© Copyright 2002 American Chemical Society
OCTOBER 1, 2002 VOLUME 18, NUMBER 20
Letters Detection of DNA Hybridization via Fluorescent Polymer Superquenching Stuart A. Kushon, Kevin D. Ley, Kirsten Bradford, Robert M. Jones, Duncan McBranch, and David Whitten* QTL Biosystems, LLC, Santa Fe, New Mexico 87507 Received July 9, 2002. In Final Form: August 6, 2002 An assay for a target single strand 20-base sequence of DNA coding for the anthrax lethal factor, based on conjugated polymer fluorescence superquenching, is reported. The assay employs a platform in which the receptor (a biotinylated complementary sequence “capture strand”) and polymer (two components: an anionic poly(phenylene ethynylene) (PPE) and a biotinylated -PPE) are co-located on streptavidinderivatized polystyrene microspheres. A conjugate of the target strand with the energy transfer quencher QSY-7 (DNA-QTL) is used to construct competition assays for the target. A direct competition assay between the target-DNA and DNA-QTL for the microsphere-bound capture is only marginally successful due evidently to greater kinetic affinity of the polymer-capture ensemble for the conjugate. However a sequential addition of target, followed by DNA-QTL affords a quantitative assay for the target by attenuation of PPE fluorescence quenching by the DNA-QTL. Likewise a direct competition in solution between the target and DNA-QTL for the biotinylated capture strand followed by addition of microspheres provides a sensitive and quantitative assay for the target single strand DNA.
Introduction Several recent studies have shown that certain classes of fluorescent polymers (including conjugated polyelectrolytes and J-aggregated cyanine pendant poly-L-lysine derivatives) are highly sensitive to quenching by small molecule energy transfer and electron-transfer quenchers which can associate with the polymers through nonspecific Coulombic and hydrophobic interactions.1-10 This phe(1) Chen, L.; McBranch, D. W.; Wang, H.-L.; Helgeson, R.; Wudl, F.; Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12287-12292. (2) Chen, L.; McBranch, D.; Wang, R.; Whitten, D. Chem. Phys. Lett. 2000, 330, 27-33. (3) Chen, L.; Xu, S.; McBranch, D.; Whitten, D. J. Am. Chem. Soc. 2000, 122, 9302-9303. (4) Wang, J.; Wang, D.; Miller, E. K.; Moses, D.; Bazan, G. C.; Heeger, A. J. Macromolecules 2000, 33, 5153-5158. (5) Harrison, B. S.; Ramey, M. B.; Reynolds, J. R.; Schanze, K. S. J. Am. Chem. Soc. 2000, 122, 8561-8562. (6) Zhou, Q.; Swager, T. M. J. Am. Chem. Soc. 1995, 117, 1259312602. (7) Lu, L.; Jones, R. M.; McBranch, D.; Whitten, D. Langmuir, in press.
nomenon of superquenching can also occur when the polymers and quenchers are collected on a surface, including supports such as microspheres or nanoparticles. Recent studies have demonstrated that the use of supported formats permits smaller molecules (oligomers or even monomer) to self-assemble into aggregates or “polymer ensembles” that exhibit superquenching equivalent to or even enhanced compared to individual polymer molecules.7,11,12 Conjugates consisting of a quencher linked to a ligand for a specific bioagent (QTL conjugates) have (8) Tan, C.; Pinto, M. R.; Schanze, K. S. Polym. Prepr. 2002, 42, 126-127. (9) Tan, C.; Pinto, M. R.; Schanze, K. S. Chem. Commun. 2002, 446447. (10) List, E. W. J.; Creely, C.; Leising, G.; Schulte, N.; Schlueter, A. D.; Scherf, U.; Muellen, K.; Graupner, W. Chem. Phys. Lett. 2000, 325, 132. (11) Lu, L.; Helgeson, R.; Jones, R. W.; McBranch, D.; Whitten, D. F. J. Am. Chem. Soc. 2002, 124, 483-488. (12) Jones, R. M.; Lu, L.; Helgeson, R.; Bergstedt, T. W.; McBranch, D. W.; Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A. 2001, 26, 1476914772.
10.1021/la026211u CCC: $22.00 © 2002 American Chemical Society Published on Web 09/04/2002
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been developed to achieve rapid and sensitive biosensing based on modulation of superquenching of these polymers or polymer ensembles in the presence of the target bioagent.1,12-15 Quantitative assays have been demonstrated based on small molecule-protein interactions, protein-lipopolysaccharride association, and proteinprotein (antibody-antibody) interactions.1,14,16 In previous examples of biosensing based on fluorescent polymer superquenching, the initial fluorescence quenching has been dependent on nonspecific association between the fluorescent polymer and the QTL bioconjugate. In this work we report an improved assay format obtained by co-locating the polymer (or polymer ensemble) and a bioreceptor onto the same support. Herein we employ streptavidin-functionalized polymer microspheres that can serve as a support for anchoring either anionic conjugated polymers (or other anionic polyelectrolytes) by adsorption or for linking biotinylated reagents, including biotinylated polymers. We have also found that polymers and receptors anchored on these spheres afford more robust assay platforms especially for studies in complex media.16 We have used this assay platform for competition assays involving turn-off or turn-on modes of fluorescence quenching. (13) Jones, R. M.; Bergstedt, T. S.; Buscher, C. T.; McBranch, D.; Whitten, D. Langmuir 2001, 17, 2568-2571. (14) Whitten, D.; Chen, L.; Jones, R.; Bergstedt, T.; Heeger, P.; McBranch, D. In Optical Sensors and Switches; Schanze, K. S., Ramamurthy, V., Eds.; Molecular and Supramolecular Photochemistry 7; Marcel Dekker: New York, 2001; Chapter 4, pp 189-208. (15) Jones, R. M.; Bergstedt, T. A.; McBranch, D.; Whitten, D. J. Am. Chem. Soc. 2001, 123, 6726-7. (16) Jones, R. M.; et al. Unpublished results.
These new formats provide particularly attractive opportunities for detection of DNA through specific hybridization processes.17-20 For example, in solution cationic fluorescent polymers associate strongly with single- or double-stranded DNA in processes that can result in strong modification of their fluorescence that render it difficult to separate specific hybridization from nonspecific interactions.21 Conversely, anionic conjugated polymers may either not associate with DNA or interact via hydrophobic interactions resulting in similar large “background effects” that hinder effective sensing.21 In the present Letter we report extension of these new platforms to the detection of single-stranded DNA. The results reported here indicate that polymer superquenching may provide the basis of improved assays for singleand double-stranded DNA that may be used either as a stand-alone assay or in conjunction with other assay procedures employing target amplification such as polymerase chain reaction. Experimental Section A sensing polyelectrolyte polymer, poly(p-phenylene ethynylene) (PPE), was supplied by K. S. Schanze and co-workers at the University of Florida.8,9 A similar PPE derivative, containing (17) Bonnet, G.; Tyagi, S.; Libchaber, A.; Kramer, F. R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6171-6176. (18) Dubertret, B.; Calame, M.; Libchaber, A. J. Nat. Biotechnol. 2001, 19, 365-70. (19) Keller, G. H.; Manck, M. M. “DNA Probes”; Stocktonton, NY, 1989. (20) Park, S.-J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 15031506. (21) Wang, D.; Rininsland, F.; Yu, W.; Heeger, P. S.; Bazan, G. C.; Whitten, D. G.; McBranch, D. W.; Heeger, A. J. Unpublished manuscript.
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Langmuir, Vol. 18, No. 20, 2002 7247 Scheme 1. Three of the Assays Described in the Texta
a Key: (A) A quenching assay where DNA-QTL is presented to MS and ALF-capture strand. Quenching is effected upon specific recognition of the DNA-QTL and the ALF-capture strand. (B) The “stepwise” assay for detection of target DNA. The MS are prepared with ALF-capture strand on the surface, the DNA-MS mixture is then mixed with variable amounts of ALF-target. After 10 min of hybridization, the ALF-capture strands on the surface of the MS are hybridized to the ALF-target. A fixed amount of DNA-QTL is then added and quenching occurs. The strength of this quenching is dependent on the amount of ALF-target that is bound. (C) The solution phase direct competition assay presents a fixed amount of DNA-QTL and variable amounts ALF-target to the ALF-capture strand in solution. The strands are allowed to hybridize for 10 min. The reaction mixture is then mixed with PPE-coated MS. The quenching that is observed is dependent on the amount of ALF-target strand that is present.
pendant carboxyl groups, was synthesized by similar procedures and was converted to a biotinylated derivative (PPE-B) by formation of amide linkages between the carboxylic acid and biotin. (See Chart 1.) NMR measurements confirmed the incorporation of biotin at a level of one per every four polymer repeat units (PRU). Polymers were coated onto streptavidin-derivatized polystyrene microspheres (MS), 0.46 µm diameter (from Bangs Laboratories), by a two-step procedure. In step one, a predetermined amount of PPE solution is added to a rapidly stirring pH 4 suspension of MS contained in a ultrafiltration cell fitted with a 0.1 µm polyvinylidiene fluoride membrane. After a 2 h incubation under ambient conditions, the adsorptively coated PPE-MS are then exhaustively diafiltered to remove any unbound polymer while simultaneously raising the pH to 7. In the second step, a defined amount of PPE-B is added to the PPEMS suspension, incubated for 2 h, and then diafiltered and exchanged into phosphate-buffered saline. Difference fluorescence spectroscopy was employed to quantify the polymer coating densities. Estimated polymer coating densities are 380 000 PRU/ MS for PPE and 83 000 PRU/MS for PPE-B. Prior to polymer over-coating, the MS possessed approximately 68 000 biotin binding sites (BBS) per MS. After coating this value dropped to about 30 000 BBS per MS, as determined from binding experiments employing a fluorescein-labeled biotin derivative.
DNA oligonucleotides and QTLs (purified by gel filtration chromatography and/or RP-HPLC) were purchased from Integrated DNA Technologies, Inc. (www.idtdna.com), and Synthegen (www.synthegen.com) and were used without further purification. Extinction coefficients for the oligonucleotides were provided by the manufacturer, and concentrations were determined on a Cary 100 Bio spectrophotometer via absorption at 260 nm. All DNA oligo quench experiments were performed in a GeminiXS plate reader (Molecular Devices, Inc.) in well scan mode at 420 nm excitation and 530 nm emission employing a 475 nm emission cutoff filter. Measurements were conducted in white polystyrene 96-well plates (Packard Biosciences Corp.) containing 3.7 × 107 microspheres in 200 µL of PBS. All experiments were performed in phosphate-buffered saline (PBS, 120 mM NaCl, 2.7 mM KCl, 10 mM NaPi).
Results and Discussion For initial studies to develop a DNA assay based on fluorescent polymer superquenching, we selected as a target analyte a 20-base sequence coding for the anthrax lethal factor (ALF-target) (Chart 1).22 A complementary (22) Hutson, R. A.; Duggleby, C. J.; Lowe, J. R.; Manchee, R. J.; Turnbull, P. C. J. Appl. Bacteriol. 1993, 75, 463-472.
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Figure 1. Stern-Volmer plot for specific quenching of the fluorescence of 1.4 × 10-7 M (repeat units) of PPE (or PPE-B) supported on latex microspheres by the hybridization of a 17mer DNA-QTL with ALF-capture oligonucleotides. Variable amounts of capture strand (indicated by open symbols) were presented to the microspheres followed by incubation with QTL. All experiments were performed at 25 °C in a 96-well plate.
sequence capture strand was constructed with biotin tethered at the 5′ end (biotinylated capture strand). Derivatives of the ALF target tethered at the 3′ terminal with the nonfluorescent, energy accepting quencher QSY-7 (Molecular Probes) (Chart 1) were selected as the initial QTL conjugates (DNA-QTL).23 Polystyrene microspheres containing streptavidin covalently coated on their surface were coated sequentially with two different polymer samples (Chart 1) as described above. As shown in Figure 1, addition of DNA-QTL to microspheres containing the polymer and the capture strand results in quenching of the polymer fluorescence. The quenching at low concentrations of capture strand becomes saturated at 20 pmol of DNA-QTL (1 × 10-7 M); from the results at low conjugate concentration, a minimum value for the Stern-Volmer quenching constant (KSV) of 2.1 × 107 M-1 can be extracted. This compares favorably with quenching constants obtained for polymer-bound beads in other assays using peptide, protein, and small-molecule based conjugates.15,16 In general, increasing the concentration of microspheres increases the dynamic range but reduces the KSV. Figure 2 shows the quenching observed in several control experiments. For example, when beads containing the same polymer composition but no biotinylated capture strand are used as the substrate, addition of DNA-QTL results only in a small amount of quenching (3-5%). Interestingly, addition of quencher QSY-7 by itself to the same polymer-bead composition does result in quenching of the polymer fluorescence. Free QSY-7 quenching most likely results as a consequence of hydrophobic association of the quencher with the polymer-bead interface. For the DNA-QTL conjugate, favorable hydrophobic interactions are evidently more than offset by the unfavorable Coulombic forces between the net negative charge on the beads and on the DNA-QTL such that little net association occurs. Similarly, as shown in Figure 2, the substitution of noncomplementary biotinylated capture strands on the same polymer-bead composition results in insignificant fluorescence quenching when the DNA-QTL is introduced. The first approach to DNA detection using the platform described above was a direct competition assay using the fluorescent polymer-biotinylated capture strand beads (23) Both 17- and 20-base sequence DNA-QTLs have been used in the present experiments: the two different length conjugates give very similar results as compared in Figure 1. The data in Figures 2 and 3 are for the 17-base DNA-QTL.
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Figure 2. Quenching of PPE fluorescence by 20 pmol of various oligonucleotides and mixtures of oligonucleotides. Minimal quenching is observed due to nonspecific interactions of the DNA-QTLs and the microsphere surfaces. The specific interaction of the DNA-QTLs and the ALF-capture strand result in significant quenching above that of the nonspecific quenching. Noncomplementary DNA oligonucleotides are denoted (NC). All experiments were performed at 25 °C in a 96-well plate (200 µL Vt per well).
Figure 3. Quench inhibition of DNA-QTL by ALF-target strands: (4) stepwise addition of ALF-target to the microsphere suspension followed by the addition of DNA-QTL; (O, and inset) direct competition of ALF-target and DNA-QTL for hybridization to LF-capture strand in solution, followed by addition of the hybridization mixture to the microsphere suspension; (0) direct competition of DNA-QTL and ALF-target for hybridization to ALF-capture strand in the presence of the microsphere suspension. All experiments were performed in a 96-well plate (200 µL Vt) at 25 °C with 3.7 × 107 microspheres, 3 pmol of ALF-capture strand, 10 pmol of DNA-QTL (17-mer), and variable amounts of ALF-target.
suspended in PBS solution at pH ) 7.0 with direct addition of a mixture of ALF-target and DNA-QTL at 25 °C. Results from a typical set of experiments are shown in Figure 3. In these experiments the amount of DNA-QTL was kept constant (5 × 10-8 M) and the concentration of the ALFtarget was varied. At this temperature it would be anticipated that the hybridization of the capture and target strands would be under kinetic control and that the “off rate” of a 15-20-base strand from the duplex would be too slow to permit equilibration.24-27 If hybridization rates for the ALF-target and DNA-QTL with the capture strand (24) Nelson, J. W.; Tinoco, I., Jr. Biochemistry 1982, 21, 5289-5295. (25) Williams, A. P.; Longfellow, C. E.; Freier, S. M.; Kierzek, R.; Turner, D. H. Biochemistry 1989, 28, 4283-4291. (26) Turner, D. H.; Sugimoto, N.; Freier, S. M. In Nucleic Acids; Saenger, W., Ed.; Springer-Verlag: Berlin, 1990; Vol. C, pp 201-227. (27) Wang, S.; Frieedman, A. E.; Kool, E. T. Biochemistry 1995, 34, 9774-9784.
Letters
were comparable, it would be anticipated that addition of ALF-target should suppress the quenching induced by hybridization of the DNA-QTL with the capture strand. In fact, for the direct competition assay with MS carried out in this manner, relatively little attenuation of quenching by the DNA-QTL is observed. A possible reason for the limited attenuation of quenching may be that the hydrophobic QSY-7 appended on the DNA-QTL may enhance association of the conjugate with the receptorpolymer ensemble and thus bias the competition in favor of the DNA-QTL. Since the “off rate” of target QTL from the duplex should be slow, a sequential or stepwise addition of ALF-target to the capture ensemble followed by addition of DNAQTL to hybridize with “free” capture strand sites on the beads was employed to afford a viable assay. The experiment involved addition of ALF-target to a suspension of beads containing the biotinylated capture strand, followed by a 10 min incubation period. DNA-QTL was then added, followed by a second 10 min incubation prior to measurement of the fluorescence. All steps were performed at 25 °C. The fluorescence of the resulting suspension did not change over a period of 24 h, indicating that there is little or no equilibration between between duplex and single strands. As shown in Figure 3, this procedure results in attenuation of the quenching of the DNA-QTL, affording a quantitative correlation of the amount of ALF-target with the degree of quenching. The quench recovery obtained at 500 fmol of ALF-target is ∼3.5% of the fluorescence of the MS suspension. If, as suggested above, the only marginal attenuation of quenching by ALF-target in the MS-based direct competition assay is attributable to QSY-7-polymer hydrophobic effects on the surface of the beads, it should be possible to construct an assay by carrying out a competitive hybridization in solution. In this case, the duplexes formed in competition for the biotinylated capture strand can subsequently attach to beads containing the fluorescent polymer via biotin-avidin association. The level of
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fluorescence quenching should again provide a quantitative indication of the amount of ALF-target. In this direct competition (solution) assay, biotinylated capture strand is incubated with mixtures containing a constant amount of DNA-QTL and variable amounts of ALF-target. After 10 min of incubation, the mixture is added to the polymer beads and the fluorescence intensity is measured. As shown in Figure 3, the level of quenching decreases with increasing concentration of ALF-target providing comparable results and sensitivity to those obtained in the stepwise assay (Figure 3 inset). The results obtained in these initial studies indicate that polymer superquenching can form the basis for simple, rapid, specific, and sensitive DNA assays. These assays offer improved or comparable sensitivity relative to other fluorescence28,29 or fluorescence resonance energy transfer17 based assays for DNA that do not involve amplification by polymerase chain reaction. The results also point to a number of potential improvements and new possibilities for assay development. The identification of quenchers that exhibit lower tendencies to associate with the fluorescent polymer-bead should enable the generation of conjugates that can be used in a direct competition assay. The wide range of tunability of conjugate and capture strand binding affinities can also provide versatility in assay format, sensitivity, and accessible target DNA. Acknowledgment. This work was supported by the Defense Advanced Research Projects Agency under Contract #MDA972-00-C-006. We thank Professor Kirk Schanze for supplies of PPE and Professor Bruce Armitage for helpful suggestions and discussions. LA026211U (28) Dudley, A. M.; Aach, J.; Steffen, M. A.; Church, G. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7554-7559. (29) Dueymes, C.; De´cout, J.; Peltie´, P.; Fontecave, M. Angew. Chem., Int. Ed. 2002, 41, 486-489.
