Screening the Sequence Selectivity of DNA-Binding Molecules Using

15 Aug 2007 - We have developed a novel competition assay that uses a gold nanoparticle (Au NP)-based, high-throughput colorimetric approach to screen...
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Anal. Chem. 2007, 79, 7201-7205

Screening the Sequence Selectivity of DNA-Binding Molecules Using a Gold Nanoparticle-Based Colorimetric Approach Sarah J. Hurst, Min Su Han,† Abigail K. R. Lytton-Jean, and Chad A. Mirkin*

Department of Chemistry and International Institute for Nanotechnology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113

We have developed a novel competition assay that uses a gold nanoparticle (Au NP)-based, high-throughput colorimetric approach to screen the sequence selectivity of DNA-binding molecules. This assay hinges on the observation that the melting behavior of DNA-functionalized Au NP aggregates is sensitive to the concentration of the DNA-binding molecule in solution. When short, oligomeric hairpin DNA sequences were added to a reaction solution consisting of DNA-functionalized Au NP aggregates and DNA-binding molecules, these molecules may either bind to the Au NP aggregate interconnects or the hairpin stems based on their relative affinity for each. This relative affinity can be measured as a change in the melting temperature (Tm) of the DNA-modified Au NP aggregates in solution. As a proof of concept, we evaluated the selectivity of 4′,6-diamidino-2-phenylindone (an ATspecific binder), ethidium bromide (a nonspecific binder), and chromomycin A (a GC-specific binder) for six sequences of hairpin DNA having different numbers of AT pairs in a five-base pair variable stem region. Our assay accurately and easily confirmed the known trends in selectivity for the DNA binders in question without the use of complicated instrumentation. This novel assay will be useful in assessing large libraries of potential drug candidates that work by binding DNA to form a drug/DNA complex. Researchers are striving to generate large libraries of DNAbinding molecules through rational design and combinatorial methods.1-5 Such classes of molecules, which include doxorubicin, daunorubicin, and amsacrine, can be used to regulate gene expression and are being developed as anticancer drugs.6 The development of high-throughput assays that can be used to quickly * To whom correspondence should be addressed. E-mail: chadnano@ northwestern.edu. Fax: (+1) 847-467-5123. † Current address: Department of Chemistry, Chung-Ang University, Seoul, Korea, 156-756. (1) Trauger, K. W.; Baird, E. E.; Dervan, P. B. Nature 1996, 382, 559-561. (2) Wemmer, D. E.; Dervan, P. B. Curr. Opin. Struct. Biol. 1997, 7, 355-361. (3) White, S.; Baird, E. E.; Dervan, P. B. Chem. Biol. 1997, 4, 569-578. (4) White, S.; Szewczyk, J. W.; Turner, J. M.; Baird, E. E.; Dervan, P. B. Nature 1998, 391, 468-471. (5) Boger, D. L.; Fink, B. E.; Hedrick, M. P. J. Am. Chem. Soc. 2000, 122, 6382-6394. (6) Yang, X.-L.; Wang, A. H.-J. Pharm. Ther. 1999, 83, 181-215. 10.1021/ac071253e CCC: $37.00 Published on Web 08/15/2007

© 2007 American Chemical Society

and efficiently evaluate these potential drug candidates is critical to progress in this area. Such gold nanoparticle (Au NP)-based methods recently have been developed by our group to screen for DNA duplex- and triplex-binding molecules both in solution and on the surface of a chip.7-9 Once a potential drug candidate is synthesized and its DNAbinding capabilities confirmed, it is then vital to determine the selectivity of its binding for particular sequences of DNA. This information is crucial in targeting specific areas of the genome in certain therapeutic schemes.10-11 Unfortunately, most methods for evaluating the sequence selectivity of libraries of potential drug candidates are often inconvenient, especially for the purposes of large-scale, high-throughput screening. Traditional biological techniques such as foot printing and affinity cleavage have thus far been the most commonly used methods.12 More recently, mass spectroscopy, competition dialysis, NMR, calorimetry, circular dichroism, and X-ray diffraction also have been used.13-17 Only recently has a high-throughput, fluorescence-based method been developed to elucidate the sequence specificity of DNA-binding molecules.18-19 Although useful, this fluorescent intercalator displacement (FID) assay has some weaknesses. First, the FID assay is an onoff system, where a relative decrease rather than an increase in signal is monitored. Second, the fluorescence signal of the reference intercalator (thiazole orange or ethidium bromide, EB) (7) Han, M. S.; Lytton-Jean, A. K. R.; Oh, B.-K.; Heo, J.; Mirkin, C. A. Angew. Chem., Int. Ed. 2006, 45, 1807-1810. (8) Han, M. S.; Lytton-Jean, A. K. R.; Mirkin, C. A. J. Am. Chem. Soc. 2006, 128, 4954-4955. (9) Han, M. S.; Lytton-Jean, A. K. R.; Mirkin, C. A. Anal. Chem. 2007, 79, 6037-6041. (10) Woynarowski, J. M. Biochim. Biophys. Acta 2002, 1587, 300-308. (11) Turner, P. R.; Denny, W. A. Curr. Drug Targets 2000, 1, 1-14. (12) Trauger, J. W.; Baird, E. E.; Mrksich, M.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118, 6160-6166. (13) Wan, K. W.; Shibue, T.; Gross, M. L. J. Am. Chem. Soc. 2000, 122, 300307. (14) Ren, J.; Chaires, J. B. Biochemistry 1999, 38, 16067-16075. (15) Pelton, J. G.; Wemmer, D. E. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 57235727. (16) Rentzeperis, D.; Marky, L. A.; Dwyer, T. J.; Geierstanger, B. H.; Pelton, J. G.; Wemmer, D. E. Biochemistry 1995, 34, 2937-2945. (17) Coll, M.; Frederick, C. A.; Wang, A. H.-J.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 8385-8389. (18) Boger, D. L.; Fink, B. E.; Brunette, S. R.; Tse, W. C.; Hedrick, M. P. J. Am. Chem. Soc. 2001, 123, 5878-5891. (19) Tse, W. C.; Boger, D. L. Acc. Chem. Res. 2004, 37, 61-69.

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Figure 1. (A) Normalized melting curves (monitored at the maximum in Au NP extinction (λ ) 524 nm)) for aggregates of NP-1 and NP-2 for varying concentrations of DAPI between 0 and 10 µM. (B) Corresponding plot of melting temperature (Tm) vs DAPI concentration.

often interferes with that of the DNA-binding molecule of interest.20 Third, the fluorescence intensities of the intercalator/DNA complex are sometimes sensitive to the DNA sequence.21 Last, in the FID method, there is some error associated with the assumption that the reference intercalator has no selectivity for any particular DNA sequence. Although intercalators such as thiazole orange and ethidium bromide are considered nonspecific binders, they in fact have slight preferences for certain sequences of DNA.21,22 Herein, we show that aggregates of DNA-functionalized gold nanoparticle probes can be used to determine the selectivity of a particular DNA-binding molecule for different sequences of hairpin DNA (HP DNA) in a way that offers some advantages over the FID method. This assay is an off-on system, where the increase in the visible signature of the Au NPs is monitored. Critically, this signal does not interfere with the signal from the DNA-binding molecules that are being evaluated. This assay also does not need reference intercalators. In addition, owing to the high extinction coefficient of Au NPs in the visible region, the readout of this nanoparticle-based assay can be analyzed with the naked eye without the need for complicated or expensive instrumentation. MATERIALS AND METHODS Au NPs (13 nm) were synthesized according to literature procedures.23 All oligonucleotides were purchased from Integrated DNA Technologies, Inc. (IDT, Coralville, IA). Dithiothreitol was purchased from Pierce Biotechnology, Inc. (Rockford, IL). DNAbinding molecules and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Melting data were collected on a Cary 500 or Cary 5000 UV-vis spectrophotometer. Au NPs were functionalized with thiolated DNA according to the protocol outlined in ref 24. RESULTS AND DISCUSSION In general, when Au NPs are modified with complementary oligomers (short, synthetic DNA sequences) and mixed, these (20) Haugland, R. P. Handbook of Fluorescent Probes and Research Products; Molecular Probes: Eugene, OR, 2002. (21) Nygren, J.; Svanvik, N.; Kubista, M. Biopolymers 1998, 46, 39-51. (22) Baguley, B. C.; Falkenhaug, E.-M. Nucleic Acids Res. 1978, 5, 161-171. (23) Frens, G. Nat. Phys. Sci. 1973, 241, 20-22. (24) Hurst, S. J.; Lytton-Jean, A. K. R.; Mirkin, C. A. Anal. Chem. 2006, 78, 8313-8318.

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nanoparticles aggregate to form network structures that are held together by multiple duplex DNA linkages.25 Au NP aggregation is characterized by a red-to-blue color transition that is the result of the red shifting and dampening of the nanoparticle surface plasmon resonance band. The aggregation is reversible, and when these aggregates are heated, the DNA links that hold them together dehybridize and the structures break apart. A melting transition for these nanoparticle-DNA structures is typically described in terms of a melting temperature (Tm) and the full width at half-maximum (fwhm) of the first derivative of the transition. The melting transitions of these nanoparticle structures are dramatically sharper (fwhm ∼1-2 °C) than those for the corresponding free DNA duplexes that are not attached to the nanoparticle surfaces (fwhm ∼10-12 °C).26 Here, Au NPs functionalized with complementary DNA-1 (3′ SH A10 AAT AAC AAT 5′) and DNA-2 (3′ SH A10 ATT GTT ATT 5′) (AT-rich sequences) denoted as NP-1 and NP-2, respectively, exhibit this characteristic melting behavior. When DNA-binding molecules, such as 4′,6-diamidino-2-phenylindone (DAPI), are added to solutions containing hybridized aggregates of NP-1 and NP-2 (1.5 nM each, 3.0 nM total Au NP), they can potentially bind to the duplex DNA linking the nanoparticles and stabilize the aggregate structures. These additional binding interactions increase the Tm of the Au NP aggregates (Figure 1). The data show that the nanoparticle Tm is sensitive to the concentration of DAPI in solution, and despite the presence of DAPI, the melting transitions of the nanoparticles remain sharp (∼1-2 °C), which allow for small differences in Tm to be distinguished. We have used the relationship between DAPI concentration and nanoparticle Tm to screen the selectivity of DAPI for six short sequences of hairpin DNA (5′ CG XXXXX CAAAAAG XXXXX CG 3′) (Scheme 1). The sequence of the loop region of these hairpins is held constant, while the 5-base duplex stem region is variable (denoted X), consisting of a different number of GC pairs (0 (all AT)-5 (all GC)). Throughout this note, the HP DNA will be referred according to the number of GC pairs in its variable region (25) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (26) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science, 1997, 277, 1078-1081.

Scheme 1. General Scheme for Screening the Sequence Selectivity of DNA Binding Molecules Using DNA-Functionalized Au NPs

(e.g., 0 GC, 1 GC, etc). When DAPI is added to a solution containing NP-1, NP-2, and the HP DNA, it can bind to either the duplex interconnects between the Au NPs or the duplex stem of the HP DNA depending on its relative affinity for each. The Tm of the nanoparticle aggregates varies as a result of the change in the effective concentration of DAPI in solution. Importantly, the Tm of the HP DNA in the presence of DAPI is much higher than that of the nanoparticle aggregates. Therefore, additional DAPI is not released from its bound state in the stem duplexes as the assay progresses. To initiate the assay, NP-1 and NP-2 each were adjusted to a concentration of 1.5 nM (3 nM total Au NP concentration, using  ) 2.40 × 108 L/(mol‚cm)), in 0.1 M NaCl, 10 mM phosphate buffer (PB) (pH 7). The concentration of DAPI and HP DNA each were brought to 5 µM (1:1 DAPI/HP DNA). After their preparation, the samples were allowed to incubate overnight (∼12 h) at room temperature in order to reach equilibrium. During this time, NP-1 and NP-2 hybridized and formed a polymeric aggregate that settled to the bottom of the cuvette, leaving a colorless supernatant. This incubation time has the potential to be shortened significantly for use in biodiagnostic applications since here the majority of the nanoparticle aggregation was complete within ∼2 h. Next, the samples were heated at a rate of 1.0 °C/min with stirring, while being monitored at the extinction maximum of the dispersed Au NPs (λ ) 524 nm). In this manner, a melting curve for the duplex DNA holding the aggregate together can be obtained (Figure 2). In the control sample, we use a DAPI concentration that leads to the maximum observable shift in Tm (Tm ) 52.9 °C). Above this concentration, the Tm remains constant, presumably because the DNA links are fully loaded with DAPI. The Tm of the nanoparticle aggregates decreases for all cases when HP DNA is added. The magnitude of the decrease, however, depends on the number of GC pairs in the HP duplex stem region. The greatest decrease in Tm is observed for the 0 GC sample (Tm ) 36.1 °C), while the 5 GC sample maintains a Au NP Tm of 51.1 °C (Figure 2, Table 1). DAPI is a known AT-specific, DNA-binding molecule and therefore has a greater affinity for the hairpins with more AT pairs (less GC pairs).14 As a result, as more AT pairs are introduced in the HP stem region, DAPI preferentially binds the HP DNA, and since less DAPI is available to react with the Au NPs, the nanoparticle Tm decreases. Tms separated by typically ∼1-3 °C were observed for HP DNA possessing the same

Table 1. Melting Temperatures (Tm) as a Function of the HP Stem Sequence for the Au NP Samples and for a Control Sample (no NPs) for the DAPI Systema

0 GC 1 GC 2 GC 3 GC 4 GC 5 GC control

stem sequence

NP Tm (°C)

HP Tm (5 µM) (°C)

ATAAT TATTA ATATG TATAC CATTG GTAAC ATCCG TAGGC GCGCT CGCGA CGCGC GCGCG na

36.1 40.3 45.8 48.2 50.7 51.1 52.9

51.0 55.5 64.8 70.7 78.4 85.8 na

HP Tm (with DAPI, 1:1) (°C)

∆ HP Tm (°C)

79.1 75.9 76.0 79.4 81.9 88.8 na

28.1 20.4 11.2 8.7 3.5 3.0 na

a The rightmost column shows the difference between the T of m the HP DNA and its Tm with DAPI in solution. na is denoted where the data in that box are not applicable.

Figure 2. Normalized melting curves for aggregates of NP-1 and NP-2 with 5 µM DAPI and 5 µM HP DNA (0 GC-5 GC, 1:1 HP/DAPI). The curve denoted Control contains no HP DNA.

number of GC base pairs but arranged in a different order (Supporting Information, Table S1). The Tm of the Au NP aggregates also can be affected by the concentration of EB (a nonspecific DNA binder) in solution (Supporting Information, Figure S1).27 However, for any given concentration, the increase in Tm for a sample containing EB is (27) Bailly, C.; Henichart, J.-P.; Colson, P.; Houssier, C. J. Mol. Recognit. 1992, 5, 155-171.

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Figure 3. Digital images of cuvettes containing 0 GC, 1 GC, 2 GC, 3 GC, 4 GC, and 5 GC samples (with DAPI) (1-6, respectively) and a control sample (no HP DNA) (7) over a range of temperatures.

less than that for a sample containing DAPI. When EB was studied with the six HP DNA using the same protocol, the Au NP Tms of all the samples differed by less than 2 °C and followed no particular trend regarding sequence (Supporting Information, Figure S2). The Tm of these samples, however, was lower than that of the control sample containing only NP-1, NP-2, and EB (5 µM). This result supports the conclusion that EB has a relatively nonspecific affinity for any of the DNA sequences studied here.27 In contrast to DAPI and EB, the Tm of the Au NP aggregates is not affected by the concentration of chromomycin A (CA) (a GC-specific binder) in solution (Supporting Information, Figure S3).14 As a result, when CA was investigated with the six HP DNA using the same protocol, the Au NP Tms of all the samples were approximately equal to the sample containing only aggregates of NP-1 and NP-2 (Supporting Information, Figure S4). This result suggests that CA is GC-specific. In order to further interrogate the sequence specificity of a GC-binding molecule, such as CA, the DNA sequences on the Au NPs can be changed from ones that are AT-rich to ones that are GC-rich. Then, the assay would proceed for these types of intercalators in an analogous fashion. Control experiments were performed where the Tm of the free HP DNA was evaluated (at λ ) 260 nm) both with and without the DNA-binding molecules of interest (no Au NPs) (for DAPI (Table 1) and EB (Supporting Information, Table S2)). A larger difference between the Tm of the free HP DNA and the Tm of the 7204

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same HP in the presence of the intercalator suggests a greater affinity of the intercalator for a given HP sequence. The results of these control experiments were well correlated with the trends that we determined from our nanoparticle assay and are consistant with literature trends based upon, for example, competition dialysis and electric linear dichroism.14,27 One additional advantage of this system is that it offers the potential for naked eye detection. For the DAPI system, with the exception of the four GC and five GC samples, which have Tms within 1 °C of each other (and so very similar binding affinities for DAPI), the binding strength of DAPI to a particular sequence of HP DNA can be compared using a temperature gradient (Figure 3). At room temperature, all of the Au NP samples in cuvettes 1-7 are aggregated and exhibit a blue-purple color. As the temperature is raised and the Tm of the aggregates is reached, the aggregates disperse and the samples become bright red in color. At 38 °C, a red color is observed only in cuvette 1 (0 GC sample), which contains the HP DNA for which DAPI has the highest binding affinity. At slightly higher temperatures, the samples in cuvettes 2-4 (1 GC-3 GC samples, respectively) undergo a color change in turn as the binding affinity of DAPI for the HP DNA of interest decreases. The color change for the samples in cuvettes 5 and 6 (4 GC and 5 GC samples, respectively) happens almost simultaneously around 52 °C. The Au NP aggregates in cuvette 7 (no HP DNA) are the most stable and melt at ∼53 °C. Using this simple, naked eye detection method,

CONCLUSION We have demonstrated a powerful method that employs DNAfunctionalized Au NPs to screen the sequence selectivity of DNAbinding molecules. This competition-based assay can differentiate between AT-specific, nonspecific, and GC-specific binding molecules and has several benefits compared to previous systems. (1) It is amenable to high-throughput screening. (2) It is an offon system. (3) It does not use reference intercalators. (4) The measured signal does not interfere with the signal from the DNAbinding molecules that are being evaluated. (5) The readout of this nanoparticle-based assay can be analyzed with the naked eye without the need for complicated or expensive instrumentation, owing to the high extinction coefficient of Au NPs in the visible region of the spectrum. Also, as highlighted in Figure 4, with this method we can directly compare the relative affinity of one intercalator for two different sequences of DNA rather than the affinity of two intercalators (1 reference and 1 sample) for one sequence of DNA, as has been accomplished previously.18,19 It is also possible that this general procedure could be extended and modified to gauge the sequence selectivity of other types of DNA binding molecules. Efforts in this direction are underway.

Figure 4. Comparison of the Au NP-based assay described in this work and the FID assay.

the trend in DAPI binding selectivity for the HP DNA was correctly determined to be 0 GC > 1 GC > 2 GC > 3 GC > 4 GC∼ 5 GC, which is consistent with the control experiments involving instrumental analysis of each HP with DAPI.

ACKNOWLEDGMENT C.A.M. acknowledges a NIH Director’s Pioneer Award, the NCI CCNE program, and the NSF-NSEC for their generous financial support. S.J.H. and M.S.H. contributed equally to this work. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 13, 2007. Accepted July 3, 2007. AC071253E

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