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2009, 113, 865–868 Published on Web 01/06/2009
Detection of Quadruplex DNA by Luminescence Enhancement of Lanthanide Ions and Energy Transfer from Lanthanide Chelates Jill L. Worlinsky and Swarna Basu* Department of Chemistry, Susquehanna UniVersity, 514 UniVersity AVenue, SelinsgroVe, PennsylVania 17870 ReceiVed: NoVember 14, 2008; ReVised Manuscript ReceiVed: December 16, 2008
Small amounts of quadruplex DNA have been detected using luminescence enhancement of aqueous lanthanide ions and energy transfer from lanthanide chelates. The 22mer human telomeric DNA, AGGG(TTAGGG)3, was detected using europium ions at concentrations as low as 20 ppb DNA. Detection with terbium ions was not possible due to the inherent weak luminescence intensities of lanthanides. Two different terbium chelates were used to overcome this challenge. When quadruplex DNA was added to the chelates there was a change in the excited-state lifetime of the chelate with subsequent energy transfer to the DNA. Experiments showed an increase in the amount of energy transferred from the chelate to the human telomeric DNA and other quadruplex sequences increased as a function of DNA concentration. DNA can form a variety of quadruplex structures. It has been known that guanine-rich (G-rich) nucleotides and polyguanine can form G-rich DNA structures containing both inter- and intramolecular guanine quartets held together by Hoogsteen base-pairs.1 These quadruplex structures have been identified in G-rich eukaryotic telomeres that are located at the ends of eukaryotic chromosomes and are crucial to the life of a cell. Single stranded telomeric DNA can form G-quadruplexes and these sequences have been studied extensively. Telomeric DNA consists of random repeats of G-rich sequences, such as (TTAGGG)n in humans.1 The study of quadruplex structures formed from G-rich DNA can be used as a model system for the functional roles played by quadruplexes in telomeric DNA. In this work, the 22mer human telomeric DNA, AGGG(TTAGGG)3 has been studied along with the 15mer DNA aptamer, d(GGTTGGTGTGGTTGG), which adopts an intramolecular quadruplex structure and is one of the most intensely studied aptamer sequences because it binds to and inhibits thrombin.2 Another DNA sequence studied in this work, the DNA 12mer d(GGGGTTTTGGGG), adopts the intermolecular quadruplex structure.2 The underlying motivation of this work is our interest in developing a methodology for the reliable detection of low concentrations of quadruplex DNA sequences using lanthanide ions and chelates. Lanthanides have been used in the past for the detection of duplex DNA. It has been reported that when lanthanides bind to duplex DNA, luminescence enhancement occurs. Quadruplex DNA such as the 15mer and 12mer form the “chair” and “basket” type quadruplex structures, respectively, and these configurations contain binding sites for metal ions.1,2 This work primarily focuses on the 22mer human telomeric DNA. We have used lanthanide ions and chelates to detect small amounts of this DNA. * To whom correspondence should be addressed. E-mail: basu@ susqu.edu.
10.1021/jp8100464 CCC: $40.75
Over the years different experimental approaches have been attempted for reliable detection of duplex and quadruplex DNA sequences using lanthanide ions.1 Guanine-rich DNA containing both inter- and intramolecular guanine quartets can form quadruplex structures.2 The luminescence of aqueous solutions of common lanthanide ions such as terbium (Tb3+) and europium (Eu3+) is weak as the transitions take place between ground and excited states that involve 4f -4f inner shell electrons. However, lanthanide chelates strongly luminesce when they are excited by ultraviolet light. These chelates are formed by the binding of different ligands and the absorption of light by the lanthanide ion results in an emission profile that is identical to that of the free lanthanide and the intensity is greater due to the greater absorption cross-section of the ligand.3,4 The ligands also serve to shield the lanthanide ions from solvent quenching.4 As a result, lanthanide chelates have been successfully developed as fluorescence probes for highly sensitive detections of various biological molecules. Ilie et al. have shown that a Tb3+-DTPA complex forms an association fluorescence complex with duplex DNA.5 This type of DNA-luminophore interaction is different from the process of intercalation which occurs when the aromatic rings of compounds such as ethidium bromide, proflavin, and various porphyrins insert themselves between the base pairs resulting in local unwinding of the nucleic acid helix. Nonetheless, the interaction between DNA and lanthanide chelates opens up an avenue by which the inherent deficiencies of lanthanide ions can be overcome and the detection of quadruplex DNA made possible. Even though these chelates exhibit stronger luminescence intensity than free lanthanides, the enhancement when bound to DNA is often difficult to measure using steady-state methods. Therefore, an alternate approach of detecting small quantities of quadruplex DNA is presented here. First, luminescence experiments involving quadruplex DNA sequences and lanthanide ions (terbium) were designed along the same lines as previously published by Panigrahi et al.1 They 2009 American Chemical Society
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Figure 1. Emission spectra showing the fluorescence enhancement when human telomeric DNA (22mer) was added to aqueous Eu3+. λex ) 395nm. The error bars are based on the instrument’s sensitivity and indicate that the enhancement is clearly measurable.
demonstrated that as the concentration of the calf thymus DNA was increased, the luminescence intensity of the lanthanide also increased. Next, we proceeded to measure the effect of adding different concentrations of the 22mer human telomeric DNA on the luminescence intensity of Eu3+ at 452.5 nm. This is shown in Figure 1. In aqueous solution, the emission band at 452.5 nm is very prominent while the usually sharp peaks in the 580-685 nm range appear to be weaker and broader, which likely occurs due to solvent quenching of the lanthanide. Overall as the concentration of DNA was increased modest enhancement was observed. Prior to annealing the samples, there was measurable and reproducible enhancement (10-15%) after adding DNA at concentrations as low as 20 ppb but no further enhancement at the higher concentrations. The enhancement was even greater after the samples were annealed, with an increase in luminescence intensity of over 70% at 800 ppb DNA concentration, higher than the 15-17% increase observed at 20 and 400 ppb DNA concentration (not shown). This indicates that the quadruplex structure may have formed more effectively as annealing is known to facilitate the formation of quadruplex structures. The quadruplex structure contains the binding sites for the lanthanides that are located in the pockets of the chair and basket type structures.2 Prior to annealing the DNA exists as single strands; therefore, the binding sites for lanthanides are not available. Enhancement of Eu3+ luminescence with duplex calf thymus DNA resulted in an approximately 25% enhancement at 800 ppb DNA concentration (not shown). Therefore, the enhancement observed with 800 ppb 22mer DNA is predominantly due to the binding of Eu3+ with the quadruplex structure. The same experiment was carried out with aqueous terbium ions as Tb3+ is known to be sensitized by duplex DNA and some quadruplex DNA (21mer human telomeric DNA).1 However, with the 22mer, the luminescence enhancement was negligible, even at 800 ppb DNA concentration and low (1 µM) Tb3+ concentration. This might be a result of the high Tb3+ to DNA ratio used in this work which would have made it difficult to observe measurable luminescence enhancement. Therefore, the energy transfer approach was undertaken to overcome any luminescence sensitivity issues. Terbium chelates formed using the ligands DTPA and TTHA were used as a vehicle to transfer energy to the DNA.4 The overall energy transfer process is illustrated in Figure 2. It is worth noting that the fluorescence profile of the chelate system is similar to that of the aqueous lanthanide and the fluorescence profile of the DNA-chelate also remains unchanged.3-5
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Figure 2. Schematic illustrating the energy transfer process from the excited lanthanide chelate to the quadruplex DNA (represented by a G-quartet), resulting in a decrease in lifetime of the lanthanide chelate.
Figure 3. Energy transfer ((2.5%) from ethidium bromide as a function of DNA concentration: calf thymus (9), 12mer (b), 15mer (2), and 22mer human telomeric DNA (1). λex ) 488nm.
In this work, a frequency-domain luminescence lifetime instrument has been used to measure excited-state lifetimes and calculate the corresponding energy transfer. Frequency-domain experiments are well suited for systems with longer lifetimes (microseconds to milliseconds), generally luminescence (in lanthanides), delayed fluorescence or phosphorescence. The change in lifetime of lanthanide chelates in the presence of small amounts (20-800 ppb) of quadruplex DNA has been used to calculate the amount of energy transferred from the chelate to DNA upon binding using Forster’s theory (eq 1)3,4
E)1-
IDA τDA )1ID τD
(1)
IDA and ID are the fluorescence intensities of the donor species (chelate), in the presence (DA) and absence (D) of the acceptor species (DNA), respectively. τDA and τD are the corresponding lifetimes. Therefore, a decrease in the lifetime of the chelate upon binding to the DNA would correspond to a finite amount of energy transferred from the chelate. Experimentally, lifetimes were calculated by first measuring the phase-shift with respect to an internal reference and applying the generally accepted rule of thumb that the modulation
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J. Phys. Chem. B, Vol. 113, No. 4, 2009 867
TABLE 1: Energy Transfer (%) from Terbium Chelates (1 µM)a 22mer-DTPA
22mer-TTHA
12mer-DTPA
15mer-DTPA
[DNA], ppb
before annealing
after annealing
before annealing
after annealing
before annealing
after annealing
before annealing
after annealing
0 20 400 800
0 12 ( 1.2 24 ( 2.4 24 ( 2.4
0 10 ( 1.0 19 ( 1.9 0
0
0 20 ( 2.0 27 ( 2.7 8 ( 0.8
0 7 ( 0.7
0 7 ( 0.7 22 ( 2.2 10 ( 1.0
0
0 42 ( 4.2 14 ( 1.4 17 ( 1.7
a
Ligands: DTPA (diethylenetriaminepentaacetic acid) and TTHA (triethylenetetraamine-N,N,N′,N′′,N′′′,N′′′-hexaacetic acid).
frequency (ν) should be approximately equal to the mean lifetime, which corresponds to a phase-shift of θ ) 45°. The lifetime was calculated using eq 2
τ)
1 2πν
(2)
The approach was first tested by measuring lifetimes and energy transfer using ethidium bromide, a common duplex DNA intercalator. Arthanari et al. showed significant enhancement in the fluorescence intensity of ethidium bromide in the presence of duplex DNA (calf thymus) and double-stranded quadruplex DNA (12mer) but not single-stranded 15mer quadruplex DNA.6 In this work, the interaction between various DNAs and ethidium bromide was investigated by measuring the lifetime of ethidium bromide, before and after addition of DNA and calculating the amount of energy transferred to the DNA. The results showed that as the amount of double-stranded DNA (calf thymus and 12mer) increased, more energy was transferred from ethidium bromide. The amount of energy transfer reached a maximum of approximately 40% with calf thymus DNA and 15% with 12mer. However, no such increase or trend was observed in the case of single-stranded DNA (15mer and 22mer human telomeric DNA). This is shown in Figure 3. This shows that this is a valid approach for detecting small amounts of quadruplex DNA, and it is possible to distinguish between single- and double-stranded quadruplex structures based on the choice of fluorophore. Energy transfer between the terbium chelates Tb3+-DTPA and Tb3+-TTHA and DNA was measured in a similar fashion. The amount of energy transferred as a function of DNA concentration changed under certain conditions (Table 1). Initially, the amount of energy transferred increased with DNA concentration but leveled off or decreased at 800 ppb DNA, both before and after annealing. It is possible that the chelate (Tb3+-DTPA) may have reached its DNA detection limit. This was also observed in the case of Tb3+-TTHA only after annealing. The trends with the double-stranded 12mer DNA and Tb3+-DTPA were similar to those obtained with the 22mer human telomeric DNA and Tb3+-TTHA (Table 1). Of the other quadruplex sequences, the energy transfer from the chelate was negative in the presence of the unannealed single-stranded 15mer aptamer, and this will be investigated in the future. There was a high amount of energy transfer when 20 ppb 15mer was added but there was a significant decrease at higher concentrations. The results with the double-stranded 12mer DNA and Tb3+DTPA were similar to those obtained with the human telomeric DNA, and there was measurable energy transfer to the DNA only after annealing. The energy transfer results illustrate two key findings. First, annealing plays an important role in the formation of quadruplex structure and subsequent detection of these DNAs with lanthanide chelates. Second, both single-stranded (22mer) and
double-stranded (12mer) quadruplex DNA were detected by at least one of the two chelates. The single-stranded 15mer DNA did decrease the lifetime of the Tb3+-DTPA chelate after annealing but the decrease did not correlate with the concentration of DNA. This may be the result of the nature of binding between the DNA and chelate. The chair-type quadruplex structure may not offer the same binding motifs as the boattype 12mer or the 22mer. The detection of both single- and double-stranded DNA by these chelates is in contrast to the detection of only double-stranded DNA by ethidium bromide. Future experiments using NMR spectroscopy will be used to ascertain the exact interaction between quadruplex DNA and the chelates. Wang et al. have previously shown that NMR can be used to determine the nature of binding of metal ions to quadruplex DNA.2 Energy transfer experiments with europium chelates were not carried out as the excitation wavelength of these chelates (576 nm) is outside the range of the laser used in this work.4 However, it should be noted that the formation of Eu3+-TTHA chelate greatly enhanced the emission intensity of the 5D0f 7F1 peak at 595 nm when compared to free Eu3+ in solution, but there was no measurable enhancement at 452 or 595 nm when the different DNAs were added, even after annealing. In conclusion, low concentrations (20 ppb or nM) of the 22mer human telomeric DNA have been detected via luminescence enhancement of europium and energy transfer from terbium chelates. The chelates detected both single- and doublestranded quadruplex DNA, unlike ethidium bromide. The chelates are known to form a fluorescence association complex with DNA, unlike ethidium bromide, which intercalates between bases and unwinds the DNA double helix. Finally, annealing has been shown to play a critical role in the formation of quadruplex structures as the binding of the lanthanide ions and chelates improved significantly once the samples were annealed. Acknowledgment. We thank Susquehanna University for financial support for this project. We also thank Dr. Haribabu Arthanari (Harvard Medical School) and Dr. Angela Sauers (Susquehanna University) for helpful discussions. References and Notes (1) (a) Galezowska, E.; Gluszynska, A.; Juskowiak, B. J. Inorg. Biochem. 2007, 101, 678–685. (b) Wang, K. Y.; Gerena, L.; Swaminathan, S.; Bolton, P. H. Nucl. Acids. Res. 1995, 23, 844–848. (c) Panigrahi, B. S.; Gajendran, N.; Suryamurthy, N. Spectrochim. Acta 2003, 59, 1905–1910. (d) Gross, D. S.; Simpkins, H. J. Biol. Chem. 1981, 256, 9593–9596. (e) Fu, P. K.-L.; Turro, C. J. Am. Chem. Soc. 1999, 121, 1–7. (2) (a) Feigon, J.; Dieckmann, T.; Smith, F. W. Chem. Biol. 1996, 3, 611–617. (b) Wang, K. Y.; Krawczyk, S. H.; Bischofberger, N.; Swaminathan, S.; Bolton, P. H. Biochemistry 1993, 32, 11285–11295. (c) Luu, K. N.; Phan, A. T.; Kuryavyi, V.; Lacroix, L.; Patel, D. J. J. Am. Chem. Soc. 2006, 128, 9963–9970. (d) Williamson, J. R. Annu. ReV. Biophys. Biomol. Struct. 1993, 23, 703–730. (e) Blackburn, E. H. Cell 1994, 77, 621–623.
868 J. Phys. Chem. B, Vol. 113, No. 4, 2009 (3) Selvin, P. R. Annu. ReV. Biophys. Biomol. Struct. 2002, 31, 275– 302. (4) (a) Selvin, P. R.; Hearst, J. E. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10024–10028. (b) Reifenberg, J. G.; Snyder, G. E.; Baym, G.; Selvin, P. R. J. Phys. Chem. B 2003, 107, 12862–12873. (c) Chen, J.; Selvin, R. R. Bioconj. Chem. 1999, 10, 311–315.
Letters (5) Ilie, M.; Mitrea, N.; Fugaru, V.; Stancu, D.; Ristea, I.; Andries, A. Farmacia 2000, 68, 33–39. (6) Arthanari, H.; Basu, S.; Kawano, T. L.; Bolton, P. H. Nucl. Acids. Res. 1998, 26, 3724–3728.
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