Reversible Ratiometric Probe for Quantitative DNA Measurements

The ratio of the acceptor-to-donor fluorescence intensities is independent of the amount of probe and provides a quantitative measure of the free targ...
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Anal. Chem. 2004, 76, 947-952

Reversible Ratiometric Probe for Quantitative DNA Measurements Jo 1 rn Ueberfeld and David R. Walt*

The Max Tishler Laboratory for Organic Chemistry, Department of Chemistry, Tufts University, Medford, Massachusetts 02155

We have designed a reversible fluorescent DNA probe that can be used to determine the concentration of singlestranded DNA in solution by a ratiometric fluorescence measurement. The probe consists of a single-stranded dual fluorescently labeled DNA molecule that adopts a stem-loop conformation in its nonhybridized state. The stem length and the length of the loop region complementary to the target were chosen to allow for reversible binding. The excitation and emission wavelengths of the two labels Cy3 and Cy5 allow for fluorescence resonance energy transfer in the closed state. Upon hybridization, the probe opens up resulting in a fluorescence intensity increase of the donor and a fluorescence intensity decrease of the acceptor. The ratio of the acceptor-to-donor fluorescence intensities is independent of the amount of probe and provides a quantitative measure of the free target concentration. The ability to monitor mRNA concentration changes in real time would be of great value for exploring gene transcription. Presently, the only method that allows localizing RNA in living cells is fluorescence in situ hybridization.1,2 The probes used for these studies, such as molecular beacons3-5 and labeled 2′-Omethyl oligoribonucleotides,6 only indicate the presence or absence of target but do not enable quantification. These probes are also limited by their virtual irreversibility due to slow dissociation rate constants. Although the hybridization kinetics of a molecular beacon with β-actin mRNA in living cells has been observed, the kinetics could only be followed for 15 min due to irreversible binding.5 This property limits the results to snapshots of the transcription dynamics. To understand the dynamics of how mRNA is synthesized and processed, it is necessary to develop probes that do not interfere with translation. Ideally, these probes would work reversibly, be stable against endonuclease activity, resist nonspecific binding to proteins, and be capable of quantitative measurements. In this paper, we present a reversible DNA * To whom correspondence should be addressed. E-mail: david.walt@ tufts.edu. (1) Dirks, R. W.; Molenaar, C.; Tanke, H. J. Histochem. Cell Biol. 2001, 115, 3-11. (2) Mitchell, P. Nat. Biotechnol. 2001, 19, 1013-1017. (3) Matsuo, T. Biochim. Biophys. Acta: Gen. Subj. 1998, 1379, 178-184. (4) Sokol, D. L.; Zhang, X. L.; Lu, P. Z.; Gewitz, A. M. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11538-11543. (5) Perlette, J.; Tan, W. H. Anal. Chem. 2001, 73, 5544-5550. (6) Molenaar, C.; Marras, S. A.; Slats, J. C. M.; Truffert, J. C.; Lemaitre, M.; Raap, A. K.; Dirks, R. W.; Tanke, H. J. Nucleic Acids Res. 2001, 29, e89. 10.1021/ac035093s CCC: $27.50 Published on Web 01/20/2004

© 2004 American Chemical Society

probe that responds dynamically to its target concentration. The free target concentration can be quantified by ratiometric fluorescence measurements. EXPERIMENTAL SECTION Oligonucleotide Design and Synthesis. The synthetic target represents a 24-nt sequence of HIV-1 DNA that encodes for the V3 loop in the glycoprotein gp 120 (positions 125-148).7 Labeled probe oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). The fluorescent dyes Cy3 and Cy5 were incorporated using standard phosphoramidite chemistry. The program mfold, available at http://www.bioinfo.rpi.edu/applications/mfold/old/dna/form1.cgi,8 was used to verify that probes and targets adopt no secondary structure under the measurement conditions. The lyophilized probes were suspended in 10 mM TRIS buffer (pH 8.3) containing 100 nM NaCl, diluted to 500 nM aliquots and stored at -20 °C. Unlabeled oligonucleotides were synthesized in the Tufts University Physiology Department (Boston, MA) using an ABI synthesizer. Stock solutions of 10 mM were kept at 4 °C. Fluorescence Measurements. Stock solutions of labeled probes were diluted to concentrations of 50-150 nM in a 3-mL cuvette equipped with a magnetic stirrer. The temperature was controlled by a Peltier element and monitored with a thermistor (0.1-mm diameter, Omega Engineering Inc., Stamford, CT). The excitation wavelength of the donor was 530 nm. Cy3 (donor) and Cy5 (acceptor) emissions were detected at 565 and 666 nm, respectively. Hybridizations were monitored with the kinetics software provided by the spectrometer manufacturer (Varian Instruments, Walnut Creek, CA). Determination of Kd, Imin,Cy5, and Imax,Cy3. These parameters were obtained from a calibration curve of probe 2. The Cy3 and Cy5 fluorescence intensities at the emission maximums of the dyes (565 nm for Cy3; 666 nm for Cy5) were fitted against a theoretical response curve which is derived as follows:. Kd for the hybridization reaction

P+TTD is given by

Kd ) [P][T]/[D]

(1)

where [P], [T], and [D] refer to the free probe concentration, Analytical Chemistry, Vol. 76, No. 4, February 15, 2004 947

the single-stranded target concentration, and the duplex concentration, respectively. With R defined as the ratio of bound probe

R ) [D]/[P]0

(2)

Table 1. Probe and Target Sequencesa name

sequence

probe 1

5′-Cy3-GCATG TAT CTC CTA TTA TTT CTC CT-Cy5CATGC-3′ 5′-Cy3-GCATG ACA CTC CTA TTA TTT CTA AA-Cy5CATGC-3′ 5′-Cy3-GCATG ACA AAC CTA TTA TTT CAA AA-Cy5CATGC-3′ 5′-ACA GGA GAA ATA ATA GGA GAT ATA-3′

probe 2

it follows for Kd

probe 3

Kd ) ([P]0 - R[P]0)([T]0 - R[P]0)/R[P]0

(3)

target

a The bases complementary to the target sequence are highlighted in boldface type.

with [P]0 and [T]0 as the total concentrations of probe and target. Expanding and rearranging eq 3 results in the quadratic equation

0 ) R2 +

Kd + [T]0 + [P]0 [T]0 R[P]0 [P]0

(4)

Substitution for R in eq 9 with eq 5 results in

[

[T]0 + Kd + [P]0

ICy5 ) Imax,Cy5 + (Imin,Cy5 - Imax,Cy5) with one of its solutions being

R)

[T]0 + Kd + [P]0 2[P]0

-

x(

)

[T]0 + Kd + [P]0 2[P]0

2

-

[T]0 [P]0

I ) IP + ID

(5)

I - IP [P] Ib - I P 0

(6)

(7)

of completely bound probe at the Cy3 (Imax,Cy3) or the Cy5 (Imin,Cy5) emission maximums. If the probe is completely bound, FRET efficiency and acceptor (Cy5) fluorescence intensity are at a minimum. If the probe is in a completely unbound state, the acceptor intensity is at a maximum. Hence, the specific equation for Cy5 as it relate to the duplex concentration is

ICy5 - Imax,Cy5 [P] Imin,Cy5 - Imax,Cy5 0

(8a)

The analogous equation for the donor (Cy3) is

[D] )

ICy3 - Imin,Cy3 [P] Imax,Cy3 - Imin,Cy3 0

(8b)

Substitution for [D] in eq 8a with eq 2 and rearrangement gives

ICy5 ) Imax,Cy5 + (Imin,Cy5 - Imax,Cy5)R

(9)

(7) Naghavi, M. H.; Salminen, M. O.; Sonnerborg, A.; Vahlne, A. Aids Res. Hum. Retroviruses 1999, 15, 485-488. (8) Zuker, M. Nucleic Acids Res. 2003, 31, 3406-3415.

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2[P]0

[

ICy3 ) Imin,Cy3 + (Imax,Cy3 - Imin,Cy3)

with IP corresponding to the fluorescence intensity of the nonhybridized probe and ID corresponding to the fluorescence intensity of the duplex. The duplex concentration can be calculated from the fluorescence intensities via eq 7, where Ib refers to the intensity

[D] )

)

[T]0 + Kd + [P]0

The measured intensity I at a certain wavelength is

[D] )

x(

2[P]0

x(

2

-

]

[T]0 [P]0

[T]0 + Kd + [P]0 2[P]0

)

[T]0 + Kd + [P]0 2[P]0

2

-

]

[T]0 [P]0

-

(10a)

-

(10b)

A solution of 100 nM dual-labeled DNA probe was titrated with complementary target at 38.8 °C. The fluorescence intensities ICy3 and ICy5 were recorded as a function of the total target concentration [T]0 and fitted against eq 10a to determine Imin,Cy5 and KD. To determine Imax,Cy3, the Cy3 intensities were fitted against the analogous eq 10b. Determination of ∆G. The free energy of the internal stem formation of probe 2 and of the dissociation of probe 2 from the target were determined after the method of Bonnet et al.9,10 The concentration of probe 2 was 50 nM. The temperature ranged from 10 to 83 °C. Target concentrations varied between 0.3 and 40 µM. RESULTS AND DISCUSSION Probe Sequence. To achieve reversibility, we chose an oligonucleotide with a sequence that forms a hairpin. If the hairpin’s loop sequence that is complementary to the target is sufficiently long, the hairpin should open and bind to the target.11,12 For a reversible probe, the free energy of the formed duplex should be of the same magnitude as the free energy of stem formation. To find a probe that binds reversibly to the target, three oligonucleotides were synthesized that contained target complementary sequences of different lengths (Table 1). The stem was 5 bp long and had a melting temperature of 58 °C as was determined with fluorescence measurements. The loops all contained 20 nucleotides. One probe (probe 1) contained a (9) Bonnet, G.; Tyagi, S.; Libchaber, A.; Kramer, F. R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6171-6176. (10) Tsourkas, A.; Behlke, M. A.; Rose, S. D.; Bao, G. Nucleic Acids Res. 2003, 31, 1319-1330. (11) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303-308. (12) Steemers, F. J.; Ferguson, J. A.; Walt, D. R. Nat. Biotechnol. 2000, 18, 9194.

Figure 2. Titration of 120 nM probe 1. Target concentrations were (a) 0, (b) 30, (c) 60, (d) 89, (e) 119, and (f) 148 nM. Figure 1. Structure of the dual fluorescently labeled probes. Probe 2 is given as an example. The bases complementary to the target sequence are highlighted in boldface type.

loop that was fully complementary (20 nt) to the target while the complementary sequences of the two other probes were 14 (probe 2) and 11 nucleotides (probe 3) long, respectively. The remaining six (probe 2) and nine nucleotides (probe 3) at the noncomplementary positions were replaced with A’s and C’s. Fluorescent Labels. To follow the conformational changes that occur during the binding process, two fluorescent labels were attached to the oligonucleotide. The labels were chosen to allow for fluorescence resonance energy transfer (FRET) to occur in the closed form. Binding of the complementary target opens the stem and interrupts the FRET such that the donor fluorescence intensity increases while the acceptor intensity decreases. The main advantage of this approach is that ratiometric fluorescence measurements are possible; i.e., the target concentration can be determined without knowing the amount of added probe. We chose the two cyanine dyes Cy3 as donor and Cy5 as acceptor. Their Fo¨rster radius is ∼5.4 nm.13,14 These dyes act as a good FRET pair with a large spectral separation between their emission maximums (∼100 nm), comparable quantum yields, and only minimal competing processes (e.g., charge-transfer processes) when both dyes are in proximity.15,16 Initial experiments in which the dyes were attached to the 3′ and 5′ ends of the oligonucleotide using a previously reported procedure,17 showed only weak resonance energy transfer in the closed form of the hairpin (Supporting Information). This low transfer efficiency was probably due to the formation of dye dimers.18 A remarkable increase in energy-transfer efficiency was obtained when the Cy5 dye was placed between the stem and the loop sequence (Figure 1). In this position, the Cy5 dye neither hinders stem formation nor inhibits target-loop hybridization. Upon titration of probe 1 with target, a decrease of FRET was observed (Figure 2) with the extent of the decrease dependent upon the target concentration. The probe has an isoemissive point at 639 nm. (13) Ishii, Y.; Yoshida, T.; Funatsu, T.; Wazawa, T.; Yanagida, T. Chem. Phys. 1999, 247, 163-173. (14) Kenworthy, A. K. Methods 2001, 24, 289-296. (15) Dietrich, A.; Buschmann, V.; Muller, C.; Sauer, M. Rev. Mol. Biotechnol. 2002, 82, 211-231. (16) Ha, T. Methods 2001, 25, 78-86. (17) Zhang, P.; Beck, T.; Tan, W. H. Angew. Chem., Int. Ed. 2001, 40, 402405. (18) Khairutdinov, R. F.; Serpone, N. J. Phys. Chem. B 1997, 101, 2602-2610.

Figure 3. Calibration of 120 nM probes 1, 2, and 3. The ratio R of the Cy3 intensity to the Cy5 intensity is shown over the total target concentration. The loop sequence of probe 1 is fully complementary to the target. Probe 2 has 14 nt complementary to the target and shows medium affinity, while probe 3 (11 nt complementary) does not bind.

Probe Calibration. The calibration curves of probes 1-3 are given in Figure 3. Here the ratio R of the fluorescence intensity at the Cy3 emission maximums (565 nm) to the intensity at the Cy5 emission maximums (666 nm)

R ) (ICy3/ICy5)

(11)

is shown to be a function of the total concentration of added target. The graph demonstrates that the fluorescence intensity ratio can be used to calibrate the probes. Probe 1 has the highest affinity while probe 3 does not bind to the target. No further experiments were carried out with probe 3. Replacement of Labeled Probe. Reversibility of target binding was first examined by observing the replacement reaction of the bound labeled probe by an unlabeled stem-loop oligonucleotide with exactly the same sequence. Fluorescence intensity changes for both labels indicated that probe 2 could be replaced by the unlabeled oligonucleotide (Figure 4). By assuming a second-order reaction, a rate constant of 0.0003 M-1‚s-1 was found for the replacement reaction at 24.1 °C. An Arrhenius plot gave an activation energy of 290 kJ‚mol-1. Probe 1 could not be replaced after hybridization to its target (results not shown) because its relatively long complementary sequence causes irreversible target binding. Hence it is not a valuable candidate for a reversible DNA probe. Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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Figure 4. Hybridization of probe 2 to the target and replacement of probe 2 with unlabeled hairpin. (A) Fluorescence intensities at the emission maximums of Cy3 and Cy5 over time. At t ) 0 and t ) 13 min, 10 nM target was added to 50 nM probe 2. After 31 min, 500 nM unlabeled oligonucleotide was added that had the same sequence as probe 2. (B) Fluorescence ratio R for the same reaction. The temperature was 24.1 °C.

Figure 5. (A) Determination of the dissociation constant Kd of the duplex and the minimal Cy5 intensity Imin,Cy5 for probe 2; 100 nM probe 2 was titrated. The Cy5 intensities (b) were fitted to eq 10a. The solid line shows the fit with Kd ) 560 nM and Imin,Cy5 ) 73.6. (θ ) 38.8 °C). (B) Calibration of probe 2. The ratio of the fluorescence intensities ICy3/ICy5 is drawn over the total target concentration.

Thermodynamic and Optical Properties of the Probe 2. The hybridization

P+STD is a 1:1 reaction with P the free probe, S the single-stranded target, and D the duplex. The concentration of free target in solution [S], when measured with a ratiometric probe, can be calculated by the equation19

R - Rmin Imax,Cy5 [S] ) Kd‚ Rmax - R Imin,Cy5

(12)

with Kd the dissociation constant of the duplex, Rmin the intensity ratio of the free probe, Rmax the maximal ratio of bound probe, Imin,Cy5 the minimal Cy5 intensity (completely bound probe), and Imax,Cy5 the maximal Cy5 intensity (free probe), respectively. To determine these values, a 100 nM comcentration of probe 2 was titrated with target. A fit of the resulting intensities against eqs (19) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985, 260, 34403450.

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10a and 10b gave Kd ) 560 nM, Rmax ) 2.215, and Imax,Cy5/Imin,Cy5 ) 1.988 (Figure 5A). Rmin was determined to be 0.397 from the spectrum of the unbound probe. From eq 11 it becomes apparent that the concentration of the target used in our study can be determined without knowing the concentration of probe 2: the ratio Imax,Cy5/Imin,Cy5 does not change with the probe concentration. The calibration curve of 100 nM probe 2 depicted in Figure 5B was taken at probe concentrations between 0 and 3.75 µM target. Within the measured range, R rises steadily and does not reach a plateau (i.e., the probe does not become saturated), indicating equilibrium between target and probe. Internal Normalization. To verify whether probe 2 is internally normalized, we first titrated a solution of 100 nM probe 2 with five aliquots of 20 nM target. Then 50 nM probe 2 was added in 10 nM aliquots. As seen in Figure 6 and as predicted by eq 11, no change in R was observed after addition of probe 2. A control experiment with a noncomplementary DNA target gave no fluorescence intensity change during the calibration. Reversibility. To show that probe 2 binds the target reversibly, 100 nM probe 2 was titrated with 100 nM target and then diluted 1:2 with buffer solution. In this case, the lower target concentration resulting from the dilution causes a decrease in R. As Figure 7

Figure 6. Proof of internal normalization. A 100 nM aliquot of probe 2 was first titrated with five aliquots of 20 nM target. The aliquots were added after 1, 4, 9, 13, and 17 min. Then 50 nM probe 2 was added in 10 nM aliquots after 22, 25, 28, 31, and 34 min. (A) During target addition, the fluorescence intensity of the donor (Cy3) increases while the acceptor intensity decreases (Cy5). Increasing probe concentration causes fluorescence intensity increases for both labels. (B) The Cy3/Cy5 ratio R increases upon target addition; however, no change in R is visible after more probe 2 is added. (θ ) 39.0 °C). The slight decrease in R after addition of probe is probably due to the minor amount of dilution.

Figure 7. Proof of reversible target binding. A 3-mL aliquot of 100 nM probe 2 was first titrated with five aliquots of 20 nM target. The aliquots were added after 1, 4, 8, 12, and 16 min. After 21 min, 1.5 mL of solution was replaced with 1.5 mL of buffer. (A) During target addition, the fluorescence intensity of the donor (Cy3) increases while the acceptor intensity decreases (Cy5). The 1:2 dilution after 21 min causes a decrease of both fluorescence intensities. (B) According to eq 11, an increase in the free target concentration should result in a higher fluorescence ratio R, while a dilution should give a lower R. The experiment was carried out at 38.3 °C. The spike in R at 22 min probably results from the lower temperature of the buffer used to dilute the solution.

shows, R drops to 0.46, which corresponds to 25 nM target-minus the diluted target concentration. The experiments presented in Figures 6 and 7 reveal fast response times; hybridization and dehybridization were both completed within a few seconds. This response time should be fast enough to monitor RNA transcription in real time as a previous study, with nonreversible molecular beacons, revealed that transcription takes place within a minute time frame.5 The two experiments reported here were carried out at 38.8 °C. Tests with probe 2 at 33.8 °C exhibited nonreversible binding while it did not bind to the target at 43.8 °C because melting of the looptarget hybrid occurred (results not shown). The strong temperature dependence of the hybridization reaction indicates a high activation energy. CONCLUSION We have designed and prepared a ratiometric DNA probe that binds its target reversibly. This reversibility was achieved by designing an oligonucleotide with a stem-loop conformation with the loop sequence only partially complementary to the target

sequence. The length of the complementary sequence was varied such that the free energy of the probe-target hybridization closely matched the free energy of the internal stem formation. A ∆G value of -9.6 ( 2.5 kJ‚mol-1 was determined for opening the stem while the free energy change of the dissociation of the hybrid probe 2-target was 14.2 ( 2.9 kJ‚mol-1. The two fluorescent labels, which undergo fluorescence resonance energy transfer in the target-unbound state, enable ratiometric measurements to be made without dependence on the probe concentration. These features suggest that this probe, or a modification, could be a probe for in vivo monitoring of transcription processes. In vivo mRNA visualization involves target detection in a very complex environment. The presence of endonucleases limits the lifetime of molecular beacons to ∼15 min.4,5 The use of phosphorothioate20 or 2′-O-methyl oligonucleotides21 could possibly result in probes that resist endonucleases for a longer time. (20) Vijayanathan, V.; Thomas, T.; Sigal, L. H.; Thomas, T. J. Antisense Nucleic Acid Drug Dev. 2002, 12, 225-233. (21) Tsourkas, A.; Behlke, M. A.; Bao, G. Nucleic Acids Res. 2002, 30, 51685174.

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The design of our reversible probe was completely empirical, which represents the current limitation of the approach. At this stage, we have not found appropriate design software that enables us to reliably compare the free energy of intermolecular probetarget hybrid formation to the intramolecular stem formation. To develop a reversible probe for a different target, a custom probe would have to be developed.

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SUPPORTING INFORMATION AVAILABLE Calibration of a probe as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review September 17, 2003. Accepted December 10, 2003. AC035093S