Proc. Natl. Acad. Sci. USAVol. 90, pp. 2994-2998, April 1993Biophysics

Observing the helical geometry of double-stranded DNA in solutionby fluorescence resonance energy transferROBERT M. CLEGG*, ALASTAIR I. H. MURCHIEt, ANNELIES ZECHEL*, AND DAVID M. J. LILLEYt*Department of Molecular Biology, Max-Planck-Institute for Biophysical Chemistry, D-W 3400 Gottingen, Federal Republic of Germany; and tCancerResearch Campaign, Nucleic Acid Structure Group, Department of Biochemistry, The University of Dundee, Dundee DD1 4HN, United Kingdom

Communicated by Gregorio Weber, December 18, 1992

ABSTRACT The efficiency of fluorescence resonance en-ergy transfer (FRET) between fluorescein and rhodaminecovalently attached to both 5' termini of a series of double-stranded DNA species (ranging from 8 to 20 bp) was measured.FRET efficiency varied with a dependence compatible withdye-to-dye distances (R) calculated on the basis of double-stranded B-DNA structure; the helical geometry of double-stranded DNA in solution is clearly evident. The experimentaldata were consistent with a 1/[1 + (RIRo)6] dependence ofFRET efficiency characteristic for the Forster dipole-dipolemechanism. The thermal dissociation of the strands of theduplex DNA species can be followed by using FRET, and fromthese data we have been able to obtain enthalpies of duplexformation in good agreement with earlier measurements usingalternative techniques. FRET measurements at very differentsalt concentrations can be accurately compared. We concludethat FRET is a reliable and valuable method for studyingstructure and conformational transitions in nucleic acids.

Nucleic acids may adopt specific and sometimes complexfolded structures that are critical for their biological function.Full determination of these structures requires the measure-ment of distances up to 80 A or more, but there are fewtechniques that allow such distances to be determined insolution. This is particularly important for nucleic acids inview of the extended helical structures involved, which canlead to underdetermination of structures by methods that canonly yield short distances. Fluorescence resonance energytransfer (FRET) is sensitive to distances in the longer sizerange and has recently proved to be very useful in the studyof nucleic acid structures, such as the solution structure ofthe four-way DNA junction (1, 2). FRET-derived distanceinformation inDNA and RNA could be complementary to theshorter distances determined by NMR. However, the appli-cation of FRET to fluorescence probes covalently linked tonucleic acid structures has been relatively infrequent (1,3-11).The rate of nonradiative energy transfer from an excited

donor molecule (D) to a nearby acceptor molecule (A)depends in a characteristic manner on the distance betweenthe two chromophores and their relative angular disposition(12-14). Depending on the D-A molecular pairs, the effi-ciency of transfer responds sensitively to relatively smallchanges of distances in the range of 10 to 80 A. Carefulevaluations of FRET experiments can yield quantitativeestimates of distances between labeled positions in macro-molecules, or in molecular aggregates, provided that certainspectroscopic parameters are known. Even if sufficient in-formation is not available to calculate exact distances, rela-tive dimensions of molecular structures can often be deduced(1, 2, 11), and in principle FRET is applicable to very complexmolecular structures. The method has been applied success-

fully to estimate intra(inter)molecular distances betweendonor and acceptor molecules in biological molecules otherthan nucleic acids (15-26), to determine molecular distribu-tions in condensed media (27-31), and to estimate distancesand distributions in a large variety of other polymer problems(26, 32).The linear geometry and regular helical structure of DNA

provides an excellent opportunity for structural investigationby the FRET technique. It is important to test whether FRETis sufficiently sensitive and accurate to observe the helicalpattern traversed by the 5' end of a DNA double helix. Bydetermining the simplest physical model to account for FRETmeasurements along a DNA double helix of varying length,the limits and constraints of FRET measurements with morecomplex structures can be appraised. In this work, we haveanalyzed the efficiency of FRET for a series of duplex DNAmolecules of lengths varying from 8 to 20 bp. The fluoro-phores, fluorescein (donor) and rhodamine (acceptor), wereattached to the 5' end of the individual strands comprisingeach duplex. The simplest model of energy transfer consis-tent with the results was used to interpret the change of theefficiency of FRET as the length of the DNA helix wasextended. We show that a simple double-helical model inconjunction with Forster dipole-dipole energy transfer ac-counts for the data, and melting transitions can be determinedaccurately with FRET.

MATERIALS AND METHODSConstruction of Fluorescently Labeled Duplex DNA Mole-

cules. This was carried out using our published procedures (2).In brief, oligonucleotides were synthesized by using ,B-cyano-ethylphosphoramidite chemistry (33). 5'-Amino groups at theend of six-carbon linkers were introduced by means of a finalcoupling step with N-methoxytrityl-2-aminohexyl-2-cyanoeth-yl-N,N-diisopropyl aminophosphite (34). After HPLC purifi-cation, oligonucleotides were reacted with the N-hydroxy-succinimide ester of tetramethylrhodamine (MolecularProbes) in dimethyl sulfoxide with 0.2M carbonate buffer (pH9.5) or with fluorescein isothiocyanate (Molecular Probes) indimethylformamide with 0.3 M carbonate buffer (pH 9.5).Conjugated DNA was purified by gel electrophoresis (2).These methods were designed to yield 100%o labeled DNA, andthis was confirmed spectroscopically. Base sequences of theoligonucleotides used in these FRET studies are (top strand ofduplex only) as follows: 5'-CCACTAGG-3' (8 bp), 5'-CCACTCTAGG-3'(10 bp), 5'-CCACTGGCTAGG-3'(12 bp),5'-CCACTGCTGCTAGG-3' (14 bp), 5'-CCACTGCACT-GCTAGG-3' (16 bp), 5'-CCACTGCACGCTGCTAGG-3' (18bp), and 5'-CCACTGCACTCGCTGCTAGG-3'(20 bp). Com-plementary combinations of fluorescein- and rhodamine-conjugated oligonucleotides were hybridized in 450 mMNaCl/24 mM sodium citrate, pH 7.0/2 mM MgCl2 by slowcooling from 65°C to 0°C. Duplex molecules were purified by

Abbreviation: FRET, fluorescence resonance energy transfer.

2994

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 8,

202

0

Proc. Natl. Acad. Sci. USA 90 (1993) 2995

electrophoresis in 20% polyacrylamide gels. Fluorescent du-plexes were recovered by electroelution or by soaking influorescence buffer at 4°C for the shorter species. The extentof labeling was verified by absorption measurements at 260,496, and 558 nm.

Preparation of the DNA Samples for Fluorescence andAbsorption Experiments. The duplex samples were dialyzedinto the fluorescence buffer (90 mM Tris borate, pH 8.3/0.1mM EDTA/100 mM NaCl). The 8-mer samples were toosmall to be dialyzed and were extracted into the correctbuffer directly out ofthe final electrophoresis gel. Absorptionmeasurements were made on these solutions from 230 to 600nm; these spectra were used to determine the concentrationof the duplexes (DNA and the probes) and, in conjunctionwith the fluorescence measurements, determined the con-sistency of labeling efficiency. All samples were measured at4.4°C; at this temperature the 8-mer concentration that wasused for the series of comparative measurements (Fig. 1) wasin the duplex form, as was verified by Tm measurements (seeFig. 3).

Spectroscopy Equipment. Absorption measurements weretaken on either a Uvicon 820 (Kontron, Zurich) or a CecilCE6600 spectrophotometer. All steady-state fluorescencespectra and anisotropy measurements were made with anSLM 8000S (SLM Aminco, Urbana, IL) instrument. Exci-tation and emission spectra were corrected for lamp fluctu-ations and wavelength variations. Polarization artifacts wereavoided by using "magic angle" conditions. Excitation andemission fluorescence spectra were acquired and processedwith a Macintosh IIci (Apple) computer. Each spectrum wasfitted to a series of polynomials to filter the random noise.The standard fluorescence spectra for FRET were collectedover a broad range of emission wavelengths (Aex = 490 nm,Aem = 500-650 nm; or Aex = 560 nm, Aem = 570-650 nm). Theabsorption of all solutions used for fluorescence measure-ments at the wavelength of excitation was always <0.015cm-1. Duplexes singly labeled with fluorescein and rhoda-mine were also synthesized; the fluorescence spectra oftheseprobes were used to decompose the doubly labeled fluores-cence spectra into donor and acceptor components, as de-scribed below.DNA Melting Curves. Melting of the duplex structures was

followed by several fluorescence methods-quantum yields,anisotropy, and FRET measurements; only the FRET ex-periments are reported here. The temperature was increasedslowly in steps of 3-5 K. The temperature was measured byinserting a microthermal resistor into a filled cuvette in asecond water-jacketted cuvette holder. The fluorescencemeasurements were always performed 10-15 min after thenew temperature was reached; no spectroscopic kineticchanges were observed at any time during the heating ex-periments. The ionic strength of the solution was adjusted byadding NaCl from a concentrated stock solution.Method of FRET. The rate of resonance energy transfer,

kT, from the singlet state of a fluorescence donor D to thesinglet state of an acceptor A is related to the scalar D-Aseparation (R) according to kT = (Ro/R)6/rD, where TD iS theemission lifetime of D in the absence of A; when R = Ro, kT= 1/TD (27). Forster has given a theoretical expression for Rovalid for dipole-dipole coupling between D and A. Ro isdirectly proportional to an orientation factor (K2) and thequantum yield of the donor (OD). The fundamental expres-sions for kT and Ro (27) are valid for particular values ofR andK2. If there is a distribution of -A configurations, theobserved efficiency is the appropriate average over themolecular ensemble. For a particular configuration of thedonor and acceptor the "'efficiency of energy transfer" E isdefined as E = 1/[1 + (R/Ro)6]. K2 is often set to 2/3 (14),which applies to rapid orientational randomization of therelative orientation between D and A. Fluorescence anisot-

ropy measurements (at 4.40C, r < 0.07 for fluorescein, r =0.25 for rhodamine) indicate that this is a good approximationfor this study.

Analysis of Data. There are several ways to analyze spec-trally the total emission measured from a solution of donorand acceptor labeled duplexes to isolate emission of theacceptor alone (11, 35). The fluorescein contribution can besubtracted at every wavelength of the total emission spec-trum of a doubly labeled molecule, from 500 to 650 nm, byusing a standard DNA-conjugated fluorescein spectrum. Theresulting rhodamine emission, containing both the emissionfrom direct excitation and energy transfer, is then divided bya fluorescence value from the same sample, exciting with awavelength where only the acceptor absorbs (e.g., 560 nm).This ratio (Eq. 1) normalizes the acceptor FRET signal for thequantum yield of rhodamine, for the concentration of theduplex molecules, and for any error in percentage acceptorlabeling. To generalize for any wavelength, we define thatrhodamine alone is excited at v" and the correspondingemission is measured at xt; the fluorescence for the FRETmeasurement is excited at v' and observed at va. The follow-ing ratio of signals is formed from the measured values,

rt _D(V) EA(VF) __A(_)(ratio)A ={E eA(i) EA(v, ) OA( , [1]

Pi and x2 are both 585 nm for all the measurements reportedhere. Superscripts D and A refer to the donor and acceptor.6D(v') and eA(V') are molar absorption coefficients of thedonor and acceptor. 4A(v) is an emission spectrum shapefunction of the acceptor; the integral over v [UfOA(v)dv] isproportional to the fluorescence quantum yield of the accep-tor. The precision of the measurement is estimated bydetermining the repeatability of the quantities discussedbelow several times on certain duplex probes. According toEq. 1, we can determine E from two separate emissionspectra on each duplex molecule [for this work, both spectraare determined from emission spectra, but they can also bemeasured from excitation spectra (2)]. These equations as-sume 100o labeling of the donor and the acceptor. A gener-alized version of these equations for any extent of labelinghas been presented (11).

RESULTS AND DISCUSSIONSpectral Characteristics of a Series of DNA Duplexes of

Different Lengths. We have measured the efficiency ofFRETfor a series of DNA duplexes (8-20 bp; in steps of 2 bp)labeled at respective 5' termini with fluorescein and rhoda-mine. The terminal 4 bp were kept constant throughout theseries for both ends of the molecules. The extracted acceptorspectra are identical with the rhodamine fluorescence of asingly labeled duplex, indicating that the energy distributionsof the D or A emission are not affected by the presence of thecomplementary A or D, respectively; the absorption spectraare likewise not disturbed. The doubly labeled moleculeswere prepared in such a way that the extent oflabeling shouldbe 100%, and a comparison of absorbance and fluorescencevalues of all doubly labeled molecules confirms that theextent of labeling is the same for all samples.FRET Reveals the Helical Geometry of DNA. The FRET

experiments for the series of doubly labeled DNA moleculesof different lengths are presented in Fig. 1 in terms of theefficiency ofFRET (E in Eq. 1). It can be seen that while theefficiency of FRET is reduced as the length of the helixincreases, the dependence is not a simple function of helixlength. The modulation of the efficiency curve, most appar-ent around the 12-bp duplex, is due to the helical geometry ofthe double-stranded DNA.

Biophysics: Clegg et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 8,

202

0

Proc. Natl. Acad. Sci. USA 90 (1993)

The solid curve in Fig. 1 is a simulation of the FRET datausing Eq. 1 according to the cylindrical geometrical model ofDNA shown in Fig. 2; the fitted parameters and the values ofthe parameters defining the DNA are given in the legend toFig. 1. These data definitely show that the efficiency ofFRETfor this series of duplexes does not vary in a manner pro-portional to 1/[1 + (3.4.N/Ro)6], where N is the number ofbase pairs in the duplex and Ro is in A; we did not expect thisto be the case, considering the helical geometry ofDNA (seeFig. 2). The helical molecular geometry of DNA produces amore complex behavior of the FRET efficiency than a simple1/[1 + (3.4.N/Ro)6] functional form, as is shown by theobvious modulation in Fig. 1. We interpret the data of Fig. 1according to the simplified schematic model of Fig. 2, whichrepresents the cylindrical properties of a DNA helix. Theactual distance between donor (D) and acceptor (A) (R in Fig.2) not only increases along the helical axis as the duplex islengthened [see the distance (N 3.4 + L) in Fig. 2], but thecomponent perpendicular to the helical axis is modulated ina sinusoidal fashion as shown in the equation in the legend forFig. 2. For short helical segments, this results in a strongnonlinear dependence ofR on the number of base pairs. Thisnonlinearity, coupled with the expression for FRET effi-ciency given in Eq. 1, leads to a characteristic undulatingdependence of the FRET efficiency when the data arepresented in terms of the number of base pairs. The best fitto these data places the average positions of the D and Afluorophores on opposite sides of the helix (4 2200 ± 200;see legend to Fig. 2); for a helix of length of9-10 bp the D andA chromophores are maximally displaced from each otherwhen they have this relative orientation. The distance be-tween D and A decreases rapidly as the helix length becomesshorter than 10 bp; one dye molecule swings around the helixtoward the complementary dye molecule, and this leads tothe extremely rapid increase in the FRET efficiency shown inthe simulation of Fig. 1. As the length of the helix increases

0.4-

0.8

0.3- 0.6

a ~~~~~~~~0.40.2

0 4 8 12 16 20X- 8\ bF ~~~~~~~~bpv0.1-\

O-,,I ---

6 8 10 12 14 16 18 20 22bp

FIG. 1. FRET efficiency of Eq. 1 is shown as a function of thenumber of base pairs in the duplexes. For all these data, the bottomstrands of the duplexes are labeled with rhodamine, and the topstrands are labeled with fluorescein. A separate series of data withreversed strand labeling gives a similar curve, and the independentfits agree within +2 A for all the values L, a, and d, and within ±15°for 4. Data have been fitted to a function based on the cylindricalgeometry of DNA (see Fig. 2 and legend). Ro has been set to 45 A.Fitted parameters are L = 13 A, 4 = 2270, and d = 19 A, and eachbase pair increases the helix length by 3.4 A; parameter a of Fig. 2has been set to 13 A. Data are presented for a series of duplexes withN = 8, 10, 12, 14, 16, 18, and 20 bp; donor and acceptor moleculesare attached to the 5' ends of the same strands throughout the seriespresented. Solid line is the expected (ratio)A from N = 8 to N = 20.Angle 4 and angular distribution of dyes projecting away from theends of the helices are probably dependent on the length of the linkerconnecting the dyes to the 5' ends of the DNA oligomers. (Inset)Simulated expected value of (ratio)A for all base-pair lengths between1 and 20.

z e

a~~a d

FIG. 2. Schematic representing helical geometry of the DNAmolecule. For this simple model, we assume that K2 remains constantfor all samples, an assumption that is corroborated by the agreementbetween the data and the simulation. VectorR originates on the donormolecule and points toward the acceptor molecule; length IRI is thedistance R of Eq. 1. Component ofR projected alongN base pairs onthe helical axis is given as (N 3.4 + L) A. This assumes that the basesare 3.4A in height; distance L accounts for the fact that the projectionsof the molecular centers of D and A onto the DNA helical axis maynot correspond with the planes of the base pairs to which they areattached. L represents the theoretical vectorial distance along thehelical axis separating D and A if they would be attached to the samebase pair (L can be negative). A and D can extend different perpen-dicular distances away from the helical axis; this is accounted for bythe distances d and a, which refer to the distances of the D and Amolecules, respectively. Polar angle 4)is the cylindrical angle betweenD and A for the case that N = 1. Distance R is given as a function ofN; N is the only variable that varies independently throughout all theseries ofdoubly labeled molecules. Eq. 1 of the text becomes (ratio)A= (e490/e5 + E490/560)E (490/e + 490o/es) [1 + ({[(3.4.N+ L)2 + (d'sin6)2 + (a - d cosO)2]1/2}/RO)6V1 according to this model,where 0 = N 36 + 4); cylinder base vector = [(a-sino)2 + (a-cos0 -

d)2]1/2 = (a2 + d2 - 2 a d cos0)2. The 1/R6 dependence of the rateof energy transfer discussed in the text is valid for Forster dipole-dipole resonance energy transfer with constant Ro provided that theFRET efficiency is independent of the orientation between the D andA dipoles. Ratios of absorption coefficients 4/eA6A = 0.072 and

/A = 0.66 were determined at 20°C from measurements of theE values of the singly labeled molecules.

from 10 bp, the helical path of the dye molecules leads to adecreasing rate of D-A separation (as a function of basepairs); that is, the distance R, and therefore the FRETefficiency, changes less rapidly for the duplexes with basepair lengths between 10 and 12.

It is important to point out that attempting to simulate thedata with a functional expression simply proportional to 1/{1+ [(3.4-N + L)/Ro]6} (that is, using a linear rather than ahelical geometry), results in Ro values of .70 A or more, andapparent total linker lengths of >45 A, to even approximatelyaccount for the data. Both of these distances are far too longto be realistic. This emphasizes the fact that it is not only theshape of the FRET efficiency dependence in Fig. 1 thatreflects the helical geometry of the DNA molecule; themagnitude of the FRET efficiency also indicates that the dyesare attached to the 10- and 12-bp duplexes on opposite sidesof a helix.FRET Measurements on DNA Are in Good Agreement with

the Forster Dipole-Dipole Mechanism of Energy Transfer. Weemphasize that the data in Fig. 1 are fully consistent with a1/[1 + (R/Ro)6] dependence of the efficiency of FRET; theshape of the curve reflects simply the helical geometry of theDNA. This expectation has also been discussed by Cardulloet al. (8), who have used FRET to detect the hybridization ofDNA in solution. It is not surprising that the DNA duplexshows this characteristic behavior, and it was not our pur-pose to check the validity of Forster's mechanism for FRET.However, considering the potential of FRET measurements

2996 Biophysics: Clegg et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 8,

202

0

Proc. Natl. Acad. Sci. USA 90 (1993) 2997

for investigating nucleic acid structuretion of the applicability and sensitivitments on a simple, well understood Dable.

In a previous attempt to correlatewith the length ofaDNA duplex (9), thethe helical geometry of DNA did not aBecause their simulations accordingform 1/{1 + [(3.4-(N - 1) + L)/Ro]6} ditheir FRET measurements, they prop(anisms of energy transfer in addition 1dipole resonance energy transfer ma!they did not specify an alternative mesupport for this, other than trivial quenccan be corrected for in Eq. 1, and in afor all our samples in Fig. 1. The majoour data can be accounted for by applidipole model together with the simplhelical DNA shown in Fig. 2.Thermal Stability of DNA Duplexes

Melting Experiments. Melting curve,observing the decrease in FRET of thiduplex (Fig. 3). Because the duplex is athe equilibrium is dependent on thechange in enthalpy of the association (Aby fitting the temperature dependencemeasured by the extent of FRET. The

0.2-

;r0.15

4-1l~a 0.1

'= 0.05

0-

-0.05-0 10 20 30

t, oc

FIG. 3. Melting curves of an 8-mer at ticentrations. For a simple bimolecular as:reaction (o) can be estimated by f = [(ratio)(ratio)W], where (ratio)° and (ratio)' are the N1) corresponding to the duplex and fully merespectively, at every temperature. In spmeasured fluorescence value in the denonmi

-s, a clear demonstra- are simulations of the difference in (ratio)A according to thety of FRET measure- best fit of the data to the simple two-state thermodynamicNA structure is desir- model (see legend of Fig. 3). Duplexes as short as 8 bp are

generally found to undergo "all or none" transitions-that is,FRET measurements the transition from helix to coil takes place as a simpleauthors reported that bimolecular association reaction (36, 37). Our fluorescence

Lccount for their data. melting experiments imply that the 8-bp molecule existsto an equation of the either as a duplex with the maximum FRET for an 8-bpd not account fully for double-stranded molecule or as completely separated singleosed that other mech- strands with zero FRET.to the Forster dipole- The simple interpretation of the melting transition as ay be active, although two-state process applies well to the data of Fig. 3 and shows-chanism. We find no that FRET provides a reasonable measure of the extent oftheching ofthe donor that reaction, 4. There is agreement between estimates of AHny case it is the same calculated from the shape of the individual FRET meltingtr characteristics of all curves and from the concentration dependence of the meltingying Forster's dipole- temperature (Tm) (see Fig. 3 Inset; Tm is defined as thee geometric model of temperature at which e = 0.5). Our AlH determination (-50.3

kcal per mol of duplex; 1 cal = 4.184 J) agrees well withs Measured by FRET expectations ( -56 kcal per mol of duplex) for a two-states were measured by melting transition, considering the nucleotide sequence ofthee doubly labeled 8-bp 8-mer according to a nearest-neighbor analysis of previousi bimolecular complex data (38). We have also studied the melting ofthe 8-bp duplexE concentration. The observed by FRET as a function of ionic strength (Fig. 4).JH) can be determined The variation of the Tm with ionic concentration displayed inof the equilibrium as Fig. 4 shows that the expected increase in the thermalsolid curves in Fig. 3 stability of even short DNA duplexes with increasing ionic

strength becomes less pronounced at higher salt concentra-tions. At higher salt concentrations, the Tm becomes essen-tially independent of the ionic strength. This has been foundfor the melting of much longer DNA at higher ionic strengths(39, 40) and has been predicted for short duplexes accordingto a theoretical study based on potentials of mean force (41).

Applicability of These Methods for Analysis of FRET Mea-surements in Nucleic Acids. We believe that the simple

1 2 3 4 method of analysis presented here has clear advantages forln(rel [duplex]) investigating similar structural and conformational (Figs. 3

and 4) problems with nucleic acids. We have analyzed morei Ai complex nucleic acid structures in a similar manner-e.g.,

four-way DNA junctions (1, 2) and bulged DNA duplexes-and it is possible to compare relative FRET efficiencies

40 50 60 determined under very different solution conditions in addi-tion to the large temperature variations in Fig. 3. There aremany modifications of FRET experiments that are particu-

hree different DNA con- larly applicable to studies of nucleic acid structures. Forsociation, the extent of example, we have varied the percentage labeling of either D'A - (ratio)l]/[(ratio)° - or A by mixing known ratios of single-stranded labeled andvalues of (ratio)A (see Eq. unlabeled molecules to form duplexes and other multi-lted DNA single strands, stranded complexes (unpublished data). In combination withnite of the fact that thel2tfrn(rnin~. ~normalized acceptor emission measurements (Eq. 1), this

%'%A,. LA-v%JR ,_ "AL,_ Al L"%, _%vsus-v vs i "Lw "AvGV J L%"a9

the reaction progresses, simulations show that this equation accountswell for the thermodynamics of the transition. Solid lines are fits ofdata according to the FRET (ratio)A. Tms are 11.2°C (*), 15.2°C (m),and 22.2°C (0). Fluorescence maximum of the fluorescein changesslightly as the temperature is decreased (blue shift of 2-3 nm). Thiscould be related to a slight variation in the dielectric constant of thesolvent as the temperature is changed. Also, preferential solvation ofthe fluorescein dye, due to the fact that the fluorescein is close to theDNA molecule, may contribute to the small shift in the emissionmaximum. This was taken into account by measuring a duplex labeledwith only the donor, fluorescein, and using the appropriate donorstandard spectrum at each temperature. This shift is not extensiveenough to change critically the value of Ro, but the dependence of thequantum yields on temperature demonstrates the advantage of usingan analysis that is relatively insensitive to the external solutionconditions-e.g., (ratio)A. Since we know the value of (ratio)A at thesufficiently high temperatures, we can determine the Tm of the duplexfrom the halfway point of the melting curve. (Inset) Plot of Tm' vs.relative duplex concentration. The slope of this plot (3.75 x 10-50C-1)can also be used to determine the AH(--50.3 kcal per mol of duplex)of the melting transition (36) (see text).

29

28-

C) 27-r.u 26 h

250

E.~24-

23-

22 _4.5 5 5.5 6 6.5 7

ln[NaCl], mM

FIG. 4. Melting transition temperature, Tm, of a doubly labeled8-mer duplex is plotted against the ln[NaCI]. Tm was determined fromanalyses similar to that shown in Fig. 3. Solid line is not a theoreticalfit but is simply to emphasize the shape of the dependence.

Biophysics: Clegg et al.

0.25ST

Dow

nloa

ded

by g

uest

on

Nov

embe

r 8,

202

0

Proc. Natl. Acad. Sci. USA 90 (1993)

provides additional requirements that the data must satisfyand checks the accuracy of estimating relative FRET effi-ciency. We have also used fluorescence anisotropy measure-ments to accurately measure nucleic acid oligomer meltingtransitions. These measurements will be presented else-where; here we only wish to emphasize the broad applica-bility of FRET for future studies on a large variety of nucleicacid structures; routine measurements of multiple nucleicacid samples in very different environments can be com-pared. The efficiency of FRET on the samples reported inFig. 1 accounts well for anisotropy and fluorescence lifetimemeasurements made on these samples. These latter studiessubstantiate the steady-state FRET measurements, as in anearlier FRET study with DNA four-way junctions (2), andwill be reported elsewhere. However, the simplicity of theanalysis according to Eq. 1 is evident and, excluding com-plications, it is robust and reliable.

CONCLUSIONSWe have shown that Forster dipole-dipole FRET can con-tribute significantly to our understanding of the structure ofnucleic acids. The helical geometry of double-stranded DNAin solution is clearly evident, and measurements of theefficiency of FRET as the length of a short DNA duplex isextended agree with our expectations based on the helicalstructure ofDNA. There are very few alternative techniquesthat can provide this information in solution. Our resultsindicate that fluorescence energy transfer in nucleic acids isquantitatively explained by Forster dipole-dipole interac-tion, and there is no requirement to postulate any additionalmechanism.The method of analysis that we have used is general,

accurate, reproducible, and self-normalizing. The experi-ments required to acquire the necessary spectroscopic dataare relatively easy to perform and are available to manylaboratories. Measurements taken under different solventconditions can be compared, and therefore structuralchanges can be followed readily. We are extending similarFRET measurements on nucleic acid structures to reactionkinetic studies (as rapid as microseconds), and the dynamicproperties of nucleic acid structures can be studied byobserving the fluorescence decay of the donor directly.Considering the ease of synthesis, the large selection offluorophores, and the robustness of the data analysis pre-sented here, we expect a rapid extension of the FRETtechnique into the field of nucleic acid structure.

We thank Dr. Stephan Diekmann for discussion, Christoph Gohlkeand Gyorgy Vamosi for comments on the manuscript, the Scienceand Engineering Research Council and Cancer Research Campaignfor financial support, and North Atlantic Treaty Organization for atravel grant.

1. Murchie, A. I. H., Clegg, R. M., von Kitzing, E., Duckett,D. R., Diekmann, S. & Lilley, D. M. J. (1989) Nature (London)341, 763-766.

2. Clegg, R. M., Murchie, A. I. H., Zechel, A., Carlberg, C.,Diekmann, S. & Lilley, D. M. J. (1992) Biochemistry 31, 4846-4856.

3. Beardsley, K. & Cantor, C. R. (1970) Proc. Natl. Acad. Sci.USA 65, 39-46.

4. Cantor, C. R. & Tao, T. (1971) in Proceedings in Nucleic Acid

Research, eds. Cantoni, G. L. & Davies, D. R. (Harper &Row, New York), Vol. 2, pp. 31-93.

5. Yang, C.-H. & Soll, D. (1974) Proc. Natl. Acad. Sci. USA 71,2838-2842.

6. Genest, D. & Wahl, P. (1983) in Time-Resolved FluorescenceSpectroscopy in Biochemistry and Biology, ed. Cundall, R. B.& Dale, R. E. (Plenum, New York), Vol. 69, pp. 523-539.

7. Eshaghpour, H., Dieterich, A. E., Cantor, C. R. & Crothers,D. M. (1980) Biochemistry 19, 1797-1805.

8. Cardullo, R. A., Agrawal, S., Flores, C., Zamecnik, P. C. &Wolf, D. E. (1988) Proc. Natl. Acad. Sci. USA 85, 8790-8794.

9. Cooper, J. P. & Hagerman, P. J. (1990) Biochemistry 29, 9261-9268.

10. Jovin, T. M., Rippe, K., Ramsing, N. B., Klement, R., Elm-horst, W. & Vojtiskova, M. (1990) in Structure and Methods,eds. Sarma, R. H. & Sarma, M. H. (Adenine, Guilderland,NY), Vol. 3, pp. 155-174.

11. Clegg, R. M. (1992) Methods Enzymol. 211, 353-388.12. Forster, T. (1946) Naturwissenschaften 33, 166-175.13. Forster, T. (1948) Ann. Phys. (New York) 2, 55-75.14. Forster, T. (1951) Fluoreszenz Organischer Verbindungen

(Vandenhoeck & Ruprecht, Gottingen, F.R.G.).15. Weber, G. (1957) Nature (London) 180, 1409.16. Weber, G. (1960) Biochem. J. 75, 335-345.17. Weber, G. (1960) Biochem. J. 75, 345-352.18. Stryer, L. & Haugland, R. P. (1967) Proc. Natl. Acad. Sci.

USA 58, 719-726.19. Stryer, L. (1968) Science 162, 526-533.20. Conrad, R. H. & Brand, L. (1968) Biochemistry 7, 777-787.21. Weber, G. & Anderson, S. R. (1969) Biochemistry 8, 361-371.22. Stryer, L. (1978) Annu. Rev. Biochem. 47, 819-846.23. Haas, E., Katchalski-Katzir, E. & Steinberg, I. Z. (1978)

Biopolymers 17, 11-31.24. Haas, E. & Steinberg, I. Z. (1984) Biophys. J. 46, 429-437.25. Gryczynski, I., Wiczk, W., Johnson, M. L., Cheung, H. C.,

Wang, C.-K. & Lakowicz, J. R. (1988) Biophys. J. 54, 577-586.26. Valeur, B. (1989) in Fluorescent Biomolecules: Methodologies

and Applications, eds. Jameson, D. M. & Reinhart, G. D.(Plenum, New York), pp. 269-303.

27. Forster, T. (1949) Z. Naturforsch. A 4, 321-327.28. Cantor, C. R. & Pechukas, P. (1971) Proc. Natl. Acad. Sci.

USA 68, 2099-2101.29. Gosele, U., Hauser, M. & Klein, U. K. A. (1975) Chem. Phys.

Lett. 34, 81.30. Gosele, U., Hauser, M. & Klein, U. K. A. (1976) Z. Phys.

Chem. (Frankfurt/Main) 99, 81-96.31. Hauser, M., Klein, U. K. A. & Gosele, U. (1976) Z. Phys.

Chem. (Frankfurt/Main) 101, 255-266.32. Mugnier, J., Pouget, J., Bourson, J. & Valeur, B. (1985) J.

Lumin. 33, 273-300.33. Beaucage, S. L. & Caruthers, M. H. (1981) Tetrahedron Lett.

22, 1859-1862.34. Connolly, B. A. (1987) Nucleic Acids Res. 15, 3131-3139.35. Fairclough, R. H. & Cantor, C. R. (1978) Methods Enzymol.

48, 347-379.36. Breslauer, K. J. (1986) in Thermodynamic Data for Biochem-

istry and Biotechnology, ed. Hinz, H.-J. (Springer, Berlin), pp.402-427.

37. Markey, L. A. & Breslauer, K. J. (1987) Biopolymers 26,1601-1620.

38. Breslauer, K., Frank, R., Blocker, H. & Markey, L. A. (1986)Proc. Natl. Acad. Sci. USA 83, 3746-3750.

39. Gruenwedel, D. W., Hsu, G. H. & Lu, D. S. (1971) Biopoly-mers 10, 47-68.

40. Gruenwedel, D. W. (1975) Biochim. Biophys. Acta 395, 246-257.

41. Soumpasis, D. M. & Jovin, T. M. (1987) in Nucleic Acids andMolecular Biology, eds. Eckstein, F. & Lilley, D. M. J.(Springer, Berlin), Vol. 1, pp. 85-111.

2998 Biophysics: Clegg et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 8,

202

0