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Adsorption and Hybridization of Oligonucleotides on Mercaptoacetic Acid-Capped CdSe/ZnS Quantum Dots and Quantum Dot-Oligonucleotide Conjugates W. Russ Algar and Ulrich J. Krull* Chemical Sensors Group, Department of Chemical and Physical Sciences, UniVersity of Toronto at Mississauga, 3359 Mississauga Road North, Mississauga, Ontario, L5L 1C6 Canada ReceiVed July 27, 2006. In Final Form: September 26, 2006 Interest in the unique optical properties of quantum dots (QDs) has resulted in the development QD-bioconjugates for imaging and diagnostics. Although these applications are numerous, considerably less is known about the interactions between QDs and biomolecules. In this work, we describe hydrogen-bonding interactions between oligonucleotides and CdSe/ZnS quantum dots capped with mercaptoacetic acid ligands. The strength of the interactions can be modulated by changes in the pH and ionic strength, the addition of formamide, and differences between ssDNA and dsDNA. Fluorescence resonance energy transfer experiments have shown that conjugated oligonucleotides adopt a conformation that lies across the surface of the QD. The hydrogen-bonding interactions also affect the kinetics of hybridization with QD-DNA conjugates and the thermal stability of QD-conjugated dsDNA. The former is analogous to conventional solid-phase hybridization, where stronger oligonucleotide adsorption leads to faster kinetics. With respect to the latter, interactions with the QD surface can sharpen the melt transition and alter the melt temperature of dsDNA. These effects are largely absent when adsorptive interactions are minimized.
Introduction Quantum dots (QDs), or colloidal semiconductor nanocrystals, have been at the center of much research in recent years. Since the first application of water-soluble QDs,1,2 an array of imaging3-8 and diagnostic9-14 applications have been explored. Concomitant to these advances has been the development of a variety of QD surface chemistries and QD bioconjugates. The former have included thiol-alkyl acid ligands,2,9-11,15-18 thin silica shells,19,20 amphiphilic polymers,21-24 and phospholipids.25 * To whom correspondence should be addressed. Phone: (905) 8285437. Fax: (905) 828-5425. E-mail:
[email protected]. (1) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013. (2) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016. (3) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631. (4) Jaiswal, J. K.; Mattoussi, H.; Mauro, J.; Simon, S. M. Nat. Biotechnol. 2003, 21, 47. (5) Stroh, M.; Zimmer, J. P.; Duda, D. G.; Levchenko, T. S.; Cohen, K. S.; Brown, E. B.; Scadden, D. T.; Torchilin, V. P.; Bawendi, M. G.; Fukumura, D.; Jain, R. K. Nat. Med. 2005, 11, 678. (6) Smith, A. M.; Gao, X.; Nie, S. Photochem. Photobiol. 2004, 80, 377. (7) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435. (8) Pinaud, F.; Michalet, X.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Iyer, G.; Weiss S. Biomaterials 2006, 27, 1679. (9) Goldman, E. R.; Clapp, A. R..; Anderson, G. P.; Uyeda, H. T.; Mauro, J. M.; Medintz, I. L.; Mattoussi, H. Anal. Chem. 2004, 76, 684. (10) Goldman, E. R.; Medintz, I. L.; Whitley, J. L.; Hayhurst, A.; Clapp, A. R.; Uyeda, H. T.; Deschamps, J. R.; Lassman, M. E.; Mattoussi, H. J. Am. Chem. Soc. 2005, 127, 6744. (11) Kim, J. H.; Morikis, D.; Ozkan, M. Sens. Actuators, B 2004, 102, 314. (12) Zhang, C. Y.; Yeh, H. C.; Kuroki, M. T.; Wang, T. H. Nat. Mater. 2005, 4, 826. (13) Chang, E.; Miller, J. S.; Sun, J.; Yu, W. W.; Colvin, V. L.; Drezek, R.; West, J. L. Biochem. Biophys. Res. Commun. 2005, 334, 1317. (14) Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, G.; Mauro, J. M. Nat. Mater. 2003, 2, 630. (15) Wang, S.; Mamedova, N.; Kotov, N. A.; Chen, W.; Studer, J. Nano Lett. 2002, 2, 817. (16) Mitchell, G. P.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1999, 121, 8122. (17) Zhou, D.; Piper, J. D.; Abell, C.; Klenerman, D.; Kang, D.-J.; Ying, L. Chem. Commun. 2005, 4807. (18) Gill, G.; Willner, I.; Shweky, I.; Banin, U. J. Phys. Chem. B 2005, 109, 23715.
The latter have included oligonucleotides,11,12,16-18 aptamers,26,27 antibodies,9,10 and proteins.14,28 Thiol-alkyl acids have been one of the most commonly used surface chemistries for water solubility and bioconjugation. Commonly used thiol-alkyl acids include mercaptoacetic acid,2,11,15 3-mercaptopropionic acid,16-18 and dihydrolipoic acid.9,10,14 The thiol-alkyl acid surface chemistry is prone to poor stability at low pH, ultraviolet photooxidation of ligands, and less efficient photoluminescence than other surface chemistries.29-31 Nonetheless, the thiol-alkyl acid surface chemistry is advantageous in that it is the most compact of the surface chemistries developed to date and is easily prepared. The former point is important in fluorescence resonance energy transfer (FRET) applications, where distance considerations are paramount. Recently, we have reported the use of mercaptoacetic acid (MAA)-capped QDs in a FRET-based approach to twocolor nucleic acid diagnostics.32 In this previous work, it was found that the diagnostic scheme was complicated by the adsorption of oligonucleotides on the MAA-capped QDs. (19) Gerion, D.; Pinaud, F.; Williams, S. C.; Parak, W. J.; Zanchet, D.; Weiss, S.; Alivisatos, A. P. J. Phys. Chem. B 2001, 105, 8861. (20) Parak, W. J.; D. Gerion, D.; Zanchet, D.; Woerz, A. S.; Pellegrino, T.; Micheel, C.; Williams, S. C.; Seitz, M.; Bruehl, R. E.; Bryant, Z.; Bustamante, C.; Bertozzi, C. R.; Alivisatos, A. P. Chem. Mater. 2002, 14, 2113. (21) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. Nat. Biotechnol. 2004, 22, 969. (22) Pellegrino, T.; Manna, L.; Kudera, S.; Liedl, T.; Koktysh, D.; Rogach, A. L.; Keller, S.; Ra¨dler, J.; Natile, G.; Parak, W. J. Nano Lett. 2004, 4, 703. (23) Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat. Biotechnol. 2003, 21, 41. (24) Nann, T. Chem. Commun. 2005, 1735. (25) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759. (26) Levy, M.; Cater, S. F.; Ellington, A. D. ChemBioChem 2005, 6, 2163. (27) Dwarakanath, S.; Bruno, J. G.; Shastry, A.; Phillips, T.; John, A.; Kumar, A.; Stephenson, L. D. Biochem. Biophys. Res. Commun. 2004, 325, 739. (28) Chang, E.; Miller, J. S.; Sun, J.; Yu, W. W.; Colvin, V. L.; Drezek, R.; West, J. L. Biochem. Biophys. Res. Commun. 2005, 334, 1317. (29) Aldana, J.; Wang, Y. A.; Peng, X. J. Am. Chem. Soc. 2001, 123, 8844. (30) Kloepfer, J. A.; Bradforth, S. E.; Nadeau, J. L. J. Phys. Chem. B 2005, 109, 9996. (31) Kim, S.; Bawendi, M. G. J. Am. Chem. Soc. 2003, 125, 14652. (32) Algar, W. R.; Krull, U. J. Anal. Chim. Acta, in press.
10.1021/la062217y CCC: $33.50 © 2006 American Chemical Society Published on Web 11/10/2006
Oligonucleotide Adsorption on CdSe/ZnS QDs Table 1. Oligonucleotides 1 2 3
4 5 6 7 8
9 10
Mixed Base Sequences NH2C6H12-5′-ATT TTG TCT GAA ACC CTG T-3′ NH2C12H24-5′-ATT TTG TCT GAA ACC CTG T-3′ 5′-ACA GGG TTT CAG ACA AAA T-3′ (fully complementary) 5′-ACA GGG TTT TAG ACA AAA T-3′ (single-base-pair mismatch) 5′-ACA GAG TTT CAG ACG AAA T-3′ (double-base-pair mismatch) 5′-ACA GGG TTT CAG ACA AAA T-3′-Cy3 Cy3-5′-ACA GGG TTT CAG ACA AAA T-3′ 5′-ATT TTG TCT GAA ACC CTG T-3′-Cy3 Homopolymer Sequences NH2C6H12-5′-AAA AAA AAA AAA AAA AAA AA-3′ 5′-TTT TTT TTT TTT TTT TTT TT-3′ (fully complementary) 5′-TTT TTT CTT TTT TCT TTT TT-3′ (double-base-pair mismatch) 5′-TTT TTT TTT TTT TTT TTT T-3′-Cy3 Cy3-5′-TTT TTT TTT TTT TTT TTT TT-3′
In the present work we explore the interactions between oligonucleotides and the surface of MAA-capped QDs using the sensitivity of FRET to the proximity between a QD donor and Cy3 acceptor. While most other reports to date have been application centered with little attention given to underlying physical mechanisms, this work is one of the first investigations into the nature of oligonucleotide-QD interactions. The behavior of nucleic acid hybrids is often manipulated by changing the ionic strength or pH of the solution, adding denaturants such as formamide, or, in the case of solid-phase hybridizations, altering the probe oligonucleotide density. In this work, the effect of these changes on (1) the adsorption and conformation of oligonucleotides on MAA-QDs, (2) the kinetics of adsorption and hybridization with MAA-QDs and QD-ssDNA conjugates, and (3) the thermal stability of hybrids as QD-dsDNA conjugates were investigated. The results obtained are described in terms of an electrostatic and hydrogen-bonding model and furthermore are not dissimilar to the current understanding of solid-phase hybridization. Given the popularity of QDs and the number of applications employing QD bioconjugates, understanding the interactions between QDs and biomolecules is of considerable importance and multidisciplinary interest. Experimental Section Reagents and Oligonucleotides. Adirondack Green CdSe/ZnS core/shell semiconductor nanocrystals (QDs) in toluene were from Evident Technologies (Troy, NY) and had an emission maximum of 524 nm and a core diameter of ca. 2.1 nm. Mercaptoacetic acid (98%), N,N-diisopropylethylamine (99.5%), and N-[3-(dimethylamino)propyl]-N′-ethylcarbodiimide hydrochloride (EDC) were from Sigma-Aldrich (Oakville, ON, Canada) and were used without further purification. Chloroform was from EM Science (Toronto, ON, Canada) and was used as received. Pico green fluorescent stain was obtained from Molecular Probes (Eugene, OR). All buffer solutions were prepared with autoclaved doubly distilled water and filtered through a 0.2 µm syringe filter for the preparation of QD solutions. Buffers included Tris-borate (TB; pH 7.4, 90 mM), borate (pH 9.5, 500 mM), acetate (pH 4.8, 100 mM), and TB containing 30%, 50%, or 75% formamide. Modified and unmodified oligonucleotides were from Integrated DNA Technologies (Coralville, IA) and were HPLC purified by the manufacturer. The oligonucleotides were dissolved in deionized water with a specific resistance of 18 MΩ cm-1. Water was deionized and purified by the Milli-Q cartridge purification system (Millipore Corp., Mississauga, ON, Canada). Nucleotide sequences are listed in Table 1 with a corresponding number. Throughout the Experimental
Langmuir, Vol. 22, No. 26, 2006 11347 Section, nucleotide sequences are referred to by boldface numbers for clarity. The “mixed base” oligonucleotide corresponds to the SMN1 sequence, which is diagnostic of spinal muscular atrophysa genetic disorder. Quantum Dot Capping and Quantum Dot-DNA Conjugate Preparation. QDs in toluene were made water-soluble by ligand exchange with MAA. Excess MAA was removed by repeated chloroform washes and ethanol precipitation from aqueous solution. The concentration of the resulting MAA-QDs was determined by absorption spectroscopy using the first absorption peak at 515 nm. DNA conjugates were prepared by mixing MAA-QDs with n equiv of amine-modified oligonucleotides in TB buffer containing EDC. This chemistry forms a stable amide bond between an MAA ligand and an oligonucleotide. Excess EDC was removed by ethanol precipitation. Typical conjugate recoveries were 70-80%, and final concentrations were determined by UV-vis absorption spectroscopy. The details of both the capping and conjugation procedures can be found in the Supporting Information. Adsorption Experiments. Adsorption experiments were conducted by preparing 1.0 µM solutions of QDs, QD-1×DNA conjugate (1), or QD-2×DNA conjugate (1) with n equiv of adsorbing Cy3-labeled nucleic acid (6 or 9), diluted with the desired buffer. Adsorbing sequences were noncomplementary to conjugated probe oligonucleotides. Buffers included TB, borate, acetate, and TB containing 30%, 50%, or 75% formamide. Adsorbing nucleic acids included single-stranded mixed base (6) and poly-T homopolymer (9) sequences, as well as a mixed base duplex (3/6) and a poly-A/poly-T duplex (7/10). Following 12 h at room temperature, fluorescence spectra were measured, and the extent of adsorption was measured as the ratio of FRET-sensitized Cy3 to QD fluorescence (560 nm:526 nm). The QD and Cy3 emission maxima are at 526 and 560 nm, respectively, and convolution of the two spectra is small at these two wavelengths. Nonetheless, the ratio of QD fluorescence at 560 and 526 nm has been subtracted from the ratio reported. Excitation was at 385 nm, where direct excitation of Cy3 is undetectable. Effects of the Cy3 Label Position and Linker Length. QDprobe conjugates were prepared using either a mixed base probe with a six- or twelve-carbon amine linker (1 or 2) or a poly-A probe with a six-carbon amine linker (7). Conjugates were mixed with 1 equiv of fully complementary target material and allowed to hybridize for 24 h. Samples prepared included 3′- and 5′-Cy3-labeled mixed base hybrids (1/4, 1/5, 2/4, 2/5) and homopolymer QD-dsDNA conjugates (7/9, 7/10). Control samples for QD measurements were unlabeled mixed base hybrids (1/3, 2/3) and homopolymer QDdsDNA conjugates (7/8). Control samples for Cy3 measurements were free dsDNA in solution with 3′- and 5′-Cy3 labels (1/4, 1/5, 2/4, 2/5, 7/9, 7/10). FRET efficiencies were determined by measuring the relative change in the donor quantum yield or fluorescence lifetime in the absence and presence of a Cy3 acceptor. Further descriptions of these methods are available in the Supporting Information. Kinetic Experiments. Kinetic experiments were done by preparing solutions 1.0 µM in QDs, QD-1×DNA conjugate (1 or 7), or QD2×DNA conjugate (1) in the desired buffer. One equivalent of adsorbing (6) or fully complementary target (5 or 9) oligonucleotide was added and mixed (vortexed) quickly. FRET-sensitized Cy3 fluorescence (excitation at 385 nm, emission at 560 nm) was then measured at 1 min increments for 4-8 h. Between fluorescence measurements there was no illumination of the sample. The kinetic curves obtained were fit with a Box Lucas model of the general form given in eq 1, y ) A(1 - e-kx)
(1)
where y corresponds to the FRET-sensitized Cy3 fluorescence as a function of time, x is the time parameter, A is a constant, and k may be considered to be a kinetic parameter describing the rate of hybridization or adsorption. It is the k parameter which is of interest and is used to indicate the rate of the process under study. Best fit curves were obtained using OriginPro 7.5 scientific graphing and analysis software (OriginLab Corp., Northampton, MA).
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Table 2. Adsorption of Cy3-Labeled DNA on QDs and QD-DNA Conjugates in Various Buffersa acetateb
TBc
TBd
boratee
TB with 75% (v/v) formamidef
QD QD-1×DNA QD-2×DNA
ssDNA Mixed Base (SMN1) Sequence 0.85 0.37 11.45 0.72 0.89 0.22 3.85 0.15 0.25 0.07
QD
dsDNA Mixed Base (SMN1) Sequence 0.24
QD QD-1×DNA QD-2×DNA QD
0.12 no ads no ads
ssDNA Poly-T Homopolymer no ads 0.54 no ads no ads 0.26 no ads no ads 0.08 dsDNA Poly-T/Poly-A Homopolymer no ads
a Adsorption is listed as the ratio of FRET acceptor (Cy3-oligonucleotide) and donor (QD) emission intensities. See the text for details. b pH 4.8, 100 mM. c pH 7.8, 15 mM. d pH 7.4, 90 mM. e pH 9.5, 500 mM. f Not listed in the table: mixed base (SMN1) ssDNA adsorption on QDs in TB with 10% formamide (0.85), 30% formamide (0.51), and 50% formamide (0.20).
Figure 1. Adsorption of mixed base Cy3-labeled oligonucleotides (5) on MAA-QDs as a function of concentration in TB (9) and borate (O) buffers. The abscissa on the right corresponds to FRET ratios obtained in TB; the abscissa on the left corresponds to FRET ratios obtained in borate. These ratios differ from those in Table 2 because a 5′-Cy3 label was used instead of a 3′ label. The former is brighter and gives better resolution for similar concentrations of oligonucleotides.
Adsorption of Oligonucleotides. The adsorption of 1 equiv of oligonucleotides onto the surface of MAA-modified CdSe/ ZnS QDs in a number of solvent systems is summarized in Table 2. These FRET ratios are semiquantitative measures of adsorption, but the trends observed are consistent and reliable. The adsorption of a mixed base oligonucleotide is dependent on the pH and ionic strength, the presence of denaturants, differences between ssDNA and dsDNA, and the number of QD-conjugated oligonucleotides. With respect to pH dependence, adsorption is very strong in acetate buffer at pH 4.8, where most surface carboxyl groups on
the surface of the QD are expected to be protonated. Adsorption is >10-fold weaker in borate buffer at pH 9.5, where most carboxyl groups are expected to be deprotonated. At neutral pH, where adsorption is intermediate, there is likely a mixture of protonated and deprotonated carboxyl groups and adsorption is roughly 3-4fold stronger than in borate buffer. Figure 1 shows the concentration-dependent adsorption on MAA-QDs in TB and borate buffer. Adsorption begins to saturate in TB buffer near 1.25 equiv of nucleic acid. In contrast, adsorption increases steadily past this point in borate buffer, confirming that a higher pH decreases the number of adsorbed oligonucleotides. Although the data in Table 2 indicate that the ionic strength affects adsorption, it is clear that pH is the critical variable. A hydrogen-bonding mechanism is suggested by the pHdependent behavior and is supported by the observation that increasing amounts of formamide in TB decrease and eventually eliminate adsorption. Formamide is well-known to disrupt hydrogen-bonding interactions, particularly with nucleic acids. A hydrogen-bonding interaction should involve the nucleobases, and such a mechanism is consistent with the observation that adsorption of dsDNA is lower than that of ssDNA. Additional evidence for the role of the nucleobases is found in the behavior of a homopolymer of thymine (poly-T). In contrast to the mixed base sequence, or a poly-A sequence (data not shown), the poly-T sequence showed no tendency to adsorb at neutral pH and a greater tendency to adsorb at basic pH. In dsDNA, the availability of the nucleobases is reduced and adsorption decreases. However, because polar groups on the nucleobases point into the grooves of the double-helix structure,34 the capacity of adsorption is not altogether eliminated. Similarly, a reduction in the availability of the QD surface due to conjugated oligonucleotides also reduces adsorption. In addition to reducing the QD surface area of adsorption, covalently attached oligonucleotides add negative charge and increase electrostatic repulsion. It should be noted that changes in the protonation state of MAA with pH also induce changes in the surface charge. Analogous to the nature of interactions between two complementary strands of nucleic acid, it appears that hydrogen bonding is the associative mechanism between MAA-QDs and oligonucleotides and that electrostatic interactions have a destabilizing effect on that interaction.
(33) Molecular Probes: Invitrogen Detection Technologies, http://probes.invitrogen.com/handbook/figures/1549.html.
(34) Adams, R. L. P.; Knowler, J. T.; Leader, D. P. The Biochemistry of Nucleic Acids, 10th ed.; Chapman & Hall: New York, 1986; Chapter 2.
Melt Curve Experiments. Samples for melt curves were prepared by mixing QD-1×DNA (1 or 7) or QD-2×DNA (1) oligonucleotide conjugate with 1 equiv of complementary or mismatched nonlabeled target oligonucleotide (3 or 8) in the desired buffer and allowing hybridization to occur over a 24 h period. In contrast to the other experiments described, melt curves were not obtained with FRETsensitized fluorescence from a Cy3-labeled target. Instead, unlabeled target was used, 1 equiv of pico green was added, and the resulting mixture was allowed to stand for at least 4 h. Pico green is a fluorescent stain which is known to have a much higher quantum yield in the presence of dsDNA compared to ssDNA.33 Samples were ultimately 1.0 µM in pico green and QD-dsDNA conjugate. Control samples were prepared similarly, but used a 1.0 µM concentration of free probe oligonucleotides in solution rather than QD-probe conjugates. QD-probe conjugates and the corresponding control sample were melted simultaneously with a temperature ramp of 0.4 °C min-1. Pico green fluorescence (excited at 500 nm, measured at 515 nm) was measured every 2 °C. Due to its stronger absorption at 500 nm and high quantum yield, the pico green fluorescence was approximately 2 orders of magnitude larger than the QD luminescence. Consequently, no deconvolution of the optical signals was needed. The raw data from the melt curves were processed as follows to yield melt curves corrected for the decrease in fluorescence with temperature. The initial 15-25 °C range was fit with an exponential function to model the change of fluorescence with temperature. The raw data were normalized to this function and corrected to have maximum and minimum values of unity and zero. The melt curves presented are the average of three replicates.
Results and Discussion
Oligonucleotide Adsorption on CdSe/ZnS QDs
Langmuir, Vol. 22, No. 26, 2006 11349 Table 3. Relative Quantum Yield (QY) and Lifetimes for QDs and Cy3 as a Function of the Cy3 Label Position (3′ or 5′), Linker Length (Six- or Twelve-Carbon), and Sequence QD rel QY
QD rel lifetime
Cy3 rel QY
QD-Amide-C6H12-Mixed Base (SMN1) Probe 5′-target-3′-Cy3 0.39 0.40 0.60 Cy3-5′-target-3′ 0.53 0.50 1.01
Cy3 rel lifetime 1.08 1.20
QD-Amide-C12H24-Mixed Base (SMN1) Probe 5′-target-3′-Cy3 0.41 0.44 0.54 1.10 Cy3-5′-target-3′ 0.47 0.53 0.98 1.20 QD-Amide-C6H12-Poly-A Homopolymer Probe 5′-target-3′-Cy3 0.93 0.81 0.69 1.20 Cy3-5′-target-3′ 0.96 0.84 0.87 1.19
Figure 2. Conformational models (approximately to scale) for dsDNA on the surface of an MAA-capped QD. The dashed line around the QDs indicates the Fo¨rster distance (taking the center of the QD as the zero distance). The weight of the arrow shown indicates the expected FRET efficiency based on the Cy3 acceptor position and linker length. Upright conformations: (a) A 3′-Cy3 label with six- and twelve-carbon linkers will yield the highest FRET efficiencies. (b) A 5′-Cy3 label with six- and twelve-carbon linkers should yield no FRET. Conformations along the QD surface should yield similar FRET efficiencies for (c) 3′- and (d) 5′-Cy3 labels, regardless of the linker length.
Conformational Insights from the Linker Length and Cy3 Label Position. Our group has previously reported32 that donoracceptor distances measured from FRET efficiencies suggest that covalently attached and adsorbed oligonucleotides have a conformation that lies along the surface of the QD. If the oligonucleotide lies along the surface of the QD, the FRET efficiency should be similar for both 3′- and 5′-Cy3 acceptor labels on the oligonucleotide, despite (1) an estimated 6.5 nm between the 3′ and 5′ termini or (2) changes in the linker length. Conversely, if an upright and nearly linear conformation were adopted, it would be expected that (1) a 3′-Cy3 acceptor would yield a high FRET efficiency and a 5′-Cy3 acceptor would yield a very low FRET efficiency and (2) changes in the linker length would modulate the FRET efficiency. These two scenarios are depicted in Figure 2, and the above arguments are made noting that the Fo¨rster distance for this system is 2.7 nm.32 The data in Table 3 clearly indicate that the mixed base oligonucleotide adopts a conformation along the surface of the QD. Both 3′- and 5′-Cy3 acceptors result in similar FRET efficiencies and are largely unaffected by switching between six-carbon and twelve-carbon linkers. As a consequence of FRET, both the 3′- and 5′-Cy3 labels show approximately 10% and 20% longer fluorescence lifetimes
than those in the bulk solution. The acceptor dye can become excited at any point during the donor lifetime, resulting in a longer apparent acceptor lifetime. The QD lifetimes for 3′- and 5′-Cy3 labels are approximately 60% and 50% shorter, respectively, indicating slightly more efficient FRET for the 3′ label. However, despite the greater FRET efficiency for the 3′-Cy3 acceptor, the increase in lifetime is greater for the 5′-Cy3 acceptor. There is also a 40-50% decrease in the relative quantum yield of the 3′-Cy3 acceptor relative to that of the free solution. No such decrease is observed in the 5′ case. The quenching phenomenon may be a result of “pinching” of the dye between the linker and the oligonucleotide when a conformation along the surface of the QD is adopted. Free poly-A/poly-T dsDNA does not adsorb on the surface of a QD from solution and thus could be expected to adopt a nearly upright conformation and show a large difference in FRET efficiency for 3′- and 5′-Cy3 acceptors. The resulting FRET efficiencies (Table 3) are much lower than those for the mixed base experiments, but the efficiencies do not quite correspond to the distances expected for an upright conformation. Nonetheless, the data do not suggest a conformation pinned along the surface. Structural bends associated with the high propeller twist angle of poly-A/poly-T sequences35,36 are likely responsible for the higher than anticipated FRET efficiency for a 5′-Cy3 acceptor. Kinetics of Adsorption and Hybridization. Adsorption and hybridization follow kinetics which can be fit with the exponential function in eq 1. Examples of kinetic curves for adsorption and hybridization can be found in the Supporting Information. Kinetic parameters for adsorption and hybridization under different buffer conditions are listed in Table 4. The data show that adsorption and hybridization occur over periods approaching 8 h in TB buffer, or longer with other buffer solutions. Hybridization periods on the order of several hours are often encountered with solidphase hybridizations such as, for example, on nylon membranes (12 h).37 More importantly, the kinetic parameters obtained for adsorption mirror the tendency for adsorption; that is, the QD conjugates and buffer conditions that yielded the lowest steadystate adsorption also yielded the slowest rates of adsorption. Adsorption was found to be fastest in acetate buffer, with kinetics more than 40-fold and 100-fold slower in TB and borate buffers, respectively. Similarly, the rate of adsorption decreased as the extent of DNA conjugation increased. The physical interactions previously invoked to explain the trends in equilibrium adsorption are readily extended to the observed kinetic trends. As the electrostatic repulsion and hydrogen-bonding capacity are (35) Travers, A. A. Annu. ReV. Biochem. 1989, 58, 427. (36) Lin, C. H.; Sun, D., Hurley, L. H. Chem. Res. Toxicol. 1990, 4, 21. (37) Aubert, D.; Toubas, D.; Foudrinier, F.; Villena, I.; Gomez, J. E.; Marx, C.; Lepan, H.; Lemaire, P.; Jacquier, P.; Bonhomme, A.; Pinon, J. M. Anal. Biochem. 1997, 247, 25.
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Table 4. Rate Parametersa Derived from Best Fit Data for Adsorption and Hybridization under Different Buffer Conditions adsorption constant, k (h-1) mixed base w/MAA-QD acetate Tris-borate borate mixed base w/MAA-QD-1×DNA acetate Tris-borate borate mixed base w/MAA-QD-2×DNA acetate Tris-borate borate
hybridization constant, k (h-1) 112 1.37 0.30 40.0 0.62 0.12 15.3 0.36
mixed base w/MAA-QD-1×DNA acetate Tris-borate borate mixed base w/MAA-QD-2×DNA acetate Tris-borate borate poly-T w/MAA-QD-1×poly-A acetate Tris-borate borate
0.64 0.21 0.29
0.12
a Adsorption and hybridization kinetic curves were fit with a Box Lucas model, y ) A(1 - e-kx), where the constant k describes the rate of the process.
decreased and increased (respectively) at lower pH, there is a corresponding increase in the likelihood of an adsorptive event, thus resulting in the observed kinetic trends. It is also observed in Table 4 that the rates of hybridization mirror the rates of adsorption, suggesting that adsorption may play a role in hybridization. For example, the rate parameters for adsorption and hybridization with QD-1×DNA conjugates are 0.62 and 0.64 h-1, suggesting that adsorption plays a key role. Experiments with dyes sensitive to the dsDNA structure have confirmed that hybridization does indeed occur in the latter case. The role of adsorption of oligonucleotides in enhancing solidphase hybridization rates is well established,38,39 where target oligonucleotide adsorption and subsequent surface diffusion to immobilized probes is a critical process leading to hybridization. We consider hybridization on a QD to be a pseudo-solid-phase hybridization due to the surface area provided by the QD which supports adsorption. Previous work has also demonstrated that hybridization kinetics are faster on larger QDs,32 confirming the importance of the QD surface area. For hybridization to occur, nucleobase-nucleobase interactions between complementary sequences must displace nucleobase-MAA interactions that result in a conformation along the QD surface. Similar to solidphase hybridization, it is likely that the target oligonucleotide first adsorbs to the surface of the QD and subsequently diffuses across the surface to the probe oligonucleotide. If the process of breaking nucleobase-ligand interactions in favor of nucleobasenucleobase interactions (hybridization) is not rapid, the adsorptive interactions would serve to maintain the proximity between the probe and target, thereby facilitating the process. With such a process, hybridization kinetics would be enhanced by stronger nonspecific adsorption as the data suggest. In the absence of nonspecific adsorption, hybridization rates should be slower since the target oligonucleotide must not only collide directly with the probe oligonucleotide, but also collide in such a manner as to allow hybridization to displace the adsorptive interactions of the probe. The data in Table 4 indicate that, for a poly-T target which does not adsorb, hybridization rates are the lowest of all the conditions studied. This supports the hypothesis that target oligonucleotide adsorption helps displace the surface adsorption of the probe oligonucleotide, which hinders hybridization. Melt Curves. It is known that differences in hybridization at a bulk interface and in free solution are not trivial.40,41 Similarly, (38) Chan, V.; Graves, D. J.; McKenzie, S. E. Biophys. J. 1995, 69, 2243. (39) Erickson, D.; Li, D.; Krull, U. J. Anal. Biochem. 2003, 317, 186. (40) Piunno, P. A. E.; Watterson, J.; Wust, C. C.; Krull, U. J. Anal. Chim. Acta 1999, 400, 73. (41) Watterson, J. H.; Piunno, P. A. E.; Wust, C. C.; Krull, U. J. Langmuir 2000, 16, 4985.
Figure 3. Melt curves for (a) mixed base hybrids (1/3) in TB buffer with (i) a two-base-pair mismatch, (ii) a one-base-pair mismatch, and (iii) fully complementary hybrids and (b) poly-A/T hybrids (7/8) with (i) a two-base-pair mismatch and (ii) fully complementary hybrids. Melt curves obtained as QD-1×dsDNA conjugates are denoted by solid squares; melt curves obtained as free dsDNA in solution are denoted by hollow circles.
one could expect that hybridization involving a QD-DNA conjugate would have nontrivial differences with hybridization in bulk solution. Figure 3a shows melt curves obtained with fully complementary and single- and double-mismatched mixed base hybrids. There is a clear difference in the melt curves obtained for dsDNA in bulk solution in comparison to the respective QDdsDNA conjugate. The QD conjugate curves are notably sharper and exhibit shifts in the melt temperature. With respect to the former, the bulk solution melt transitions occur over temperature ranges greater than 20 °C while the melt transitions for QDdsDNA conjugates occur over temperature ranges less than 20 °C. The most notable sharpening of the transition is for the fully complementary duplex, with transition ranges of approximately 24 and 12 °C for bulk solution and QD conjugate dsDNA,
Oligonucleotide Adsorption on CdSe/ZnS QDs
Figure 4. Melt curves for (a) mixed base hybrids (1/3) as QD2×dsDNA conjugates in TB buffer with (i) a two-base-pair mismatch, (ii) a one-base-pair mismatch, and (iii) fully complementary hybrids and (b) mixed base hybrids (1/3) as QD-1×dsDNA conjugates in borate buffer with (i) a two-base-pair mismatch and (ii) fully complementary hybrids. Melt curves obtained as QD-dsDNA conjugates are denoted by solid squares; melt curves obtained as free dsDNA in solution are denoted by hollow circles.
respectively. With respect to the melt temperatures, shifts of -2.3, +1.3, and +2.2 °C are observed for fully complementary, single-base-pair-mismatched, and double-base-pair-mismatched duplexes as QD conjugates. The features described above could arise from a decrease in oligonucleotide degrees of freedom due to QD conjugation, or could be more specifically related to the interactions between the QD surface and nucleic acid sequences. To confirm that the latter were responsible, the melt curves in Figure 4 were obtained with (a) a QD-2×DNA conjugate in TB buffer and (b) a QD1×DNA conjugate in borate buffer. Both conditions were established to significantly reduce interaction between the QD surface and nucleic acid. These melt curves more closely resemble those obtained for free dsDNA under the same conditions. There is no substantial sharpening of the melt transition or melt temperature elevation for mismatched dsDNA. These observations indicate that the surface interactions are largely responsible for the change in hybridization behavior compared with that of the free solution. In addition, melt temperature depressions ranging from 0.5 to 2.2 °C are observed. This suggests that, when adsorptive interactions are reduced, the negative charge density on the surface of the QD and/or due to additional oligonucleotide probes has a slight destabilizing effect on the dsDNA. The sharpening of melt transitions for QD-dsDNA conjugates, as well as the melt temperature elevation for mismatched QDdsDNA, can be rationalized within a simple physical description of the melt transition and adsorptive interactions. It is wellknown that oligonucleotides exhibit breathing behavior in which
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sections of the hybrid, particularly near the termini or a mismatch, transiently denature and re-form the hybrid. On the surface of an MAA-modified QD, adsorptive interactions between the QD surface and DNA must be considered. Breathing of the dsDNA on the surface of the QD provides a mechanism by which strong interactions with ssDNA can occur. As the melt temperature is approached, breathing becomes more prominent and interactions between the hybrid and the surface increase. Eventually, the combined phosphate backbone repulsion and ssDNA-QD surface adsorptive interactions will exceed the attractive interactions between the remaining segments of duplex. The net effect is that the adsorptive interactions are able to help “pull” the duplex apart and sharpen the melt transition by “grabbing” segments of the duplex that breathe and thus prevent the duplex from reforming. Melt curves obtained via FRET with Cy3-labeled target oligonucleotide (rather than using unlabeled target and dye sensitive to dsDNA) show no defined melt transition, indicating that the target melted onto the surface of the QD, in agreement with this model. The slight elevation of the melt temperature for mismatched hybrids can be explained by similar arguments. Breathing is enhanced in the vicinity of a mismatch. However, under conditions where the attractive interactions between complementary nucleobases remain dominant, adsorptive interactions near a mismatch may have the effect of stabilizing the overall hybrid, thereby shifting the entire melt curve toward higher temperature. Such an effect would be strongest for a greater number of mismatches and absent for a fully complementary duplex, as has been observed experimentally. Melt curves obtained with the poly-A/poly-T duplex are shown in Figure 3b. A priori, these appear similar to those obtained with the mixed base duplex in Figure 3a. However, closer inspection reveals that the nature of the curves is intermediate between those in Figures 3a and 4. For the poly-A/poly-T duplex, the QD conjugate melt transitions are again sharper than those of free dsDNA in solution, but this effect is most pronounced for the second half of the transition. Due to the weak interaction with the poly-A/poly-T duplex, the surface can only interact strongly with the poly-A sequence once breathing and destabilization of the hybrid is large, resulting in the observed “second half” sharpening. The reduced adsorptive interaction is likely also responsible for the failure to observe any elevation of the melt temperature for the doubly mismatched AT duplex.
Conclusions Adsorptive interactions between oligonucleotides and MAAcapped QDs have been studied and rationalized in terms of a hydrogen-bonding model. The adsorption of a mixed base sequence was found to decrease with increasing pH, decreasing ionic strength, the addition of formamide, and the presence of conjugated oligonucleotides and between ssDNA and dsDNA. These adsorptive interactions appear to result in a conformation that lies across the surface of the MAA-QD for mixed base oligonucloetides. Weaker adsorptive interactions associated with thymine result in a more upright conformation as observed with a poly-A/poly-T hybrid. The kinetics of adsorption and hybridization were also dependent on the strength of adsorptive interactions. Hybridization also appears to be similar to conventional solid-phase hybridization since greater target oligonucleotide adsorption results in faster hybridization. Finally, adsorptive interactions were found to sharpen the melt transition from dsDNA to ssDNA. For mixed base mismatched hybrids, melt temperatures were also found to be elevated by 1-2 °C. It is thought that the adsorptive interactions function to pull the hybrid apart and sharpen the melt transition while also stabilizing
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the mismatches. These effects are not apparent when adsorptive interactions are minimized. The description of QD-oligonucleotide conjugate behavior described herein is expected to be useful in the further advancement of imaging and diagnostic applications. Acknowledgment. We acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support of this work and provision of a fellowship to W.R.A. We also thank Professor Virginijus Barzda, Dr. Arkady
Algar and Krull
Major, and Dr. Paul Piunno for the use of their lifetime instrumentation and facilities. Supporting Information Available: Detailed experimental procedures for conjugate preparation and quantum yield and lifetime measurement, additional discussion on the role of the ionic strength and the adsorption of poly-T, and examples of kinetic curves for adsorption and hybridization. This material is available free of charge via the Internet at http://pubs.acs.org. LA062217Y
