions, and the assay of DNA denaturation.For flow cytometry, until recently much of the nucleicacid-based technology was available only for researchapplications such as the PCR-driven fluorescent in situhybridization (FISH) method (76). With color-coded beadtechnology, it is possible to prepare DNA hybridizationand ligand binding interactions. The principle is the sameas that utilized by immunoassays. The ligand-ligate interac-tion occurs not between an antigen and an antibody butbetween oligonucleotides in a hybridization situation.

The human genome project is driving the developmentof high-speed and high-output DNA sequencing and analy-sis methods. Currently, the Sanger dideoxy chain-termina-tion method is accepted as the technique of choice for alllarge-scale sequencing projects (87,88). The dideoxy se-quencing involves the use of 28,38-dideoxynucleosidetriphosphates (ddNTPs), which lack a 38-hydroxyl group.In this method, the single-stranded DNA to be sequencedserves as the template strand or in vitro DNA synthesis: a

synthetic 58-end-labeled oligodeoxynucleotide is used asthe primer. Four separate polymerization reactions areperformed, each with a low concentration of one of thefour ddNTPs in addition to higher concentrations of thenormal deoxynucleoside triphosphates (dNTPs). In eachreaction the ddNTP is randomly incorporated at thepositions of the corresponding dNTP; such addition ofddNTP terminates polymerization because the absence of38 hydroxyl prevents addition of the next nucleotide. Thenewly synthesized DNA can be fluorescently labeled withany of four differently end-labeled fluorescent primers (91)or terminators (85). The mixture of terminated fragmentsfrom each of the four reactions is subjected to gel orcapillary electrophoresis in parallel; the separated frag-ments then are detected using appropriate excitation(usually laser) and detection systems. The sequence of theoriginal DNA template strand can directly be read from theresulting chromatogram. The detection sensitivity is lim-ited by the spectroscopic properties of the availablefluorescent dyes for labeling the sequencing fragments.Ideally each of the four dyes should exhibit strong absorp-tion at a common laser wavelength, have an emissionmaximum at a distinctly different wavelength, and intro-duce the same relative electrophoretic mobility shift of theDNA sequencing fragments. Because these criteria areinconsistent with the spectroscopic properties of singlefluorescent dye molecules, Ju and coworkers have de-signed and synthesized FRET-based primers for applicationto four-color DNA sequencing (51). These primers carry afluorescein derivative (FAM [5-carboxyfluorescein]) at the58 end as a common donor and other fluorescein (JOE[28,78-dimethoxy-48,58-dichloro-6-carboxyfluorescein]) andrhodamine (TAMRA [N,N,N8,N8-tetramethyl-6-carboxyrho-damine] or ROX [6-carboxy-X-rhodamine]) derivatives at-tached to a modified thymidine residue within the primersequence as acceptors. By adjusting the donor-acceptorspacing through the placement of the modified thymidinein the primer sequence the authors were able to generatefour primers, all having strong absorption at a commonexcitation (488 nm), similar electrophoretic mobility, andfluorescence emission maxima at 525, 555, 580, and 605nm. The FRET efficiency of these primers ranges from 65%to 97%. The fluorescence intensity of these FRET primersis 2- to 6-fold greater than that of the correspondingprimers or fragments labeled with single dyes allowingDNA sequencing with one-fourth of DNA template re-quired (51,52). This approach has successfully been com-bined with capillary electrophoresis chips for ultra-high-speed DNA sequencing (125) and applied for rapid sizingof short tandem repeat alleles (118–120). The same groupof investigators modified their method for FRET primersynthesis by constructing fluorescent primers using auniversal ET cassette that can be incorporated by conven-tional synthesis at the 58-end of an oligonucleotide primerof any sequence. In this cassette, the donor and acceptorfluorophores are separated by a polymer spacer (S6)formed by six 18,28-dideoxyribose phosphate monomers(50). The resulting primers using FAM as a donor and FAM,JOE, TAMRA or ROX as acceptors display well-separated


acceptor emission spectra with 2- to 12-fold enhancedfluorescence intensity relative to that of the correspondingsingle dye-labeled primers (49,50). To improve match inelectrophoretic mobility of extended DNA fragments withthose of the DNA sequencing fragments extended formother ET primers, the acceptor JOE was replaced by 5- and6-carboxyrhodamine-6G (R6G). With single-strandedM13mp18 DNA as the template, a typical run with the newFRET primer with the other FRET primers on a capillarysequencer provided DNA sequences with 99% accuracy inthe first 620 bases (43). The next improvement in FRETprimers was the introduction of cyanine dyes with largeabsorption cross-sections as donor chromophores. Thenew ET primers have 3-(e-carboxypentyl)-38-ethyl-58,58-dimethyloxacarbocyanine (CYA) at the 58-end as a com-mon energy donor and FAM, R6G, TAMRA, and ROX dyesas acceptors. With 488-nm excitation, the fluorescenceintensity of these four FRET primers is 1.4- to 24-foldstronger than that of the corresponding primers only withsingle acceptor dye (44,45). The CYA-ROX primers offerbetter combination of acceptor fluorescence emissionintensity and spectral purity than primers using BODIPYanalogs as energy transfer pairs (43). The improvement ofthe spectroscopic properties of fluorescent tags providedby FRET primers has led to a quantum advance in thedetection capabilities in DNA sequencing. These FRETprimers are generally applicable to all analyses that employfluorescent primers and should increase the sensitivity andthroughput (50).

Polymerase chain reaction has gained wide acceptanceas an exponential nucleic acid amplification technique andis used very frequently for research and clinical applica-tions. Although PCR is mainly used as a qualitative tool,there is a need for obtaining semiquantitative or evenquantitative information. To eliminate the application ofradioactive primers or radioactive nucleotides, detectionsystems based on fluorescence have been elaborated forquantitation the products of PCR reaction. Chan andcoworkers 58-end-labeled a PCR primer with the europiumchelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) (12). After performing thePCR and separating the PCR products with gel electropho-resis the gel was immersed into a Eu31 solution. Duringsoaking the Eu31 diffused into the gel formed a fluorescentcomplex of long fluorescence lifetime with BCPDA. Thiscomplex was quantified by scanning the gel with atime-resolved fluorometric reader. Because the BCPDAand Eu31 are not fluorescent themselves, the backgroundsignal was very low, and the detection limit was about 5 ngof DNA (12). The europium in combination with an otherchelator (with trisbipyridine cryptate) has successfullybeen applied in PCR reaction for the detection of humanpapillomavirus type 16 DNA in clinical smears. In thisapproach, nested PCR was performed with use of biotinyl-ated and 2,4-dinitrophenol (DNP)-labeled oligonucleotidesand then the biotinylated strands were anchored toavidin-coated microtiter plates. DNA hybrids containingboth the biotinylated strand and the DNP- labeled strandwere labeled with TBP(Eu31)-labeled anti-DNP antibody.

PCR product was quantified by measuring fluorescenceaccording to the principle of time-resolved fluorometry(65). In the above instances only the end products of thePCR reaction were measured, although there is a demandfor continuous monitoring the DNA amplification process.Toward this end, Wittwer and coworkers have developedthree fluorescent approaches for monitoring rapid cycleDNA amplification (122). The reaction was followed byeither measuring the fluorescence of SYBR Green dyeincorporating into double-stranded DNA; monitoring thedecrease in fluorescein quenching by rhodamine afterexonuclease cleavage of a dual-labeled hydrolysis probe;or quantitating the resonance energy transfer betweenfluorescein and Cy5 dyes by adjacent hybridization probes.The specificity of the last two methods using the FRETprinciple was much better than that of the first method.With hydrolysis probes, fluorescence continues to in-crease after the plateau phase is reached, whereas withhybridization probes, fluorescence decreases during theplateau phase. Despite these limitations, initial templatecopy number can be quantified easily by measuringfluorescence at each amplification cycle (122). Based onthis principle Wittwer and coworkers developed a commer-cial microvolume fluorimeter (LightCyclery) with rapidtemperature control interrogating 1 to 10 µl samples inglass capillaries (123). In this instrument a completeamplification and analysis requires only 10 to 15 min(123).

Detection of point mutation and deletion or insertion ofbasepairs in specific DNA fragments can accurately beachieved via DNA sequencing. If only the presence or theabsence of such alterations is to be detected, monitoringDNA denaturation could be the right solution. Hiyoshi andHosoi used fluorescein-11-dUTP and rhodamine-4-dUTP tolabel complementary DNA strands using PCR (42). Thecomplementary strands thus labeled were then annealedby incubation at a constant temperature, and the energytransfer efficiency was monitored during DNA denatur-ation induced by heat or alkali treatments. The methodproved to be a simple and efficient procedure for monitor-ing DNA denaturation for use in automated detection anddiagnosis systems (42).

Chen and Kwok have developed a novel detectionstrategy that allows the rapid analysis of single nucleotidepolymorphisms in a homogeneous assay, eliminating theneed for product separation and the use of radioactivity(15). Their approach combines the specificity of enzy-matic discrimination between the two alleles of a singlenucleotide polymorphism in a template-directed primerextension reaction and the sensitivity of fluorescenceresonance energy transfer. DNA fragments containingpolymorphic sites are incubated with a 58-fluorescein-labeled primer (designed to hybridize to DNA templateadjacent to the polymorphic site) in the presence of allelicdye (ROX)-labeled dideoxyribonucleoside trisphosphates.The dye-labeled primer is extended one base by thedye-terminator specific for the allele present on thetemplate. FRET occurs when the dye-labeled ddNTP isincorporated into the sequencing primer in the presence


of DNA polymerase and target DNA. Fluorescence inten-sity of the two dyes in the reaction mixture is analyzeddirectly without separation or purification. This template-directed dye-terminator incorporation assay is highly sensi-tive and specific and suitable for automated genotyping oflarge numbers of samples (15). The method has success-fully been applied to detect mutations in the cystic fibrosistransmembrane conductance regulator gene (14).

DNA tests will no doubt be performed more and moreby clinical rather than research laboratories, utilizing themultiplexed assays that require minimal laboratory skills,and manual handling will be crucial to the practice ofmedicine in the future.

NEW DEVELOPMENTS

Successful application of FRET is highly promoted byintroduction of modern instrumentation in fluorescencedetection systems. The advantage of fluorescence lifetimeimaging (FLIM) over conventional steady-state fluores-cence microscopy results from the fact that fluorescencelifetimes are usually independent of the fluorophore con-centration, photobleaching, and other artifacts that affectfluorescence intensity measurements (89). Time-resolvedfluorescence microcopy enables mapping of fluorescencelifetimes, which is vital for the determination of the spatialdistribution of probe molecules. In addition, it can also beused to enhance contrast between fluorescence arisingfrom distributions of different probe species having similarspectral characteristics and provides a means of discrimi-nating against background autofluorescence. Because mea-suring changes in the fluorescence lifetime can monitorFRET efficiency, combination of FLIM with FRET measure-ments will open new fields for this type of approach.Whereas the first FLIM instruments were relatively slow,the latest developments in this field greatly enhanced theresolution time-domain FLIM measurement (89).

Application of near-field scanning optical microscopy(NSOM) extends the sensitivity of FRET to the singlemolecule level by measuring FRET between single donorand single acceptor molecules. NSOM is a relatively newtechnique that allows optical measurements with sub-wavelength resolution (37). It is based on a probe havingvery small (sub-wavelength) aperture that is placed inclose proximity (less than 10 nm) to the sample underinvestigation. By using the probe as an excitation source,fluorescence of single molecules can be detected. Anotherimportant aspect of NSOM is that the optical radiation inthe near field has an electric field component along itsdirections of propagation. This allows mapping the transi-tion dipole moment orientation of a single molecule inthree dimensions. Combination of FRET and NSOM offersmany potential advantages when distance and orientationinformation is required on a molecular level (37).

It has been shown that photobleaching FRET measure-ments can be used to monitor intercellular proximity inorder to assess spatial organization of interacting proteinsin the contact region of two ‘‘communicating’’ cells (2).Interactions between CD8 and MHC class I molecules andalso between LFA-1 and ICAM-1 molecules has successfully

been studied using fluoresceinated and rhodaminatedmAbs. The geometry of these protein contacts based onFRET data was consistent with the observed blockingeffects of monoclonal antibodies (directed against theinteracting proteins) on the cytolytic activity of CTLs (2).Further development in this technique will be the introduc-tion of confocal microscopy in studying intercellularinteractions.

A new development in the field of photobleachingenergy transfer measurements is the introduction of accep-tor photobleaching (4). In this approach the increase inthe fluorescence intensity of donor is measured followingthe local photochemical destruction of the energy accep-tor. Donor fluorophores engaged in FRET exhibit anincrease in quantum yield after acceptor photo-destruc-tion, reflect as an increase of fluorescence. Photodestruc-tion of the acceptor molecules can be performed in aregion of interest (i.e., at any part of a cell). FRET efficiencycan be calculated by taking the ratio of donor imagesbefore and after photobleaching without external calibra-tion. In practice, experiments can be performed with aconfocal microscope equipped with a multiple laser sys-tem providing lines corresponding to the absorptionmaxima of both donor and acceptor. The techniquepermits the exploitation FRET in the study of the process-ing and interactions of biomolecules in cells (4).

CONCLUSIONS

Since the first description of the phenomenon, thenumber of applications of FRET has been increasedenormously in diverse research fields in the last fewdecades. A unique feature of FRET is its capability todetect, quantitatively, molecular interactions, over dis-tances of tens of angstroms. Improving the sensitivity ofthe detection systems in specrofluorometers, flow cytom-eters, and microscopes widen the field in which FRET canbe applied. Improvements in FRET analysis, especially intemporal resolution of fluorescence data have increasedconsiderably the possibilities for FRET measurements. Inaddition, introduction of new fluorescent dyes with betterphotophysical properties also promotes the application ofFRET. Developments in microscopic techniques in theperformance and availability of sophisticated image dataacquisition and image analysis have also made it possibleto contemplate more quantitative FRET experiments inoptical microscopes. Introduction of confocal detectionarrangement will also reinforce the FRET approach. Theseimprovements in microscopic techniques open up newareas in biological problems that otherwise could not beinvestigated with the classical FRET method. Spatiallyresolved FRET measurements in an image provide impor-tant information about the distribution of molecular-scaleinteractions over distances of microns, helping in under-standing the intricate interactions in functioning biologicalsystems. For example, cell-cell interactions can be studiedin a very special way using microscopic FRET technique,because the protein-protein interactions in the area ofcell-cell contact can be investigated selectively.


These improvements and inventions will continue tofacilitate the innovative applications of FRET in clinicallaboratory and basic research.

ACKNOWLEDGMENTS

We thank Francis Mandy for critical reading of themanuscript and for his valuable suggestions.

LITERATURE CITED1. Armand S, Drouillard S, Schulein M, Henrissat B, Driguez, H: A

bifunctionalized fluorogenic tetrasaccharide as a substrate to studycellulases. J Biol Chem 272:2709–2713, 1997.

2. Bacso Z, Bene L, Bodnar A, Matko J, Damjanovich S: A photobleach-ing energy transfer analysis of CD8/MHC-I and LFA-1/ICAM-1 interac-tions in CTL-target cell conjugates. Immunol Lett 54:151–156, 1996.

3. Bacso Z, Matko J, Szollo9si J, Gaspar R, Damjanovich S: Changes inmembrane potential of target cells promotes cytotoxic activity ofeffector T lymphocytes. Immunol Lett 51:175–180, 1996.

4. Bastiaens PIH, Majoul IV, Verveer PJ, Soling H-D, Jovin TM: Imagingthe intracellular trafficking and state of the AB5 quaternary structureof cholera toxin. EMBO J 15:4246– 4253, 1996.

5. Beavis AJ, Pennline KJ: ALLO-7: A new fluorescent tandem dye foruse in flow cytometry. Cytometry 24:390–394, 1996.

6. Bene L, Balazs M, Matko J, Most J, Dierich M, Szollo9si J, DamjanovichS: Lateral organization of the ICAM-1 molecule at the surface ofhuman lymphoblasts: A possible model for its co-distribution withthe IL-2 receptor, class I and class II HLA molecules. Eur J Immunol24:2115–2123, 1994.

7. Bene L, Szollo9si J, Balazs M, Matyus L, Gaspar R, Ameloot M, Dale RE,Damjanovich S: Major histocompatibility complex class I proteinconformation altered by transmembrane potential changes. Cytome-try 27:353–357, 1997.

8. Bickett DM, Green MD, Wagner C, Roth JT, Berman J, McGeehanGM: A high throughput fluorogenic substrate for stromelysin (MMP-3). Ann NY Acad Sci 732:351–355, 1994.

9. Bodnar A, Jenei A, Bene L, Damjanovich S, Matko J: Modification ofmembrane cholesterol level affects expression and clustering of classI HLA molecules at the surface of JY human lymphoblasts. ImmunolLett 54:221–226, 1996.

10. Bouvier J, Schneider P, Malcolm B: A fluorescent peptide substratefor the surface metalloproteinase of Leishmania. Exp Parasitol76:146–155, 1993.

11. Catipovic B, Talluri G, Oh J, Wei T, Su X-M, Johansen TE, Edidin M,Schneck JP: Analysis of the structure of empty and peptide-loadedMajor Histocompatibility Complex molecules at the cell surface. JExp Med 180:1753–1761, 1994.

12. Chan A, Diamandis EP, Krajden M: Quantification of polymerasechain reaction products in agarose gels with a fluorescent europiumchelate as label and time-resolved fluorescence spectroscopy. AnalChem 65:158–163, 1993.

13. Chang I-N, Lin J-N, Andrade JD, Herron JN: Photoaffinity labeling ofantibodies for applications in homogeneous fluoroimmunoassays.Anal Chem 67:959–966, 1995.

14. Chen X, Zehnbauer B, Gnirke A, Kwok P-Y: Fluorescence energydetection as a homogeneous DNA diagnostic method. Proc Natl AcadSci USA 94:10756–10761, 1997.

15. Chen X, Kwok P-Y: Template-directed dye-terminator incorporation(TDI) assay: A homogeneous DNA diagnostic method based onfluorescence resonance energy transfer. Nucleic Acid Res 25:347–353, 1997.

16. Clark ID, Macmanus JP, Szabo AG: A protease assay using time-resolved lanthanide luminescence from an engineered calciumbinding protein substrate. Clin Biochem 28:131–135, 1995.

17. Clegg RM: Fluorescence energy transfer. Curr Opin Biotech 6:103–110, 1995.

18. Clegg RM: Fluorescence Resonance Energy Transfer (FRET). In:Fluorescence Spectroscopy and Microscopy. Wang XF, Hermann B(eds). J. Wiley and Sons, New York, 1996. Chemical Analysis Series.Vol. 137:179–252.

19. Dale RE, Eisinger J, Blumberg WE: The orientational freedom ofmolecular probes. The orientation factor in intramolecular energytransfer. Biophys J 26:161–194, 1979.

20. Dale RE, Novros J, Roth S, Edidin M, Brand L: Application of ForsterLong-Range Excitation Energy Transfer to the Determination ofDistributions of Fluorescently-Labelled Concanavalin A-ReceptorComplexes at the Surfaces of Yeast and of Normal and MalignantFibroblasts. In: Fluorescent Probes, Beddard GS, West A (eds).Academic Press, London, 1981, pp 159–189.

21. Damjanovich S, Gaspar R, Pieri C: Dynamic receptor superstructureat plasma membrane. Q Rev Biophys 30:67–106, 1997.

22. Damjanovich S, Tron L, Szollo9si J, Zidovetzki R, Vaz WLC, RegateiroF, Arndt-Jovin DJ, Jovin TM: Distribution and mobility of murinehistocompatibility H-2Kk antigen in the cytoplasmic membrane. ProcNatl Acad Sci USA 80:5985–5989, 1983.

23. Damjanovich S, Vereb G, Shaper A, Jenei A, Matko J, Starink JPP, FoxGO, Arndt-Jovin, DJ, Jovin TM: Structural hierarchy in the clusteringof HLA class I molecules in the plasma membrane of humanlymphoblastoid cells. Proc Natl Acad Sci USA 92:1122–1126, 1995.

24. Dewey TG, Hammes GG: Calculation of fluorescence resonanceenergy transfer on surfaces. Biophys J 32:1023–1035, 1980.

25. Dickson EFG, Pollak A, Diamandis P: Time-resolved detection oflanthanide luminescence for ultrasensitive bioanalytical assays. JPhotochem Photobiol B: Biol 27:3–19, 1995.

26. Doody MC, Sklar LA, Pownall HJ, Sparrow JT, Gotto AM Jr, Smith LC:A simplified approach to resonance energy transfer in membranes,lipoproteins and spatially restricted systems. Biophys Chem 17:139–152, 1983.

27. Estep TN, Thompson TE: Energy transfer in lipid bilayers. Biophys J26:195–208, 1979.

28. Fairclough RH, Cantor CR: The use of singlet-singlet energy transferto study macromolecular assemblies. Methods Enzymol 48:347–379,1978.

29. Forster T: Energiewanderung und Fluoreszenz. Naturwissenschaften6:166–175, 1946.

30. Forster T: Zwischenmolekulare Energiewanderung und Fluoreszenz.Ann Phys (Leipzig) 2:55–75, 1948.

31. Fulton RJ, McDade RL, Smith PL, Kienker LJ, Kettman JR: Advancedmultiplexed analysis with the FlowMetrix system. Clin Chem 43:1749–1756, 1997.

32. Ghosh SS, Eis PS, Blumeyer K, Fearon K, Millar DP: Real time kineticsof restriction endonuclease cleavage monitored by fluorescenceresonance energy transfer. Nucleic Acid Res 22:3155–3159, 1994.

33. Goudreau N, Guis C, Soleilhac J-M, Roques BP: Dns-Gly-(p-NO2)Phe-bAla, a specific fluorogenic substrate for neutral endopeptidase24.11. Anal Biochem 219:87–95, 1994.

34. Guo C, Dower SK, Holowka D, Baird B: Fluorescence resonanceenergy transfer reveals interleukin (IL)-1-dependent aggregation ofIL-1 type I receptors that correlates with receptor activation. J BiolChem 270:27562–27568, 1995.

35. Gutierrez-Merino C: Quantitation of the Forster energy transfer fortwo dimensional systems. I. Lateral phase separation in unilamellarvesicles formed by binary phospholipid mixtures. Biophys Chem14:247–257, 1981.

36. Gutierrez-Merino C: Quantitation of the Forster energy transfer fortwo dimensional systems. II. Protein distribution and aggregationstate in biological membranes. Biophys Chem 14:259–266, 1981.

37. Ha T, Enderle DF, Ogletree DF, Chemla DS, Selvin PR, Weiss S:Probing the interaction between two single molecules: fluorescenceresonance energy transfer between a single donor and a singleacceptor. Proc Natl Acad Sci USA 93:6264–6268, 1996.

38. Haver VM, Audino N, Burris S, Nelson M: Four fluorescencepolarization immunoassays for therapeutic drug monitoring evalu-ated. Clin Chem 35:138–140, 1989.

39. Hemmila I: Fluoroimmuoassay and immunofluorometric assays. ClinChem 31:359–370. 1985.

40. Hemmila I, Malminen O, Mikola H, Lovgren T: Homogeneoustime-resolved fluoroimmunoassay of thyroxin in serum. Clin Chem34:2320–2322, 1988.

41. Hirschfeld T: Quantum efficiency independence of the time inte-grated emission from a fluorescent molecule. Applied Optics 15:3135–3139, 1976.

42. Hiyoshi M, Hosoi S: Assay of DNA denaturation by polymerase chainreaction-driven fluorescent label incorporation and fluorescenceresonance energy transfer. Anal Biochem 221:306–311, 1994.

43. Hung S-C, Ju J, Mathies RA, Glazer AN: Energy transfer primers with5- or 6- carboxyrhodamine-6G as acceptor chromophores. AnalBiochem 238:165–170, 1996.

44. Hung S-C, Ju J, Mathies RA, Glazer AN: Cyanine dyes with highabsorption cross section as donor chromophores in energy transferprimers. Anal Biochem 243:15–17, 1996.

45. Hung S-C, Mathies RA, Glazer AN: Comparison of fluorescenceenergy transfer primers with different donor-acceptor dye combina-tions. Anal Biochem 255:32–38, 1998.

46. Jenei A, Varga S, Bene L, Matyus L, Bodnar A, Bacso Z, Pieri C, GasparR, Farkas T, Damjanovich S: HLA class I and II antigens are partiallyco-clustered in the plasma membrane of human lymphoblastoidcells. Proc Natl Acad Sci USA 94:7269–7274, 1997.

47. Jovin TM, Arndt-Jovin DJ: Luminescence digital imaging microscopy.Ann Rev Biophys Biophys Chem 18:271–308, 1989.


48. Jovin TM, Arndt-Jovin DJ: FRET microscopy: Digital imaging offluorescence resonance energy transfer. Application in cell biology.In: Kohen E, Ploem JS and Hirschberg JG (eds.), Microspectrofluorom-etry of single living cells, Academic Press, Orlando, 1989, pp.99–117.

49. Ju J, Glazer AN, Mathies RA: Cassette labeling for facile constructionof energy transfer fluorescent primers. Nucleic Acid Res 24:1144–1148, 1996.

50. Ju J, Glazer AN, Mathies RA: Energy transfer primers: A newfluorescence labeling paradigm for DNA sequencing and analysis.Nature Med 2:246–249, 1996.

51. Ju J, Kheterpal I, Scherer JR, Ruan C, Fuller CW, Glazer AN, MathiesRA: Design and synthesis of fluorescence energy transfer dye-labeledprimers and their application for DNA sequencing and analysis. AnalBiochem 231:131–140, 1995.

52. Ju J, Ruan C, Fuller CW, Glazer AN, Mathies RA: Fluorescence energytransfer dye-labeled primers for DNA sequencing and analysis. ProcNatl Acad Sci USA 92:4347–4351, 1995.

53. Jurgens L, Arndt-Jovin D, Pecht I, Jovin TM: Proximity between thetype I receptor Fce (FceRI) and the mast cell function-associatedantigen (MAFA) studied by donor photobleaching fluorescenceresonance energy transfer microscopy. Eur J Immunol 26:84–91,1996.

54. Kam Z, Volberg T, Geiger B: Mapping of adherent junction compo-nents using microscopic resonance energy transfer imaging. J CellSci 108:1051–1062, 1995.

55. Khanna PL, Ullman EF: 48,58-Dimethoxy-6-carboxyfluorescein: Anovel dipole-dipole coupled fluorescence energy transfer acceptoruseful for fluorescence immunoassays. Anal Biochem 108:156–161,1980.

56. Kindzelskii AL, Laska ZO, Todd RF, Petty HR: Urokinase-typeplasminogen activator receptor reversibly dissociates from comple-ment receptor type 3 (aMb2; CD11b/CD18) during neutrophilpolarization. J Immunol 156:297–309, 1996.

57. Kronick MN, Grossman PD: Immunoassay techniques with fluores-cent phycobilliprotein conjugates. Clin Chem 29:1582–1586, 1983.

58. Lakowicz JR: Principles of Fluorescence Spectroscopy, PlenumPress, New York, 1983, pp. 303–339.

59. Lazarovits AI, Osman N, Le Feuvre CE, Ley SC, Crumpton MJ: CD7 isassociated with CD3 and CD45 on human T cells. J Immunol153:3956–3964, 1994.

60. Lansdorp PM, Smith C, Safford M, Terstappen LW, Thomas TE: Singlelaser three immunofluorescence staining procedures based on en-ergy transfer between phycoerythrin and cyanine-5. Cytometry12:723–730, 1991.

61. Lee KB, Matsuoka K, Nishimura SI, Lee YC: A new approach to assayendo-type carbohydrases: bifluorescent-labeled substrates for gly-coamidases and ceramide glycanases. Anal Biochem 230:31–36,1995.

62. Lee SP, Censullo ML, Kim HG, Knutson JR, Han MK: Characterizationof endonucleolytic activity of HIV-1 integrase using a fluorogenicsubstrate. Anal Biochem 227:295–301, 1995.

63. Liburdy RP: Antibody induced fluorescence enhancement of aN-(3-pyrene) maleimide conjugate of rabbit anti-human immunoglobu-lin G: Quantitations of human IgG. J Immunol Methods 28:233–242,1979.

64. Lim CS, Miller JN, Bridges JW: Energy-transfer immunoassay: A studyof the experimental parameters in an assay for human serumalbumin. Anal Biochem 108:176–184, 1980.

65. Lopez E, Chypre C, Alpha B, Mathis G: Europium(III) trisbibyridinecryptate label for time- resolved fluorescence detection of polymer-ase chain reaction products fixed on a solid support. Clin Chem39:196–201, 1993.

66. Matayoshi ED, Wang GT, Krafft GA, Erickson J: Novel fluorogenicsubstrates for assaying retroviral proteases by resonance energytransfer. Science 247:954–958, 1990.

67. Mathis G: Rare earth cryptates and homogeneous fluoroimmunoas-says with human sera. Clin Chem 39:1953–1959, 1993.

68. Mathis G: Probing molecular interactions with homogeneous tech-niques based on rare earth cryptates and fluorescence energytransfer. Clin Chem 41:1391–1397, 1995.

69. Matko J, Edidin M: Energy transfer methods for detecting molecularclusters on cell surfaces. Methods Enzymol 278:444–462, 1997.

70. Matko J, Szollo9si J, Tron L, Damjanovich S: Luminescence spectro-scopic approaches of cell surface molecules. Quart Rev Biophys21:479–544, 1988.

71. Matyus L: Fluorescence resonance energy transfer measurements oncell surfaces. A spectroscopic tool for determining protein interac-tions. J Photochem Photobiol B: Biol 19:67–73 1992.

72. Matyus L, Bene L, Heiligen H, Rausch J, Damjanovich, S: Distinctassociation of transferrin receptor with HLA class I molecules onHUT-102B and JY cells. Immunol Lett 44:203–208, 1995.

73. McDade RL, Fulton RJ: True multiplexed analysis by computer-enhanced flow cytometry. Med Dev Diag Indust 19:75–82, 1997.

74. Mitra RD, Silva CM, Youvan DC: Fluorescence resonance energytransfer between blue- emitting and red-shifted excitation derivativesof the green fluorescent protein. Gene 173:13–17, 1996.

75. Morrison LE: Time-resolved detection of energy transfer: Theory andapplication to immunoassays. Anal Biochem 174:101–120, 1988.

76. Mosiman VL, Patterson BK, Canterero L, Goolsby C: Reducingcellular autofluorescence in flow cytometry: An in situ method.Cytometry 30:151–156, 1997.

77. Neppert J, Muller-Eckhardt C: Monoclonal antibodies to human classI antigens cocap class II antigens. Tissue Antigens 24:187–189, 1984.

78. Neppert J, Muller-Eckhardt C: Interdependent membrane mobility ofhuman MHC coded antigens detected by monoclonal antibodies tovarious epitopes on class I and class II molecules. Tissue Antigens26:51–69, 1985.

79. Niles DW, Silvius JR, Cohen FS: Resonance energy transfer imaging ofphospholipid vesicle interaction with a planar phospholipid mem-brane. Undulations and attachment sites in the region of calcium-mediated membrane-membrane adhesion. J Gen Physiol 107:329–351, 1996.

80. Oi VT, Glazer AN, Stryer L: Fluorescent phycobiliprotein conjugatesfor analyses of cells and molecules. J Cell Biol 93:981–986, 1982.

81. Ozinskas AJ, Malak H, Joshi J, Szmacinski H, Britz J, Thompson RB,Koen PA, Lakowicz JR: Homogeneous model immunoassay ofthyroxine by phase modulation fluorescence spectroscopy. AnalBiochem 213:264–270, 1983.

82. Pennington MW, Thornberry NA: Synthesis of a fluorogenic interleu-kin-1b converting enzyme substrate based on resonance energytransfer. Peptide Res 7:72–76, 1994.

83. Poo H, Fox B, Petty HR: Ligation of CD3 triggers transmembraneproximity between LFA-1 and cortical microfilaments in a cytotoxicT cell clone derived from tumor infiltrating lymphocytes: A quantita-tive resonance energy transfer study. J Cell Physiol 159:176–180,1994.

84. Poo H, Krauss JC, Mayo-Bond L, Todd RF, Petty HR: Interaction ofFcg receptor type IIB with complement receptor type 3 in fibroblasttransfectants: Evidence from lateral diffusion and resonance energytransfer studies. J Mol Biol 247:597–603, 1995.

85. Prober JM, Trainor GL, Dam RJ, Hobbs FW, Robertson CW, ZagurskyRJ, Cocuzza AJ, Jensen MA, Baumeister K: A system for rapid DNAsequencing with fluorescent chain- terminating dideoxynucleotides.Science 238:336–341, 1987.

86. Roederer M, Kantor AB, Parks DR, Herzenberg LA: Cy7PE andCy7APC: Bright new probes for immunofluorescence. Cytometry24:191–197, 1996.

87. Sanger F, Coulson AR: A rapid method for determining sequences inDNA by primed synthesis with DNA polymerase. J Mol Biol 94:441–448, 1975.

88. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467, 1977.

89. Scully AD, Ostler RB, Phillips D, O’Neill P, Townsend KMS, ParkerAW, MacRobert AJ: Application of fluorescence lifetime imagingmicroscopy to the investigation of intracellular PDT mechanisms.Bioimaging 5:9–18, 1997.

90. Selvin PR: Fluorescence resonance energy transfer. Methods Enzy-mol 246:300–334, 1995.

91. Smith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, Connell CR,Heiner C, Kent SB, Hood LE: Fluorescence detection in automatedDNA sequence analysis. Nature 321:674–679, 1986.

92. Snyder B, Freire E: Fluorescence energy transfer in two dimensions.A numeric solution for random and nonrandom distributions. Bio-phys J 40:137–148, 1982.

93. Stack MS, Gray RD: Comparison of vertebrate collagenase andgelatinase using a new fluorogenic substrate peptide. J Biol Chem264:4277–4281, 1989.

94. Stryer L: Fluorescence energy transfer as a spectroscopic ruler. AnnuRev Biochem 47:819–846, 1978.

95. Szabo G, Pine PS, Weaver JL, Rao PE, Aszalos A: CD4 changesconformation upon ligand binding. J Immunol 149:3596–3604,1992.

96. Szabo G, Pine PS, Weaver JL, Rao PE, Aszalos A: The L-selectin (Leu8)molecule is associated with the TcR/CD3 receptor: Fluorescenceenergy transfer measurements on live cells. Immunol Cell Biol.72:319–325, 1994.

97. Szabo G, Weaver JL, Pine PS, Rao PE, Aszalos A: Cross-linking of CD4in a TCR/CD3- juxtaposed inhibitory state: A pFRET study. Biophys J68:1170–1176, 1995.


98. Szollo9si J, Damjanovich S: Mapping of Membrane Structures byEnergy Transfer Measurements. In: Mobility and Proximity in Biologi-cal Membranes, Damjanovich S, Szollo9si J. Tron L, Edidin M (eds).CRC Press, Boca Raton, 1994, pp. 49–108.

99. Szollo9si J, Damjanovich S, Balazs M, Nagy P, Tron L, Fulwyler MJ,Brodsky FM: Physical association between MHC class I and class IImolecules detected on the cell surface by flow cytometry energytransfer. J Immunol 143:208–213, 1989.

100. Szollo9si J, Damjanovich S, Mulhern SA, Tron L: Fluorescence energytransfer and membrane potential measurements monitor dynamicproperties of cell membranes: A critical review. Prog Biophys MolBiol 49:65–87, 1987.

101. Szollo9si J, Horejsi V, Bene L, Angelisova P, Damjanovich S: Supramo-lecular complexes of MHC class I, MHC class II, CD20, and tetraspanmolecules (CD53, CD81, and CD82) at the surface of a B cell line JY.J Immunol 157:2939–2946, 1996.

102. Szollo9si J, Matyus L, Tron L, Balazs M, Ember I, Fulwyler MJ,Damjanovich S: Flow cytometric measurements of fluorescenceenergy transfer using single laser excitation. Cytometry 8:120–128,1987.

103. Szollo9si J, Tron L, Damjanovich S, Helliwell SH, Arndt-Jovin DJ, JovinTM: Fluorescence energy transfer measurements on cell surfaces. Acritical comparison of steady-state and flow cytometric methods.Cytometry 5:210–216, 1984.

104. Taliani M, Bianchi E, Narjes F, Fossatelli M, Urbani A, Steinkuhler C,De Francesco R, Pessi A: A continuous assay of hepatitis C virusprotease based on resonance energy transfer depsipeptide sub-strates. Anal Biochem 240:60–67, 1996.

105. Tampe R, Clark BR, McConnell HM: Energy transfer between twopeptides bound to one MHC class II molecule. Science 254:87–89,1991.

106. Tron L: Experimental Methods to Measure Fluorescence ResonanceEnergy Transfer. In: Mobility and Proximity in Biological Mem-branes, Damjanovich S, Szollo9si J, Tron L, Edidin M (eds). CRC Press,Boca Raton, 1994, pp. 1–47.

107. Tron L, Aszalos A, Balazs M, Mulhern SA, Szollo9si J, Damjanovich S:On the biophysics of transmembrane signalling. Mol Immunol25:1075–1080, 1988.

108. Tron L, Szollo9si J, Damjanovich S: Proximity measurements of cellsurface proteins by fluorescence energy transfer. Immunol Lett16:1–9, 1987.

109. Tron L, Szollo9si J, Damjanovich S, Helliwell SH, Arndt-Jovin DJ, JovinTM: Flow cytometric measurement of fluorescence resonance en-ergy transfer on cell surfaces. Quantitative evaluation of the transferefficiency on a cell-by-cell basis. Biophys J 45:939–946, 1984.

110. Ullman EF, Khanna PL: Fluorescence excitation transfer immunoas-say (FETI). Methods Enzymol 74:28–60, 1981.

111. Ullman EF, Schwarzberg M, Rubenstein KE: Fluorescent excitationtransfer immunoassay. A general method for determination ofantigens. J Biol Chem 251:4172–4178, 1976.

112. Uster PS, Pagano RE: Resonance energy transfer microscopy: Obser-vations of membrane-bound fluorescent probes in model mem-branes and in living cells. J Cell Biol 103:1221–1234, 1986.

113. Uster PS, Pagano RE: Resonance energy transfer microscopy: Visualcolocalization of fluorescent lipid probes in liposomes. MethodsEnzymol 171:850–857, 1989.

114. Vignali DAA, Carson RT, Chang B, Mittler RS, Strominger JL: The twomembrane proximal domains of CD4 interact with the T cellreceptor. J Exp Med 183:1097–2107, 1996.

115. Waggoner AS, Ernst LA, Chen LA, Rechtenwald DJ: PE-Cy5. A newfluorescent antibody label for three-color flow cytometry with singlelaser. Ann NY Acad Sci 677:185–193, 1993.

116. Wang GT, Chung CC, Holzman TF, Kraft GA: A continuous fluores-cence assay of renin activity. Anal Biochem 210:351–359, 1993.

117. Wang W, Liang TC: Fluorogenic peptides containing only a-aminoacids. Biochem Biophys Res Commun 201:835–840, 1994.

118. Wang Y, Hung S-C, Linn JF, Steiner G, Glazer AN, Sidransky D,Mathies RA: Microsatellite-based cancer detection using capillaryarray electrophoresis and energy-transfer fluorescent primers. Elec-trophoresis 18:1742–1749, 1997.

119. Wang Y, Ju J, Carpenter BA, Atherton JM, Sensabaugh GF, MathiesRA: Rapid sizing of short tandem repeat alleles using capillary arrayelectrophoresis and energy-transfer fluorescent primers. Anal Bio-chem 67:1197–1203, 1995.

120. Wang Y, Wallin JM, Ju J, Sensabaugh GF, Mathies RA: High-resolutioncapillary array electrophoretic sizing of multiplexed short tandemrepeat loci using energy-transfer fluorescent primers. Electrophore-sis 17:1485–1490, 1996.

121. Wei A-P, Blumenthal DK, Herron JN: Antibody-mediated fluores-cence enhancement based on shifting the intramolecular dimer tmonomer equilibrium of fluorescent dyes. Anal Chem 66:1500–1506, 1994.

122. Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP: Continuousfluorescence monitoring of rapid cycle DNA amplification. BioTech-niques 22:130–138, 1997.

123. Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA, Balis UJ:The LightCycleo: A microvolume multisample fluorimeter with rapidtemperature control. BioTechniques 22:176–181, 1997.

124. Wolber PK, Hudson BS: An analytic solution to the Forster energytransfer problem in two dimensions. Biophys J 28:197–210, 1979.

125. Woolley AT, Mathies RA: Ultra-high-speed DNA sequencing usingcapillary electrophoresis chips. Anal Biochem 67:3676–3680, 1995.

126. Worth RG, Mayo-Bond L, van de Winkel JGJ, Todd FR, Petty HR: CR3(aMb2; CD11b/CD18) restores IgG-dependent phagocytosis in trans-fectants expressing a phagocytosis-defective FcgRIIA (CD32): tail-minus mutant. J Immunol 157:5660–5665, 1996.

127. Wu P, Brand L: Resonance energy transfer: Methods and applica-tions. Anal Biochem 218:1–13, 1994.

128. Youn HJ, Terpetschnig E, Szmacinski H, Lakowicz JR: Fluorescenceenergy transfer immunoassay based on a long-lifetime luminescentmetal-ligand complex. Anal Biochem 232:24–30, 1995.

129. Zarewych DM, Kindzelskii AL, Todd RF, Petty HR: LPS induces CD14association with complement receptor type 3, which is reversed byneutrophil adhesion. J Immunol 156:430–433, 1996.

130. Zelenko O, Neumann U, Brill W, Pieles U, Moser HE, Hosteenge J: Anovel fluorogenic substrate for ribonucleases. Synthesis and enzy-matic characterization. Nucleic Acid Res 22:2731–2739, 1994.

131. Zheng Y, Shopes B, Holowka D, Baird B: Conformations of IgE boundto its receptor FceRI and in solution. Biochemistry 30:9125–9132,1991.

132. Zhou MJ, Poo H, Todd III RF, Petty HR: Surface-bound immunecomplexes trigger transmembrane proximity between complementreceptor type 3 and the neutrophil’s cortical microfilaments. JImmunol 148:3550–3553, 1992.


Application of fluorescence resonance energy transfer in the clinical laboratory: Routine and...

Documents

Transcript of Application of fluorescence resonance energy transfer in the clinical laboratory: Routine and...