Detection of Single Nucleotide Mismatches via Fluorescent Polymer

Toward a Highly Sensitive Fluorescence Sensing System of an Amphiphilic Molecular ... Cationic Conjugated Polymers for Optical Detection of DNA Methyl...
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Detection of Single Nucleotide Mismatches via Fluorescent Polymer Superquenching† Stuart A. Kushon,‡ Kirsten Bradford,‡ Violeta Marin,§ Chris Suhrada,§,| Bruce A. Armitage,§ Duncan McBranch,‡ and David Whitten*,‡ QTL Biosystems LLC, Santa Fe, New Mexico 87507, Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213-3890, and Department of Chemistry and Biology, University of California, Los Angeles, California 90095 Received February 24, 2003. In Final Form: April 23, 2003 An improved assay for the detection of a 20-mer DNA sequence coding for the Anthrax Lethal Factor sequence that utilizes fluorescence superquenching and peptide nucleic acids (PNAs) is reported. The basis for this assay is a microsphere sensor that is coated with both Neutravidin (a biotin-binding protein) and biotinylated poly(phenylene)ethynylene (a fluorescent conjugated polymer). A 15-mer PNA tethered through its N-terminus to a biotin serves as a capture ligand for DNA oligonucleotides. When mixed, the PNAs and microspheres form a strong complex through the biotin-avidin interaction, creating a sensor for DNA detection. The 20-mer DNA target strand and a 17-mer DNA-QTL (QTL ) quencher tether ligand, where the quencher is a QSY-7 label at the 3′-terminus of the DNA strand) of a similar sequence to the target strand are then used to develop an assay for DNA detection. A sequential addition of target, followed by DNA-QTL, yields a sensitive and selective assay for DNA detection. This study compares PNA to DNA in the ability to perform as a capture ligand, evaluates the importance of assay temperature, and illustrates resolution of single base-pair mismatches using this novel detection platform.

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 smallmolecule energy transfer and electron-transfer quenchers that can associate with the polymers through nonspecific Coulombic and hydrophobic interactions.1-10 This phenomenon 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 sup* Author to whom correspondence should be addressed. † This paper is dedicated to the memory of David O’Brien, a great colleague, mentor, and friend, and is a part of the Langmuir special issue in honor of him. We acknowledge his influence on our research and especially his pioneering work in the use of photochemistry and photophysics for the characterization of supramolecular assemblies. ‡ QTL Biosystems LLC. § Carnegie Mellon University. | IGERT Summer Intern at QTL Biosystems from the University of California. (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. W.; 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, 112, 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 2002, 18, 7706-7713. (8) Tan, C.; Pinto, M. R.; Schanze, K. S. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 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, 132-138.

ported formats permits smaller fluorescent molecules (oligomers or even monomers) 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 (quencher tether ligand, QTL, conjugates) have been developed to achieve rapid and sensitive biosensing based on modulation of the superquenching of these polymers or polymer ensembles in the presence of the target bioagent.1,12-15 Quantitative assays have been demonstrated on the basis of small molecule-protein interactions, protein-lipopolysaccharride association, proteinprotein (antibody-antibody) interactions, and DNA hybridization reactions.1,14,16 These new formats provide particularly attractive opportunities for the detection of DNA through specific hybridization processes.17-20 In our previous work, we illustrated a DNA detection assay that was based on fluorescent polymer superquenching.16 That system used (11) Lu, L.; Helgeson, R.; Jones, R. M.; McBranch, D.; Whitten, D. G. J. Am. Chem. Soc. 2002, 124, 483-488. (12) Jones, R. M.; Lu, L.; Helgeson, R.; Bergstedt, T. S.; McBranch, D. W.; Whitten, D. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1476914772. (13) Jones, R. M.; Bergstedt, T. S.; Buscher, T. C.; McBranch, D.; Whitten, D. Langmuir 2001, 17, 2568-2571. (14) Whitten, D. G.; Chen, L.; Bergstedt, T. S.; Heeger, P.; McBranch, D. Sensors and Optical Switches. In Molecular and Supramolecular Photochemistry; Schanze, K. S., Ramamurthy, M., Eds.; Marcel Dekker: New York, 2001; Vol. 7, pp 189-208. (15) Jones, R. M.; Bergstedt, T. S.; McBranch, D.; Whitten, D. J. Am. Chem. Soc. 2001, 123, 6726-6727. (16) Kushon, S. A.; Ley, K.; Bradford, K.; Jones, R. M.; McBranch, D.; Whitten, D. Langmuir 2002, 18, 7245-7249. (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, 365-370. (19) Keller, G. H.; Manck, M. M. DNA Probes; Stocktonton: New York, 1989. (20) Park, S.-J.; Taton, T. A.; Mirkin, C. A. Science (Washington, D.C., 1883-) 2002, 295, 1503-1506.

10.1021/la034323v CCC: $25.00 © 2003 American Chemical Society Published on Web 05/31/2003

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a carboxylic acid functionalized polystyrene microsphere with streptavidin covalently linked to the surface. The biotin-binding sites on the streptavidin facilitated the simultaneous coating of biotinylated polyphenylene ethynylene (PPE-B) and DNA capture strands. This sensor served as a robust platform because of the strong interactions between biotin and biotin- binding proteins (i.e., avidin, streptavidin, and Neutravidin). The basis for the assay involved the complexation of a biotinylated DNA “capture strand” bearing a sequence complementary to a region of sequence coding for the Anthrax Lethal Factor (ALF) to the surface of the microsphere sensor. Addition of the target strand would not attenuate the fluorescence of the microsphere, whereas addition of a quencher-labeled target strand would reduce the fluorescence of the sensor. In this paper, we present a second general format for microsphere sensors that are capable of employing superquenching for the detection of DNA, and we evaluate the sensor in its ability to discriminate single nucleotide mismatches. Furthermore, we incorporate a peptide nucleic acid (PNA) capture strand into our sensor and compare its sensing properties relative to those of a DNAbased capture strand. PNAs are a class of synthetic oligonucleotides that replace the negatively charged sugar phosphodiester backbone of DNA with an N-(2-aminoethyl)glycine backbone, while retaining the naturally occurring nucleobases for Watson-Crick base pairing recognition.21 Duplexes of PNA and DNA (or RNA) have higher thermal and thermodynamic stabilities than DNADNA or RNA-RNA duplexes.21,22 This is presumably (21) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norde´n, B.; Nielsen, P. E. Nature (London) 1993, 365, 566-568. (22) Ratilainen, T.; Holme´n, A.; Tuite, E.; Haaima, G.; Christensen, L.; Nielsen, P. E.; Norde´n, B. Biochemistry 1998, 37, 12331-12342.

because of the lack of a negative charge on the backbone of the PNA monomer. Furthermore, PNAs are known for being more selective than their DNA or RNA counterparts, and, hence, they are a natural choice for a sensor aiming to discriminate single nucleotide polymorphisms. PNA has been used successfully as a hybridization agent in a number of applications ranging from diagnostics to antisense.23-26 The results presented here show that signal amplification by superquenching can be applied to a PNAbased sensor for DNA that is capable of detecting single nucleotide mismatches within 20 min for subpicomolar amounts of the DNA target. Experimental Section A polyelectrolyte polymer, poly(para-phenylene ethynylene) (PPE), similar to those introduced by Schanze and co-workers at the University of Florida, was used for these studies.8,9 This derivative, containing pendant carboxyl groups, was synthesized by similar procedures and was converted to a biotinylated derivative (PPE-B) by the 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). The polymers were coated onto quaternary ammonium functionalized polystyrene microspheres (MS), 0.55 µm diameter (from Interfacial Dynamics Corporation), by a two-step procedure. In step one, a predetermined amount of PPE-B solution is added to a solution of Neutravidin so that the final ratio of PRUs to biotin-binding protein is 5:1; this solution is incubated under (23) Agrawal, S.; Kandimalla, E. R. Mol. Med. Today 2000, 6, 72-81. (24) Nielsen, P. E. Curr. Opin. Struct. Biol. 1999, 9, 353-357. (25) Doyle, D. F.; Braasch, D. A.; Simmons, C. G.; Janowski, B. A.; Corey, D. R. Biochemistry 2001, 40, 53-64. (26) Wang, G.; Xu, X.; Pace, B.; Dean, D. A.; Glazer, P. M.; Chan, P.; Goodman, S. R.; Shokolenko, I. Nucleic Acids Res. 1999, 27, 28062813.

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Table 1. Thermodynamic Parameters for Duplexes of PNA-Cap and Targets Used in This Study duplexes

-∆G298K (kcal/mole)

-∆H (kcal/mole)

-T∆S298K (kcal/mole)

Tm (°C); Ct ) 4 µM

Tm (°C); Ct ) 30 µM

∆Tm (°C); Ct ) 30 µM vs PNA-ALF

PNA-ALF PNA-TTM PNA-GTM PNA-CTM PNA-DM PNA-QTL

15.4 ( 0.2 13.8 ( 0.2 12.4 ( 0.2 12.3 ( 0.1 10.2 ( 0.1 13.9 ( 0.2

79.3 ( 2.9 76.4 ( 3.0 62.4 ( 2.6 62.3 ( 2.5 48.6 ( 0.7 63.7 ( 2.6

63.8 ( 2.7 62.5 ( 2.9 49.9 ( 2.5 50.0 ( 2.4 38.4 ( 0.7 49.8 ( 2.4

56.2 ( 0.3 49.1 ( 0.2 48.1 ( 0.2 47.2 ( 0.3 38.0 ( 0.1 56.3 ( 0.7

42.6 36.4 31.9 31.0 19.7 41.1

6.2 10.7 11.6 22.9 1.5

Chart 2. Oligonucleotides Used in This Study

ambient conditions for 30 min. In the second step, the polymer/ protein mixture is added to the polystyrene microspheres and incubated for 2 h at pH ) 7, then diafiltered and exchanged into phosphate-buffered saline (PBS). Difference fluorescence spectroscopy was employed to quantify the polymer and protein coating densities. The estimated polymer coating density is 4.75 × 106 PRU/MS, and the estimated protein coating density is 9.5 × 105 Neutravidins/MS for PPE-B. Upon coating of the polymer/ protein mixture onto the surface of the microspheres, the spheres were determined to have ∼1.3 × 105 biotin-binding sites per sphere, as was determined from the binding experiments employing a fluorescein-labeled biotin derivative. DNA oligonucleotides and QTLs (purified by gel filtration chromatography andreverse-phase 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. The PNA probe PNA-Cap (Chart 2) was synthesized according to a standard solid-phase synthesis protocol.27,28 Boc/Z-protected PNA monomers were purchased from Applied Biosystems (http:// www.appliedbiosystems.com). Boc-protected MBHA-lysine resin was used, and the synthesis was performed on a 100 mg resin scale. Biotin was purchased from Molecular Probes, Inc. (www.probes.com) and used as was received. It was coupled onto the PNA amino terminus as the last step in the synthesis using the standard synthesis conditions, except that the coupling was allowed to proceed for 12 h. The PNA oligomer was characterized by reverse-phase HPLC and MALDI-TOF spectrometry (calculated mass 4531.0, observed mass 4529.0) and was used without further purification. Extinction coefficients for PNA monomers were C ) 6600 M-1 cm-1; T ) 8600 M-1 cm-1; and A ) 13 700 M-1 cm-1. Stock solutions of PNA were prepared in 10 mM sodium phosphate (pH ) 7.4), and concentrations were determined using the absorbance at 260 nm and 80 °C employing 260 ) 170 900 M-1 cm-1. All DNA oligonucleotide quench experiments were performed in a GeminiXS plate reader (Molecular Devices, Inc.) in the 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 1 × 107 sensing microspheres in 200 µL of PBS (120 mM NaCl, 2.7 mM KCl, 10 mM NaPi, pH ) 7.4) at 25 °C. All experiments were performed under these conditions, unless otherwise noted. Quench inhibition experiments were performed (27) Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K. H.; Hansen, H. F.; Koch, T.; Egholm, M.; Buchardt, O.; Nielsen, P. E.; Coull, J.; Berg, R. H. J. Pept. Sci. 1995, 3, 175-183. (28) Koch, T. In Peptide Nucleic Acids; Nielsen, P. E., Egholm, M., Eds.; Horizon Scientific Press: Norfolk, U.K., 1999; pp 21-37.

using sensing microspheres coated with 1.2 × 104 capture strands (DNA-Cap or PNA-Cap). The spheres are then incubated for 10 min with variable amounts of the ALF target sequence (0.5, 1, 2, 5, 10, 20, 50, and 100 pmol). Finally, the quench is resolved by the incubation of 10 pmol of DNA-QTL with the microspheres for 10 min. Percent quenching is determined by comparison of the unquenched microspheres versus the quenched microspheres [i.e., -(F - Fo)/Fo]. Error bars are shown as one standard deviation, and in some cases the error bars fall within the size of the data point symbol. Duplexes were prepared in PBS buffer by mixing equimolar amounts (2 µM) of the appropriate strands. The samples were heated at 95 °C and equilibrated for 5 min. UV-visible absorbance at 260 nm was recorded every 0.5 °C as the samples were cooled to 10 °C and then heated again to 95 °C at a rate of 1 °C/min. Experiments were done in triplicate, and the values in Table 1 reflect the averages of those experiments. The thermodynamic parameters were determined from the curve-fitting data from the heating ramps according to the method described by Marky and Breslauer. These were then used to extrapolate the melting temperatures to the lower concentration used for the fluorescence assay (30 nM) by an analysis of the concentration dependence of the oligonucleotide hybrid Tm’s by Marky and Breslauer. A linear relationship between 1/Tm and ln Ct can be plotted for the duplex formation where the slope of the line is R/∆H, where R is the ideal gas constant and the intercept of the line is [∆S R ln(4)]/∆H.29

Results Sensor Design. The sensor is comprised of three components (Scheme 1): 0.55 µm polystyrene microspheres with an ammonium functionalized surface, a biotinylated PPE-B (doping level is 25%), and Neutravidin. The biotinylated PPE is precomplexed with Neutravidin at a 5:1 ratio of PRUs to Neutravidin (an empirically determined ratio that provided the strongest superquenching effect). The PPE-B/Neutravidin complex is then adsorbed to the microsphere surface via mixing in water at pH ) 7 for 1 h followed by washing (via ultrafiltration). Electrostatic interaction of the negatively charged PPE-B with the positively charged microsphere surface results in an intensely fluorescent microsphere sensor. The amount of polymer loaded per microsphere is 4.75 × 106 PRU, which equates to a parking area per PRU of 2 × 102 Å2. Capture Strand Optimization and Loading. A biotinylated PNA (PNA-Cap) or DNA (DNA-Cap) strand is used to capture both the 20-mer ALF target DNA (ALFDNA)30 and a 3′-QSY-7-labeled DNA sequence containing 17 of the 20 bases in the ALF sequence (DNA-QTL). Our earlier study indicated that DNA oligonucleotides of 20 bases in length provided excellent capture performance in our experimental range. However, the DNA capture strand was shown to perform poorly in mismatch discrimination studies (see below). PNAs are known to show improved affinity and selectivity toward natural oligonucleotides.22 Hence, a 15-mer PNA with a biotin tag was (29) Marky, L. A.; Breslauer, K. J. Biopolymers 1987, 26, 16011620. (30) Hutson, R. A.; Duggleby, C. J.; Lowe, J. R.; Manchee, R. J.; Turnbull, P. C. J. Appl. Bacteriol. 1993, 75, 463-472.

Single Nucleotide Mismatches Scheme 1. Sensor Fabricationa

a A 5:1 mixture of Neutravidin and PRUs is complexed. The polymer-protein complex is then deposited on the surface of an ammonium-functionalized microsphere through electrostactic interactions.

prepared to serve as a capture strand in our further mismatch studies. The oligonucleotides used in this study are shown in Chart 2. PNA oligonucleotides hybridize most favorably to DNA oligonucleotides when the N-terminus of the PNA is aligned with the 3′-terminus of the DNA.21 The biotin was placed on the N-terminus of the PNA so that upon hybridization of the PNA capture strand (PNA-Cap) to DNA-QTL the biotin and the QSY-7 quencher would be on the same end of the PNA-DNA duplex. Likewise, the DNA capture strand (DNA-Cap) creates the same orientation with its biotin on the 5′-terminus. With the duplexes in this orientation, the binding of the biotin to the Neutravidin on the surface of the microsphere also orients the QSY-7 toward the surface of the PPE-coated microsphere, and efficient energy transfer is effected. This forms the basis for a DNA detection assay. It is important to determine the proper amount of DNA or PNA capture strands to place on the surface of the microsphere sensor. If the density of the capture strands is too high, entanglement could occur, resulting in a reduction of the strands capable of hybridization. Furthermore, the addition of too much capture strand to a sensor could result in free capture strands that could bind DNA-QTL in solution (as opposed to the surface of a microsphere). This would not lead to energy transfer and ultimately leads to an attenuated sensitivity. To determine the optimal number of capture strands, we utilize 1 × 107 microspheres per well and add a variable amount of the capture strand (PNA or DNA) to the suspension (0-20 pmol; 0-1.2 million capture strands per microsphere). This is followed by the addition of a fixed amount of DNAQTL. Figure 1 indicates that when no PNA capture strand is present, only a very limited amount of quenching is present. However, as the amount of capture strand is increased, a dramatic increase in quenching becomes apparent. This increase peaks at around 2 pmol (120 400 strands per microsphere) and then begins to decrease with increasing amounts of capture strand. Similar results were obtained with the DNA capture strand. With these results

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in mind, the sensors are loaded with 1.2 × 105 capture strands for all of the experiments that follow. Quench Sensitivity. The amount of quencher-labeled DNA to be used in an assay is critical. The hydrophobic nature of the QSY-7 tag on DNA-QTL (Chart 1) can lead to nonspecific interactions that increase with increasing amounts of the quencher. We determine the largest dynamic range of quenching by loading the sensors with an appropriate amount of capture strand and then titrating with DNA-QTL. Scheme 2 (top) depicts the basic sensing strategy. Figure 2 shows representative data for such a titration when a capture strand is present and for a similar titration when no capture strand is present. On the basis of these data, the dynamic range of quenching (the difference between nonspecific and specific quenching) is optimal at either 5 or 10 pmol of DNA-QTL. We opted for 10 pmol of DNA-QTL for the development of the DNA detection assay. DNA Detection and Single Mismatch Detection via a DNA Capture Strand. The procedure for the detection of target DNA is as follows: The microsphere suspension (with the DNA capture strand already bound) is loaded into a series of microwells. The suspension is then presented with variable amounts (0.5, 1, 2, 5, 10, 20, 50, and 100 pmol; 200 µL total volume) of the ALF target DNA and incubated for 10 min at room temperature. The plate is then read to determine an initial fluorescence intensity. Finally, 10 pmol of DNA-QTL is added to the suspension. The plate is incubated at room temperature for 10 min and read. This strategy (shown at the bottom of Scheme 2) allows for a target-dependent fluorescence read out. As the target strands hybridize to the available capture strands, those sites are blocked and DNA-QTL cannot hybridize to the surface, thus creating a “turn-on” assay for target DNA detection. Our previous publication addresses the necessity for the stepwise addition of the target strand and QTL.16 We have devised a series of mismatched target oligonucleotides (Chart 2) that have single and double nucleotide mismatches. The single mismatches (GT-Mis, TTMis, and CT-Mis) were incorporated into the middle of the duplex, whereas the doubly mismatched sequence (2XMis) incorporates the mismatches off center to either end of the duplex. Figure 3 illustrates the performance of the sensor with a DNA-based capture strand in detecting the ALF target and the series of mismatches (inset). The sensitivity of the assay to the perfect complement DNA is ∼500 fmol. However, the selectivity of this assay is inadequate in that only the double mismatch (2X-Mis) and one of the single mismatched targets (GT-Mis) are resolved (albeit poorly) from the perfect complement, whereas the CT-Mis and TT-Mis sequences are not resolved at all. DNA Detection and Single Mismatch Detection via a PNA Capture Strand. The poor resolution of mismatches by DNA-Cap strand required us to use an improved capture strand for the purposes of mismatch detection. A PNA-based capture strand was selected as an agent to improve our mismatch resolution. The PNACap sequence has only 15 bases for hybridization whereas DNA-Cap sequence has 20. We expected, on the basis of the improved thermal stability of PNA-DNA duplexes, that this reduction in length would not reduce the sensitivity of the sensor (i.e., reduce the thermodynamic stability of the hybrids). The PNA-Cap strand was used in place of the DNA-Cap strand to determine the sensor sensitivity to the ALF target and to the mismatched target sequences. The results of the assay are shown in Figure 4. It is obvious from these data that the PNA-Cap strand

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Figure 1. Variable loading of the microsphere sensor with the PNA capture strand (PNA-Cap). A total of 1 × 107 microspheres were loaded with PNA-Cap (1-20 pmol) and exposed to 10 pmol of a complementary DNA with a QSY-7 label (DNA-QTL). Scheme 2

dramatically improves the mismatch resolution of this system. The double mismatch sequence provides no increase in fluorescence whatsoever, indicating a failure to hybridize to the PNA capture strand. However, two of the single base mismatches (GT-Mis and CT-Mis) are resolved poorly, and the TT-Mis sequence is not resolved to any reasonable degree, meaning that under these conditions the assay would not be useful for discriminating single nucleotide polymorphisms. Thermodynamic Analysis. The results of our mismatch detection studies indicate that at room temperature the single mismatched sequences bind to the DNA and PNA capture probes with high affinity, leading to false positive readings in our assay. Nevertheless, the mispaired bases in these duplexes should lead to lower thermal denaturation (i.e., melting) temperatures, meaning that performing the assay at a higher temperature, ideally between the melting temperature of the fully matched duplex and that of the mismatched duplex, would lead to a better discrimination of the mismatched sequences. Therefore, we measured the melting temperatures using the absorbance at 260 nm (Table 1). These results suggest that performing the assay between 49 and 56 °C should permit the selective detection of only the fully matched sequence. However, the concentration at which the melting temperatures are measured (4 µM) is necessarily much higher than that used for the fluorescence assay (30 nM).

To extrapolate the melting temperatures for the lower concentrations, we determined the thermodynamic parameters by curve fitting according to the method of Marky and Breslauer. These extrapolated Tm values are also given in Table 1 and indicate that the assay should be performed between 36 and 42 °C. Note that the double mismatch sequence has a melting temperature of only 20 °C at this concentration, illustrating why virtually no hybridization is detected at room temperature. Improved Mismatch Discrimination. We proceeded to perform our assay at an elevated temperature of 40 °C, using the PNA-Cap strand. Again, the experiments were as follows. The plates were loaded with the microsphere suspension (with 2 pmol of PNA-Cap bound) and incubated within the plate reader at 40 °C. The suspension is then presented with variable amounts (0.5, 1, 2, 5, 10, 20, 50, and 100 pmol) of the ALF target DNA and incubated for 10 min at 40 °C. The plate is then read to determine an initial fluorescence intensity. Finally, 10 pmol of DNAQTL is added to the suspension. The plate is then incubated at 40 °C and read. The results (shown in Figure 5) display good mismatch resolution for all of the target strands with an order of discrimination being 2X-Mis > GT-Mis ) CT-Mis > TTMis. The specificity of this assay is clear (15% differences in signal) down to 500 fmol of the target. Discussion. The microsphere sensor is prepared so that the coating of PPE-B will be approximately one monolayer. This approach has led to the strongest superquenching effects in the materials that we have used to date. A variety of characterization steps are performed on each microsphere batch that is prepared. Our intent is to generate stable microsphere-based sensors that can be used in a variety of environments, from high-throughput screening to the clinical lab. The microspheres are analyzed for biotin-binding site capacity through incubation with biotin-4-fluorescein, filtering through a 0.1 µM membrane, and monitoring the amount of biotin-4-fluorescein in the filtrates via an absorbance measurement. The microspheres used in this study bear 1.3 × 105 biotin-binding sites per microsphere. As a matter of quality control, the microspheres are also incubated under various conditions

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Figure 2. Specific versus nonspecific interactions of DNA-QTL: incubation of the microspheres (no capture strand loaded) and microsphere sensors (3 pmol of DNA-Cap loaded) with variable amounts of DNA-QTL.

Figure 3. Mismatch analysis utilizing a microsphere sensor loaded with a DNA-based capture strand (DNA-Cap).

such as PBS at 70 °C, PBS/10 µM bovine serum albumin, and 90% water/10% DMSO. Analysis indicates that the PPE-Neutravidin complex remains bound under all of these conditions and that there is less than a 5% fluctuation in the fluorescence output of the microsphere sensors. In the preparation of the microsphere sensors, we have found that an optimal loading of capture strands (whether they be PNA or DNA) on the microsphere surface scales roughly with the number of biotin-binding sites on the surface of a microsphere. Our empirical estimate of the most effective number of binding sites per microsphere (for sensing) is 1.2 × 105 (Figure 1), while our estimate of the number of biotin-binding sites available on the

microsphere is 1.3 × 105. Consideration of the radius of gyration of a single-stranded DNA indicates that, at this density, the strands should not intermingle on the surface of the microsphere. The radius of gyration is R ) (2bIo)0.5, where b is the persistence length of a DNA monomer (∼5 Å) and Io is the chain length (i.e., 20-mer).31 In the case of our system, this equates to ∼14 Å, whereas the persistence length of the entire DNA chain is ∼100 Å. On a 0.55 µm microsphere with 1.3 × 105 biotin-binding sites, the parking area of the biotin-binding sites is 7.3 × 102 Å2, the end-to-end distance of which is much larger than (31) Shivashankar, G. V.; Libchaber, A. Curr. Sci. 1999, 76, 813816.

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Figure 4. Mismatch analysis utilizing a microsphere sensor loaded with a PNA-based capture strand (PNA-Cap).

Figure 5. Mismatch analysis at elevated temperatures utilizing a microsphere sensor loaded with a PNA-based capture strand (PNA-Cap). Experiments were performed at 40 °C with a total well volume of 200 µL.

the radius of gyration of the strands that we employ. On the basis of this analysis, it is likely that the number of biotin-binding sites is the single controlling element in determining the optimal number of capture strands to load on a microsphere. The reduction in sensitivity above 1.2 × 105 capture strands per microsphere is probably due to the hybridization of DNA-QTL to the capture strand in solution rather than on the surface of the microspheres, which would not lead to attenuation of the fluorescence of the microspheres. The fact that the reduction in sensitivity is weak (Figure 1; i.e., only 50% at fourfold excess of capture strands in solution) indicates that DNAQTL bears a stronger affinity for the surface-bound capture strands relative to the capture strands in a solution. We postulate that this may be because of an

increase in the DNA-strand hydrophobicity upon the addition of the QSY-7 label. The second critical aspect of assay formulation is the determination of the amount of DNA-QTL to use in the assay. In this case, we aimed at using an amount of quencher that gave us the largest dynamic range of quenching. Figure 2 describes an experiment where we titrate variable amounts of DNA-QTL into both a microsphere loaded with the capture strand and a microsphere with no capture strand loaded. Titration up to ∼10 pmol of DNA-QTL indicates that the specific quench signal increases relative to the nonspecific signal. Above this level, the specific quenching does not improve with added QTL, and any increases in the quench signal are due to nonspecific interactions of DNA-QTL with the micro-

Single Nucleotide Mismatches

sphere surface. If we aim to use this as a “turn-on” style assay, the background signal is the signal that is not quenched by the addition of DNA-QTL. Hence, the addition of large amounts of DNA-QTL may improve our signal-to-noise ratio by reducing the background signal, even though quenching due to nonspecific interactions cannot be recovered. In our current system, the dynamic range of quenching is ∼50% of the total fluorescence of the microsphere with a nonspecific background of 15% quenching. In our previous paper, we reported somewhat smaller nonspecific quenching using a COOH-modified polystyrene microsphere, which bears an overall negative charge at pH ) 7. The current formulation of microspheres is likely to bear a slight positive charge as the result of an NR4+ group parking area of 95 Å2 on the surface of the microsphere. If this is the case, it accounts for the increase in the nonspecific interactions with the negatively charged DNA-QTL. The capacity to generate sensors that bear different overall charges may provide us with versatility in the production of other assays for biological targets.32 The DNA detection assay that we have devised occurs as a two-step analysis via a plate reader. The microsphere sensor is incubated with the DNA-Cap strand prior to experimentation. A suspension of microsphere sensors is loaded into a 96-well plate, and variable amounts of the target are added to the suspension and allowed to incubate for 10 min. The initial fluorescence of the suspension is read via a plate reader, and DNA-QTL is then added and incubated for 10 min to allow for signal resolution. The targets that we have used for this assay are an ALF sequence, three single and one double mismatch of this sequence. Our aim is to generate a simple assay that will allow for the detection of single nucleotide polymorphisms in the shortest period of time, with moderate sensitivity. Our initial results (Figure 3) using a DNA-based capture strand indicate the poor mismatch resolution that is obtained using a DNA-based capture strand under the conditions of room temperature. Only the GT-Mis and the 2X-Mis sequences are resolved from the perfect complement in any way. However, both materials exhibit a quench-recovery effect and, hence, must be binding to the capture probe at the experimental temperature. As a result of this poor resolution, we opted to use a PNA capture strand. PNAs are well-known for their improved affinity and selectivity relative to naturally occurring oligonucleotides. Initial studies on mismatches in PNA-DNA duplexes by Egholm et al. indicate that a single mismatch in the DNA sequence can result in destabilizations on the order of 8-20 °C relative to a perfectly complementary DNA.21 In addition, studies in the use of PNA as an antisense reagent by Doyle et al. indicate that two mismatches in a PNA sequence can prevent any PNA-DNA hybridization with 15-18-mer PNAs.25 It was our hope that the use of a biotinylated PNA-based capture strand would improve our capacity to detect single base mismatches in DNA sequences. We synthesized and used a biotinylated 15-mer PNA for this purpose (PNA-Cap). Figure 4 depicts the result of changing a 20-mer DNA capture strand to a 15-mer PNAbased capture strand. It is clear that the mismatch (32) Preliminary results using a variety of microspheres bearing different overall surface charges have indicated that the nonspecific interactions of DNA-QTL increases with increasing positive charge and are essentially absent in the presence of negatively charged microspheres. Furthermore, the nonspecific response generated by a microsphere surface is dominated by the charge of the polymer that is coated onto the microsphere surface. For example, all the spheres that we have coated with a positively charged polymer exhibit strong nonspecific binding with DNA regardless of the overall charge of the microsphere/polymer complex.

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resolution improves significantly. The double mismatch sequence is now completely resolved, but all of the single mismatch sequences show quench recovery. At 10 pmol of the target, there is a 10% difference in the signal recovery of the perfect complement and all three single mismatch sequences. Although this result indicates resolution of all of the mismatches, we decided that a thermodynamic analysis may allow for further improvements. Our thermodynamic analysis of the system was aimed at indicating an appropriate temperature for the analysis of single base mismatches in this system. It is known that mismatches in a DNA duplex cause reductions in the thermal stability of the duplex being formed. However, if the reduction in the thermal stability does not reduce the melting temperature below the ambient level, the duplex can still be formed. Hence, in the analysis of mismatches it may be useful to perform the analysis at a temperature where the perfect complement is stable and the mismatches are unstable. Table 1 shows the results of the melting curve analysis for the set of duplexes formed by the PNA-Cap strand and the target and mismatches used in this study. The order of the mismatch stability is TTMis > GT-Mis > CT-Mis > 2X-Mis. This indicates that the most difficult mismatch to detect in this system is the TT-Mis sequence, whereas the 2X-Mis will be the easiest to resolve. This is born out of the order of resolution at room temperature indicated in Figure 4. The melting curve analysis provides us with information concerning the bimolecular interaction of the PNA-Cap strand with the series of targets at a concentration that is much higher than the conditions that our assay probes. As a result, the melting temperatures that we should observe in our system will be somewhat lower than those observed at a total strand concentration of 4 µM. Using the thermodynamic parameters that we have obtained, we extrapolate the melting temperatures of our hybrids to near our working concentrations and find that an operational temperature below 41.1 °C (the calculated melting temperature of the DNA-QTL/PNA-Cap hybrid) and above 36.4 °C (the calculated melting temperature of the TT-Mis/PNA-Cap hybrid) is required for improved mismatch resolution. An experimental scan of that temperature range provides us with the empirical result that 40 °C is an improved temperature for mismatch analysis (Figure 5). The increase in temperature allows us to detect all four mismatches with ∼15% differences in the total signal at 500 fmol within 20 min. The results obtained in this study indicate that polymer superquenching can form the basis for a simple, rapid, sensitive, and selective assay for DNA detection and mismatch analysis using a standard plate reader for detection. It is important to note that a variety of other excellent detection systems for DNA have recently been presented that indicate sensitivity limits between 10 pM and single-molecule analyses.33-36 However, in comparing these approaches with the present assay a number of considerations must be made. First, some of these techniques require complex instrumentation, such as confocal fluorescence microscopes, for detection.33 Our system uses a simple plate reader. Additionally, we have opted to use a commonly used buffer (PBS) for the (33) Osourne, M. A.; Furey, W. S.; Klenerman, D.; Balasubramanian, S. Anal. Chem. 2000, 72, 3678-3681. (34) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954-10957. (35) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science (Washington, D.C., 1883-) 2000, 289, 1757-1760. (36) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science (Washington, D.C., 1883-) 2002, 297, 1536-1540.

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simulation of a biologically relevant environment, where others use water34 or extremely high-salt buffers (1.2 M NaCl)35 to improve the thermodynamics of the hybridization reactions that they study (PNA-DNA interactions are favored by low-salt conditions, whereas DNA-DNA interactions are favored by high-salt conditions). Finally, our system requires only the hybridization of the target strand or DNA-QTL for a signal to be resolved, whereas others require a post-hybridization resolution of the signal, yielding a time benefit for our assay.35 We have explored the versatility of new sensor formats, and our new understanding of the overall sensor charge on nonspecific interactions will allow for the generation of improved sensors for this application and others. This assay offers

Kushon et al.

comparable or improved sensitivity to other conventional fluorescence-based assays for DNA,17,37,38 which do not involve amplification by a polymerase chain reaction, and also allows for the rapid detection of single nucleotide mismatches. Acknowledgment. This work was supported by the Defense Advanced Research Projects Agency under Contract No. MDA972-00-C-006. We thank Dr. Kevin Ley for the synthesis of PPE-B. LA034323V (37) Dudley, A. M.; Aach, J.; Steffen, M. A.; Church, G. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7554-7559. (38) Dueymes, C.; De´cout, J.; Peltie´, P.; Fontecave, M. Angew. Chem., Int. Ed. 2002, 41, 486-489.