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Extending Frster resonance energytransfer measurements beyond 100 using common organic fluorophores:enhanced transfer in the presence ofmultiple acceptors

Badri P. Maliwal

Sangram RautRafal FudalaSabato DAuriaVincenzo M. MarzulloAlberto LuiniIgnacy GryczynskiZygmunt Gryczynski

wnloaded From: http://biomedicaloptics.spiedigitallibrary.org/ on 03/07/2013 Terms of Use: http://spiedl.org/terms

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Extending Frster resonance energy transfermeasurements beyond 100 using commonorganic fluorophores: enhanced transfer inthe presence of multiple acceptors

Badri P. Maliwal,a Sangram Raut,a Rafal Fudala,a Sabato DAuria,b Vincenzo M. Marzullo,b,c Alberto Luini,cIgnacy Gryczynski,a,d and Zygmunt Gryczynskia,eaUniversity of North Texas Health Science Center, Department of Molecular Biology and Immunology, Center for Commercialization ofFluorescence Technologies, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76106bLaboratory for Molecular Sensing, IBP-CNR, Via Pietro Castellino, 111 80131 Naples, ItalycTelethon - Institute of Genetics and Medicine, Via Pietro Castellino, 111, 80131 Naples, ItalydUniversity of North Texas Health Science Center, Department of Cell Biology and Genetics, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76106eTexas Christian University, Department of Physics and Astronomy, TCU Box 298840, Fort Worth, Texas 76129

Abstract. Using commercially available organic fluorophores, the current applications of Frster (fluorescence)resonance energy transfer (FRET) are limited to about 80 . However, many essential activities in cells are spatiallyand/or temporally dependent on the assembly/disassembly of transient complexes consisting of large-size macro-molecules that are frequently separated by distances greater than 100 . Expanding the accessible range for FRET to

150 would open up many cellular interactions to fluorescence and fluorescence-lifetime imaging. Here, wedemonstrate that the use of multiple randomly distributed acceptors on proteins/antibodies, rather than the useof a single localized acceptor, makes it possible to significantly enhance FRET and detect interactions between thedonor fluorophore and the acceptor-labeled protein at distances greater than 100 . A simple theoretical model forspherical bodies that have been randomly labeled with acceptors has been developed. To test the theoreticalpredictions of this system, we carried out FRET studies using a 30-mer oligonucleotide-avidin system that waslabeled with the acceptors DyLight649 or Dylight750. The opposite 5-end of the oligonucleotide was labeledwith the Alexa568 donor. We observed significantly enhanced energy transfer due to presence of multiple accep-tors on the avidin protein. The results and simulation indicate that use of a nanosized body that has been randomlylabeled with multiple acceptors allows FRET measurements to be extended to over 150 when using commerciallyavailable probes and established protein-labeling protocols. 2012 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI:10.1117/1.JBO.17.1.011006]

Keywords: DNA; enhanced transfer efficiency; FRET; interactions at distances greater than 100 ; multiple acceptors.

Paper 11216SS received May 3, 2011; revised manuscript received Aug. 15, 2011; accepted for publication Aug. 22, 2011; publishedonline Jan. 19, 2012.

1 IntroductionFrster (fluorescence) resonance energy transfer (FRET) isextensively used in biochemical and biomedical research.13

As a spectroscopic ruler, FRET has been an enormously use-ful tool for monitoring fundamental processes such as proximityrelationships between interacting biomolecules and conforma-tional changes within single molecules.4,5 Radiationless interac-tions between a donor and a single acceptor depend on theFrster radius (R0) and the inverse 6th power of the donoracceptor separation. R0 can be theoretically calculated if one

knows the overlap integral, quantum yield of the donor, the rela-tive orientation of the emission transition moment of the donor,the absorption transition moment of the acceptor (the so-calledorientation factor, 2), and the refractive index, n, of the mediumseparating the donor and acceptor. The overlap integral reflectsthe spectral overlap between donor emission and chromophoreabsorption of the acceptor. The orientation factor can vary

between 0 and 4, and in dynamic systems where molecularmobility is high the orientation factor can be assumed to be23. Until recently, the use of commercially available organicfluorophores has allowed the observation of R0 as large as 65to 75 , and experimentally feasible FRET signals can be mea-sured at distances (i.e., separations) of up to about 100 to 110 .This is about 1.4- to 1.5-times R0. The primary reason for this isthe fact that donoracceptor interactions rapidly vanish withincreasing probe separation (1r6), and for separations greaterthan 1.5R0 the transfer efficiency is below 10%. For largerdonoracceptor separations, potential artifacts and errorsthat are typically present in experiments can easily overwhelmthe expected effects, especially when employing cellular micro-scopy.6,7 Recently available reactive forms of far-red probes withboth high extinction coefficients and good quantum yields canextend the practical R0 to about 80 and the observation win-dow to around 110 to 120 .8,9

Presently, one typically labels specific sites using a singledonor on a given macromolecule and a single acceptor on

Address all correspondence to: Zygmunt Gryczynski, Texas Christian University,Department of Physics and Astronomy, TCU Box 298840, Fort Worth, Texas76129. Tel: 817 257 4209; E-mail: [email protected] 0091-3286/2012/$25.00 2012 SPIE

Journal of Biomedical Optics 011006-1 January 2012 Vol. 17(1)

Journal of Biomedical Optics 17(1), 011006 (January 2012)


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another macromolecule in order to monitor intermoleculardistances during interactions (e.g., physiological processes).Using FRET-based fluorescence microscopy, great strides havebeen made in the study of protein interactions in cells that occuron the scale of 8 to 9 nm.13,5,10,11 However, there are stillmany important spatial/proximal interactions that occur at thecellular level, such as those related to Golgi bodies involvedin trafficking,12 that taking place at distances much greater than

100 . These interactions cannot be studied using FRET withthe commercially and widely available fluorophores.

For some time there has been ongoing research to increasethe distances monitored by FRET to beyond 100 . One of theapproaches that has been used to extend FRET to greaterdistances is the use of high quantum-yield and very long-lifelanthanide donors. Their ms luminescence lifetime also allowsthe suppression of nanosecond fluorescence from both theacceptor and the background, enabling the observation ofacceptor-sensitized emission with time-gated detection. Thelong lifetime of this emission enables the observation of signif-icantly smaller changes in the lifetime induced by FRET, allow-ing researchers to monitor interactions that occur at distances

much greater than R0. Lanthanides are also quite useful becauseof their remarkably sharp line emission that is suitable for multi-plexing. ForR0 approaching 70 that is matched to the properorganic fluorophore acceptors, it is possible to measure dis-tances over 110 using lanthanide donors.1317 These reportsindicate that using lanthanide as the donor, it is possible toreliably measure distances about 1.60- to 1.65-times R0. Theseresults compare quite favorably with the practical limit of1.5-times R0 that can be obtained using traditional organicfluorophores. Using acceptors such as quantum dots (QD) andphycobiliproteins (PE), which possess significantly higherabsorption and molar extinction coefficients, R0 values of 90 to100 have been measured when using lanthanide donors.1820

Therefore, in principle, it should be possible to monitor FRET at

distances less than 150 under practical experimental condi-tions when using acceptable donoracceptor pairs. However,it should be noted that both chelated lanthanide donors andacceptors, such as QD and PE molecules, are significantly largerthan conventional organic acceptor molecules. Because the mea-sured donoracceptor separation refers to the distance betweenthe centers of the interacting entities, a practical consequence oftheir relatively larger sizes is that the donor and acceptor mole-cules will occupy up to 50 to 60 of the measured distance.This will result in limiting the effective observation distance toaround 100 under most experimental conditions.20 Morgner etal. used this disadvantage in an imaginative application todevelop a simple and inexpensive FRET method to monitor thesize of QDs, the results of which compare favorably with the

results of more demanding transmission electron microscopy.20The molar extinction coefficients of the organic sensitizer

molecules used in lanthanide-based probes are typically lessthan 20; 000 M1 cm1. This, together with the intrinsic mslanthanide lifetimes, results in a significantly smaller photonflux that limits the applications of cellular microscopybasedFRET. Also, the need to excite in the UV/near-visible spectrum(typically between 325 and 360 nm) is also a significantdrawback.

Another promising approach designed to extend measurableFRET to beyond 100 involves gold and silver nanoparticles.

Gold nanoparticles quench fluorescence and the process hasbeen described as nanomaterial surface energy transfer (NSET).However, the underlying quenching mechanism shows a com-plex dependence on the size of the gold particle, and themechanism also changes with the amount of separation betweenthe fluorophore and gold nanoparticle. As things stand, a greatdeal has yet to be learned about how gold nanoprobe distancesare dependent on the quenching of fluorescence before this

method becomes as prevalent and routine as FRET.2123 Silvernanoparticle-associated surface plasmons are also known toenhance FRET.2426 Because silver nanoparticle plasmons aredependent on particle size, shape, and relative orientation of thechromophore in relation to the particle, it is more likely that suchinteractions will be incorporated into assays and simple detec-tion systems than quantitative FRET applications.

The original Frster formalism has been adopted to describefluorescence energy transfer involving single and multipledonors and acceptors, the various dimensionalities of the sys-tem, and cases of restricted geometry, to name a few.4,27,28 Atthe beginning of 1990s, we explained that the experimentallyobserved enhanced energy transfer of tryptophan moieties tofour heme units in hemoglobin is the result of the summationof individual radiative transfer rates involving each hemeacceptor.2931 More generally, the observed FRET, in the caseof a single donor and multiple acceptors, is the sum of the indi-vidual transfer rates to each acceptor. In addition, a more recentpublication describes a generalized theoretical framework ofFRET for oligomeric protein complexes that are labeled usingmultiple donors and acceptors.32 There are also some recentpublications that take advantage of enhanced transfer efficiencydue to multiple acceptors to optimize the experimental FRETdesign. Emphasis, however, seems to be on the developmentof more sensitive analytical assays and detection systems.3235

Our review of the available literature suggests that theobserved energy transfer efficiency from a single donor to an

acceptor can be enhanced by confining multiple acceptors toa suitably sized single protein/antibody/small particle in theplace of a single acceptor, as is routinely done. The use of asmall protein body with multiple acceptors can be a convenientway to study the interactions that occur at larger separations, asfrequently happen during protein assembly/disassembly in cells.Because the random labeling of antibodies, or fragments ofantibodies, with multiple dyes is now standardized and can becontrolled with good precision, it is likely that such labeledproteins will find wide use in the study of macromolecularinteractions that occur at distances greater than 100 .

2 ExperimentalA 30-mer oligonucleotide with biotin on the 3-end and Alexa-

568 (A568) on the 5-end and its complimentary strand werepurchased from Invitrogen (Grand Island, New York) asHPLC-purified samples. The sequence of donor oligonucleotidewas as follows:

A568-5-GAAGCTTAGATAAATAATTTGATAACGTAC-Biotin

High-purity avidin was purchased from SigmaAldrich(St. Louis, Missouri), and the reactive forms of the fluorophoreacceptors, DyLight-649 (DL649) and DyLight-750 (DL750),were purchased from Thermo Fisher (Rockford, Illinois). Tolabel avidin, typically 0.5 mL of 2 to 3 M of 50 mM borate

Maliwal et al.: Extending Frster resonance energy transfer measurements beyond 100 using common organic : : :



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buffer (pH 8.5) is mixed with the N-hydroxysuccinimide (NHS)ester of the probes (less than 1% dimethylformamide byvolume), allowed to react for 3 h, and then the labeled proteinis separated from the free probe by passing it through a SephadexG-25 (Sigma-Aldrich) desalting column. We used NHS esters ata concentration of 8- to 15-times the protein concentration,which roughly translates to the use of twice the number of reac-tive probes relative to the number of acceptors we intended to

label. The molar extinction coefficient of 103; 000 M1 cm1at 280 nm was used to calculate the avidin concentration ofthe solution. The molar extinction coefficients and 280-nm cor-rection factors used to calculate the extent of labeling were 250000/0.037 for DL649 and 220,000/0.02 for DL750, respectively.The extent of labeling is reported as the number of fluorescentacceptors per avidin tetramer molecule and represents the aver-age number of labels per protein (i.e., avidin tetramer).

In order to hybridize single strands (ss), the donor-labeledoligonucleotide and the complimentary oligonucleotide(1.15access) solutions in 5 mM Tris (pH 7.5) and 50 mmNaCl were mixed and equilibrated in a 65 C water bath for15 min. The resulting double-stranded (ds) oligonucleotide sam-ples were allowed to slowly cool to room temperature. For the dsoligonucleotide solutions, appropriate amounts of labeled avidinwere added, gently shaken, and equilibrated for 1 h. The finalconcentrations were 100 nM labeled oligonucleotide and160 nM avidin tetramer. Because an excess of avidin tetramerwas used, we expected the dominant complex to be made up ofonly one ds oligonucleotide bound to a given avidin tetramer.

Steady-state fluorescence spectra were recorded using a CaryEclipse spectrofluorometer (Varian Inc., Australia) using verti-cally polarized excited light, and emission was observed at themagic angle (i.e., 54.7 deg). The excitation wavelength was535 nm, and the slit widths were set to 10 nm. The labeled avi-din absorption spectra were recorded using a Varian Cary 50spectrophotometer.

Lifetime measurements were carried out using a customizedFT 200 (Picoquant GmbH) time-correlated single photon-counting (TCSPC) fluorometer equipped with HamamatsuR3809U-50 microchannel plate photomultiplier (MCP) and a535-nm laser diode as the excitation source. The instrumentresponse function was less than 50 ps.36

3 Theoretical ModelFor our application, we considered a system consisting of a sin-gle donor and multiple acceptors that were randomly positionedon a globular protein/antibody. The point donor and proteinlabeled with multiple acceptors are connected via a rigid linkof a given length. The distance, s, is defined as the separationbetween the center of the donor and the closest point of

approach to the protein surface (Fig. 1). In this simple model,we considered the protein to be a sphere of radius r (where its

diameterD is equal to 2r). The acceptor molecules are randomlypositioned on the surface of the sphere. For a given acceptorlocation on the protein surface j, the separation betweenthe donor and the acceptor at the position j will be Rj. The dis-tance Rj depends on the separation s, the radius of globular pro-tein, and the angle that describes the acceptor location on thesphere with respect to the center of the sphere. Rj can then becalculated as:

Rj fs r1 cos j2 r sin j

2g12

Under these conditions, the closest location of the acceptor isequal to the separation s and the furthest location is equal tothe distance ofs 2r. The transfer rate kj to a single parti-cular acceptor at an arbitrary position j can be calculated as:

kj 1

0

R0

Rj

6

;

where 0 is the lifetime of the donor in the absence of anacceptor and R0 is the characteristic Frster distance for

the given donor

acceptor pair. R0 is defined as:R0 8.79 10

3QD2n4J16 ;

where QD is the quantum yield of the donor, J is the over-lap integral, n is the refractive index of the medium separat-ing the donor and acceptor, and 2 is the orientation factor,which depends on the relative orientation of the donor-emission transition moment and acceptor-absorption transi-tion moment. Because all other parameters that define R0 areidentical for all acceptors and can be predicted with reason-able accuracy, the orientation factor is undefined and mayvary from one acceptor to another. In the simplest case ofhighly mobile donors and acceptors (i.e., a rapidly rotating

donor and an acceptor within the fluorescence lifetime of thedonor), the 2 value can be approximated as 23.37,38 Beforeassuming the dynamic average of 2 (i.e., 23), typicallyone should estimate the probes mobility by measuring thefluorescence anisotropy of the donor and acceptor in thesystem.37,38

Next, we calculated the average transfer efficiency to theacceptors that were randomly positioned on the protein surface.First, we assumed that each position on the protein is equallyprobable. Also, for simplicity, we assumed that each donorand acceptor have significant mobilities, thus we were able toassume that the orientation factor is 2 23 for each acceptorposition. In cases where there is only one acceptor per protein,the expected energy transfer efficiency will be in the range of the

following:

kt 1

0

R0

s

6

and1

0

R0

s 2r

6

:

These values correspond to closest (left term) and farthest(right term) locations. To calculate the apparent/averagetransfer rate to one acceptor that was randomly positionedon the protein surface, we should consider all positions onthe spheres surface as equally probable. The transfer ratesare additive, and for any given configuration of acceptors the

Fig. 1 A model system of a single donor, D (), and randomly distributedacceptors, A (),on the surface of a spherical body (e.g., protein).




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cumulative rate is the simple sum. The details of averagingare similar to those used for calculating the FRET efficiencyfrom a single donor to a planar acceptor.2931 In the case of asphere (as shown in Fig. 1) for a given position j, the prob-ability of finding an acceptor at a this position is proportionalto the surface element, Sj 2dj 2r sin j,where is the angle element as shown in Fig. 1. Any posi-tion on the sphere around the perimeter dj r sin j is

equally probable. In our approximation, we assumed thatthe orientation factor 2 is independent of the location onthe sphere and equal to 23. Since the sum of all surfaceelements Sj yields the surface of the sphere 4r2, themeasured average transfer of an ensemble of pairs wherethe single acceptor is randomly positioned on the surfaceis given by the following sum:

kt

PjSjkj

4r2:

Forn independent acceptors, the total transfer efficiency willbe KT nkt. The measured FRET efficiency of a random

ensemble of n acceptors can be calculated as:

ET nkt

nkt 10

kT

kT 10:

Using this simple approximation, we simulated the transferefficiency of a system where the separation is 120 . Theacceptors are labeled to the protein with a diameter 40 ,and the characteristic R0 of the donoracceptor system is80 . For a single donoracceptor system separated by120 , the FRET efficiency will be about 8.1%. However,when the same acceptor is randomly positioned on thissphere, the average transfer efficiency drops to about 3.7%.

This significant drop is a consequence of the size of the pro-tein used to label the acceptors. The effect for most positionsis that each individual acceptor is at a much larger distancethan the distance of closest approach s. What is interestingand encouraging is that with as few as three randomly placedacceptors the calculated average FRET is almost 10%. Withsix random acceptors it further increases to almost 19%.

Figure 2 shows the predicted transfer efficiencies as a func-tion of separation s for a different number of acceptors. R0 hasbeen assumed to be 80 , and the size (diameter) of the labeledprotein is 40 . The dashed line represents the dependence of asingle donoracceptor pair (i.e., no protein body). It is encoura-ging to observe that the presence of even five randomly placedacceptors results in a significantly enhanced FRET in spite of thefact that protein size adds to the actual separation between the

donor and the acceptors. The effect is more pronounced at largerseparations. For this particular set of parameters, even at aphysical separation of 150 , the presence of five acceptorsper protein results in a FRET efficiency of 5.5%, and for 10acceptors the FRET efficiency is 10.4%. It should be notedthat 150 is the closest approach of a labeled protein, and theactual distance from the donor to the center of the acceptor-labeled protein is 170 . The precise limit of measurable FRETdepends on many experimental and instrumental factors. Whatis obvious from these simulations is that regardless of the experi-mental limitations of a given situation, the presence of multipleacceptors will yield a higher FRET signal and extend the rangeof the effective interactions. Fortunately, labeling large proteins,such as antibodies, with up to 10 dyes does not seem to perturbits binding capabilities and functions.

Another aspect we would like to address is the effect of thesize of used protein (antibody). In Fig. 3 we present simulationsof expected FRET as a function of protein size (D 2r) whenusing various numbers of acceptors. For this, we assumed aseparation of 120 and, again, R0 is assumed to be 80 .In the case of a small protein (D 20 ), the presence ofsix acceptors will result in a FRET efficiency of 25.1%. Thistransfer efficiency is almost three times larger than that of asingle donoracceptor pair that is separated by just 120 .An increase in the protein size to 35 , the size of a typicalminiantibody, decreases the FRET efficiency to 20.1%. Furtherincreasing the size of the protein to 55 , the size of a typical fab

fragment of an antibody, still results in a comfortable 15.4%transfer efficiency. It is important to remember that for verylarge proteins (e.g., antibodies), a greater labeling efficiency canbe achieved without affecting protein functions. In the case of a55 diameter-labeled protein, increasing the number of accep-tors to eight resulted in an easily measurable 19.5% transfer effi-ciency at a separation distance 120 (corresponding to 147.5

Fig. 2 Transfer efficiency as a function of separation s for a differentnumber of acceptors (1, 5, and 10) that are deposited on the surfaceof a spherical body with a diameter of 40 . Dashed line representsa single acceptor. Assumed Frster radius of 80 .

Fig. 3 Transfer efficiency as a function of the protein diameter D for adifferent number of acceptors.




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from the donor to the center of the labeled protein). Interestingly,even a gigantic protein of 150 with 10 acceptors will show atransfer rate of approximately 10%. It should be noted that whenwe state that a certain size is comparable to an experimentallyrelevant protein, we are matching their respective volumes. Forexample, a sphere of 55 will have a volume comparable to thatfor a 70- to 75--long prolate ellipsoid, such as a fab fragment.

4 ResultsIn order to experimentally test our model, we used the ds 30 mer,as previously described by Hyduk et al.16 The measured length

of this 30-mer ds oligonucleotide was about 100 and the donorand acceptor molecules add another 10 to the effectivedonoracceptor separation.1216 Earlier studies16 confirmedthe length of this ds oligonucleotide and its rigidity. In our case,the avidin molecule adds a minimum of 25 . We used Alexa-568 (A568) as the donor and DyLight-649 (DL649) or DyLight-750 (DL750) as the acceptor. The calculated R0 are 71.5 and61 for the A568-DL649 and A568-DL750 pairs, respectively.

Each donoracceptor system yields significantly differenttransfer efficiencies for each oligonucleotide length. Figure 4shows the steady-state emission spectrum of a donor-labeledds oligonucleotide and the absorption of acceptor-labeled avi-din. The absorption axis is normalized to the molar extinctioncoefficients of the acceptor probes. The calculated FRETefficiencies, in the case of a single donor and a single acceptorthat are tethered at opposite ends of this ds oligonucleotide, are alittle less than 0.03 (3%) for DL750 and about 0.07 (7%) forDL649.

Table 1 presents the results of the fluorescence lifetime mea-surements. A representative example of the measured intensitydecays is shown in Fig. 5. The donor-only intensity decay(A568) is made up of three lifetime components. The longest-lived component is around 3.5 ns and makes up nearly two-thirds of the population. This lifetime is similar to the reportedfluorescence lifetime of free A568 in buffer.8 The other two life-time components are about 1.3 and 0.2 ns, respectively, eachcontributing up to 15% to 20% of the A568 population. It isnot uncommon for single-lifetime fluorophores to present

Fig. 4 Fluorescence spectrum of donor A568 ds oligonucleotide (solidgreen) and the absorption spectra of avidin-DL649 (dashed red) andavidin-DL750 (dotted blue).

Table 1 Measured fluorescence lifetimes and transfer efficiencies of the three experimental systems. The last column presents thecorresponding calculated transfer efficiencies of the presented systems.

Sample R0 ns Amplitude < > ns 2 Measured transfer Calculated

Oligo-Donor 0.20 (0.171) 3.179 0.933

1.38 (0.197)

3.45 (0.632)

Donor-DL649 71.5 0.20 (0.180) 3.029 0.935 0.055 0.070

1.35 (0.207)

3.31 (0.613)

Donor plus 61.0 0.20 (0.184) 2.893 0.944 0.09 0.081

Avidin-DL750 1.33 (0.223)

(A 8) 3.19 (0.593)

Donor plus 71.5 0.20 (0.203) 2.793 0.928 0.12 0.123Avidin-DL649 1.31 (0.245)

(A 4.5) 3.13 (0.552)

Donor-plus 71.5 0.20 (0.211) 2.724 0.925 0.143 0.156

Avidin-DL649 1.36 (0.257)

(A 6) 3.08 (0.532)

A: number of labeled acceptors.Calculation are based on a distance of 110 forDL649, a distance of closest approach of 105 forsystemswith avidin(100 DNA donor), and a 55 - diameter of the labeled protein.




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additional lifetime components when attached to DNA due tovarious quenching mechanisms. In the presence of labeledavidin, we observed both a decrease in the longest lifetimevalues as well as an increase in the amplitudes of the shorterlifetime components. This is expected because FRET decreasesthe overall photon count, and the photons from the populationswith the shortest lifetimes will effectively make up a larger por-tion of the overall photon flux. The intensities of the averagedlifetimes are also given in the Table 1. The donor-only averagelifetime is 3.179 ns. At first, we tested the system using a singleacceptor (DL649) that was labeled at the 5-end of biotin. Theaverage lifetime decreased to 3.035 ns, which corresponds to aFRET efficiency of about 5.5%. In the presence of acceptorla-

beled avidin, we measured 2.893 ns for DL750 and 2.793 and2.724 ns for DL649. The extent of FRET was about 9% in thecase of the A568-DL750 pair (A 8) and 12% and 14.2% in thecases of A568-DL649 with 4.5 and 6 acceptors, respectively. Itis clear that the presence of multiple acceptors increases thetransfer efficiency regardless ofR0 under the presented experi-mental conditions. As expected, we observed a higher transfer asthe R0 value increased, as well as with an increasing number ofacceptors. We only present an analysis of FRET based onchanges in the average intensity of the fluorescence lifetime.The exact fluorescence lifetimes recovered from the experimentsare presented in Table 1. These results are in good agreementwith the theoretical predictions, but it would be preferableto have a single-lifetime donor in order to help minimize

uncertainty.To estimate the orientation factors, we measured the aniso-

tropy decay of the A568-labeled ds oligonucleotide that wasbound to avidin. The majority (greater than 75%) of anisotropydecay was associated with fast rotational motions of 0.35 to2.74 ns, yielding a low steady-state anisotropy for the donor.Because the average donor lifetimes of the measured systemsare greater than 2.7 ns, we can reasonably conclude that theAlexa probe is freely rotating in solution when it is attached toone point on the DNA molecule. We also checked the anisotro-pies of the acceptors at low labeling efficiencies when energy

migration between acceptors is minimal and, therefore, doesnot affect anisotropy. The measured steady-state anisotropiesfor excitation wavelengths of 630 nm (DL649) and 730 nm(DL750) were below 0.2, indicating significant mobility ofacceptors molecules when bound to avidin. In conclusion, thesetwo observations sufficiently justify the use of2 23 as theorientation factor.

5 DiscussionAs our simple model based on the Frster theory predicted, wewere indeed able to observe enhanced energy transfer when asingle donor and multiple acceptors were placed on an avidinmolecule. The measured transfer efficiencies correlate very wellwith the modeled predictions. The size of avidin is similar to thesize of a fab fragment of an antibody. The extent of the increasein transfer efficiency is encouraging, and it is possible to furtherincrease the number of acceptors and still have a functional pro-tein. Good agreement between the model and the experimentaldata for all three cases indicates that, in spite of the manyassumptions made in the model, it is able to describe the ensem-ble behavior very well. By measuring the emission anisotropies

of the donor and acceptor, we confirmed that probe mobility ishigh and the orientation factors of each acceptor in relation todonor averages is close to the assumed value of 2/3. Also, wewere only able to determine the average labeling efficiency(i.e., average number of acceptors per avidin tetramer). How-ever, we realize that this labeling is statistical, and that in areal system we have to deal with a distribution (typically aLorentzian distribution) of proteins that are labeled with differ-ent numbers of acceptors. For example, in the case of a labelingefficiency of 6, the number of proteins that are labeled with sixdyes will constitute more than 35% of entire population. Thetotal number of proteins with five to seven dyes will constituteover 70% of entire population. More importantly, the number ofprotein molecules without a dye will be below 1%, and the

number of antibodies with a single dye will be only 1.3% ofthe population. Therefore, almost 98% of protein moleculeshave two or more acceptors. The FRET efficiency dependson the number of acceptors, but our experimental resultsshow that the lower the FRET efficiency of a fraction of mole-cules with a smaller number of acceptors is compensated by anequivalent fraction with a higher than average number of accep-tors. It should be beneficial to use a larger labeling efficiency inorder to avoid contributions from pairs without acceptor-labeledproteins. Interestingly, a labeling efficiency of 6 already showsthat only 1% of the proteins are unlabeled, and we conclude thatlabeling efficiencies larger than 4 are experimentally acceptable.Good agreement between the calculated and measured FRETvalues confirms that one can use proteins labeled with many

acceptors to detect intermolecular interactions at large distances.At large labeling efficiencies, even quantitative measurements ofseparation distances much greater than 100 should be possibleas long as the protein size is known and the labels are randomlydistributed. In practical terms, this is not a major hurdle.

A typical experiment in cellular FRET often uses fluorescentantibodies to localize different proteins in a multicomponentcomplex. We fully realize that our model is at best a simpleapproximation. Proteins are typically ellipsoidal and not sphe-rical. Also, the initial labeling of lysine residues may not betotally random because lysine reactivities are known to differ

4

103

(Counts)

D

101

102

Intensity

IRF

15 20 25 30 35 40 45X2=0.934

0

4

als

X2=0.924

-4

15 20 25 30 35 40 45-4

0

4Resid

Time [ns]

Fig. 5 Time-resolved fluorescence intensity decays of donor anddonoracceptor complexes.




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between proteins. However, once we reach several labels perprotein, there is a good chance that labeling will begin toapproach a random distribution. These results suggest that moredetailed experiments are warranted that involve more than oneseparation distance, more than one acceptor for a given donor inorder to vary the R0 values, and a larger degree of acceptor label-ing in order to help us refine this simple approach. It would alsobe of interest whether such a multiparameter, global approach

will allow the independent determination of the size of theacceptor body along with the distance of the closest approach.

The ultimate measurable distance is likely to be determinedby the experimental and instrumental setup. The detection andresolution limit depends on many variables.1,6,7,36,38 We arecertain that for any given system, the use of multiple, ratherthan single, acceptors will enhance the transfer efficiency andallow for larger distances to be measured. There are also severalcommercially available reactive Near Infra Red probes that canbe used to create a R0 of about 80 to 85 . These values areobtained with practical considerations in mind, such as Stokesshift for fluorescent donors and adequate separation betweendonor florescence maxima and the beginning of acceptor fluor-escence. Based on simulations and these experimental results,we are confident that FRET measurements can now be easilyextended to about 140 to 150 with over 10% FRET efficiencyusing these donoracceptor pairs with multiple acceptors. Anyfuture developments that improve R0 will only help in the inves-tigation of even larger distances. There are also some other pro-mising potential acceptors that can serve the same purpose. Forexample, very small silica nanoparticles and dendrimers (whichare only a few nm in size) can bring many probes and a verylarge molar extinction coefficient to a very small area andcan be functionalized for use as acceptor labels. In practicalterms, these particles are similar to QDs and PE, though smaller.One of the parameters governing transfer efficiency is the size ofthe acceptor-labeled protein/macromolecule/particle. The smal-

ler the particle the greater the transfer efficiency for any given R0and number of acceptors. The RET of lanthanides has manyuseful attributes and this approach should be equally applicablefor the use of lanthanides as potential donors. One of the less-realized advantages of lanthanides is their single lifetime, whichmakes the interpretation of data simpler.

The enhancement of FRET due to the presence of multipleacceptors has been explored for the development of sensitiveanalytical assays for some time. The primary aim of thisresearch is to lower the detection limit and make assays moresensitive. Only recently, it seems, have FRET-based distancemeasurements over 100 come into focus, as reported in recentpublications.33,34 Our results also suggest that not only can weenhance FRET by using multiple acceptors, but under certain

conditions we can also quantitatively measure changes inseparations much larger than 100 . The primary way to mea-sure these distances has always been to use labels with a singledonor and a single acceptor. An equally promising aspect of ourapproach is that it not only employs commercially available andwidely used probes, but it also fits in nicely with existing andwidely used cell-based fluorescence microscopy. Oftentimes,fluorescence lifetime imaging is used to monitor changes inseparation during various cellular processes. We expect thisapproach to be adapted within the framework of FRET-basedcellular florescence microscopy.

Acknowledgments

This work was supported by grants from the National Institutesof Health (R21 CA149897 and 1R01EB012003) (Z.G.).

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