Anal. Chem. 2008, 80, 3342-3349
Raman Multiplexers for Alternative Gene Splicing Lan Sun, Chenxu Yu,† and Joseph Irudayaraj*
Department of Agricultural and Biological Engineering and Bindley Bioscience Center, Purdue University, 225 South University Street, West Lafayette, Indiana 47907
Nonfluorescent labels were used for an array-format multiplex detection of alternative splice junctions of breast cancer susceptibility gene 1 (BRCA1) by surfaceenhanced Raman scattering (SERS). A four-plex detection scheme using nonfluorescent labels was demonstrated for DNA sequences specific to four BRCA1 alternative splice variants: ∆(11q) (the last 3309 nt deleted from exon 11), ∆(9, 10) (exon 9 and 10 deleted), ∆(5) (exon 5 deleted), and ∆(5q, 6) (the last 22 nt of exon 5 and the entire exon 6 is deleted). This is the first proof-of-concept study to apply SERS-based detection using nonfluorescent labels to investigate alternative gene splicing. Detection sensitivity of up to 1 fM was demonstrated for the Raman labels chosen to clearly discriminate the splice junctions via specific target identification. The proposed approach has the potential to become a highly sensitive and selective tool for comprehensive alternative splicing profiling of BRCA1 or other genes relevant to specific diseases. Breast cancer is the most frequently diagnosed (26%) and the second most common fatal cancer in women (15%) according to Cancer Statistics 2007 presented by American Cancer Society. Breast cancer susceptibility gene 1 (BRCA1) was noted as one of the two major genes that cause a genetic susceptibility to breast cancer. Pre-mRNA splicing has been observed to be altered in various types of cancer.1,2 Changes in alternative splicing profile of BRCA1 gene has already been associated with malignant transformation in breast cancer studies.3,4 However, due to the vast number of alternative splice variants and tissue specific5 and cell type specific6 pattern of the relative expression levels of these variants, it is still difficult to use alternative splicing profiling as a diagnostic tool for breast cancer. To fully understand the role of genes such as BRCA1 in tumor development in relation to alternative splicing patterns, sensitive schemes with a high degree * To whom correspondence should be addressed. E-mail:
[email protected]. Phone: 765-494-0388. Fax: 765-496-1115. † Current address: Department of Agricultural and Biosystems Engineering, 206 Davidson Hall, Iowa State University, Ames, IA 50011. (1) Caballero, O. L.; Souza, S. J. d.; Brentani, R. R.; Simpson, A. J. G. Dis. Markers 2001, 17, 67-75. (2) Baudry, D.; Faussillon, M.; Cabanis, M.-O.; Rigolet, M.; Zucker, J.-M.; Patte, C.; Sarnacki, S.; Boccon-Gibod, L.; Junien, C.; Jeanpierre, C. Oncogene 2002, 21, 5566-5573. (3) Orban, T. I.; Olah, E. Biochem. Biophys. Res. Commun. 2001, 280, 32-38. (4) Mixon, M.; Kittrell, F.; Medina, D. Oncogene 2000, 19, 5237-5243. (5) Fortin, J.; Moisan, A.-M.; Dumont, M.; Leblanc, G.; Labrie, Y.; Durocher, F.; Bessette, P.; Bridge, P.; Chiquette, J.; Laframboise, R. Biochim. Biophys. Acta 2005, 1731, 57-65. (6) Favy, D. A.; Lafarge, S.; Rio, P.; Vissac, C.; Bignon, Y.-J.; Bernard-Gallon, D. Biochem. Biophys. Res. Commun. 2000, 274, 73-78.
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of multiplexing capability need to be devised so that alternative splicing profiles of normal and tumorigenic cells across all tissues, cell types, disease states, and stages of development could be assessed. Although sequencing of full-length cDNA is the most reliable technique for alternative splicing study,7 it is labor-intensive and expensive. Alternatively, bioinformatic approaches have been applied to identify a vast number of alternative splice variants; however, the identified variants still require further experimental verification. Conventional northern blots and reverse transcription polymerase chain reaction (RT-PCR) methodologies can be applied for alternative splicing investigation, but these methods are not convenient for high-throughput multiplexing. Although multiprobe assays can be performed by a nuclease protection assay (NPA), the lack of information on transcript size and probe flexibility and safety issues due to autoradiographic visualization pose constraints. Oligonucleotide-based microarrays have been developed to provide alternative splicing data on a large scale.8,9 However, data accuracy needs to be improved before widespread adoption for disease diagnostics. Although few recent methodologies have been reported to monitor multiple alternative splicing events, such as polymerase colony (polony) technology10 and fiber-optic arrays,11 tedious iterative schemes are required in some phase of the study. In general, the present experimental methods to investigate alternative splicing are limited in their multiplexing capability, which is critical to providing high-density information with minimal assay time, sample volume, and cost. Among the current multiplex detection methods for nucleic acids involving gene chip technology12,13 and several nanostructure-based platforms, such as luminescent quantum dots,14,15 multimetal microrods intrinsically encoded with submicrometer stripes,16 and self-assembled DNA nanotiles carrying nucleic acid (7) Kalnina, Z.; Zayakin, P.; Silina, K.; Linej, A. Genes, Chromosomes Cancer 2005, 42, 342-357. (8) Hu, G. k.; Madore, S. J.; Moldover, B.; Jatkoe, T.; Balaban, D.; Thomas, J.; Wang, Y. Genome Res. 2001, 11, 1237-1245. (9) Johnson, J. M.; Castle, J.; Garrett-Engele, P.; Kan, Z.; Loerch, P. M.; Armour, C. D.; Santos, R.; Schadt, E. E.; Stoughton, R.; Shoemaker, D. D. Science 2003, 302, 2141-2144. (10) Zhu, J.; Shendure, J.; Mitra, R. D.; Church, G. M. Science 2003, 301, 836838. (11) Yeakley, J. M.; Fan, J.-B.; Doucet, D.; Luo, L.; Wickham, E.; Ye, Z.; Chee, M. S.; Fu, X.-D. Nat. Biotechnol. 2002, 20, 353-358. (12) King, H. C.; Sinha, A. A. JAMA, J. Am. Med. Assoc. 2001, 286, 2280-2288. (13) Yang, Y. H.; Speed, T. Nat. Rev. Genet. 2002, 3, 579-588. (14) Chan, W. C. W.; Maxwell, D. J.; Gao, X.; Bailey, R. E.; Han, M.; Nie, S. Curr. Opin. Biotechnol. 2002, 13, 40-46. (15) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631-635. (16) Nicewarner-Pena, S. R.; Freeman, R. G.; Reiss, B. D.; He, L.; Pena, D. J.; Walton, I. D.; Cromer, R.; Keating, C. D.; Natan, M. J. Science 2001, 294, 137-141. 10.1021/ac702542n CCC: $40.75
© 2008 American Chemical Society Published on Web 03/15/2008
probes and barcoded fluorescent dyes,17 surface-enhanced Raman scattering (SERS)-based DNA probes using metallic nanostructure substrates display unique advantages: the intrinsic sharp fingerprint signals (∼1 nm full width at half-maximum (fwhm))18,19 compared to conventional fluorescent labels (50-100 nm)18,20 or quantum dots (25-40 nm)21 lead to multilabel readouts at a single excitation wavelength;22 complex statistics and deconvolution required to resolve multiple fluorescence spectra could be avoided; SERS probes can be chosen from a wide range of labeling candidates including both fluorophores and nonfluorophores;23 single-molecule sensitivity is possible;24,25 photobleaching and blinking behavior suffered by fluorescent labels are avoided. A duplex Raman detection using HEX and rhodamine has been demonstrated by Graham et al. to genotype the mutational status of the cystic fibrosis transmembrane conductance regulator gene in an amplification refractory mutation system (ARMS).26 Multiplex approaches to detect dissimilar DNA and RNA targets using fluorescent probes by Raman have been proposed by Cao et al.27 Most of the previous SERS studies for DNA detection used commonly available fluorophores as Raman labels,26-30 but none have developed a procedure for multiplex detection of DNA targets using nonfluorescent Raman tags. When using fluorophores, the problem of obscure Raman scattering due to the displacement of fluorophores by biological media was noted.23 And the chemistry to incorporate a fluorophore into a modified oligonucleotide, although complex and costly, has been implemented.27,28,30 In an earlier report, we developed a probe design to tether singlestranded DNA and nonfluorescent Raman tags separately onto gold nanoparticles (DNA-AuP-RTag) and demonstrated that up to eight probes can be identified in a mixture.31 In fact due to the probe design, the number of RTags is not limited by the number of DNA molecules attached to Au nanoparticles to create an inherent signal amplification mechanism. Therefore, utilizing DNA-AuP-RTag probes, a highly multiplexed and highly sensitive and specific DNA detection methodology could be developed to detect multiple BRCA1 alternative splice variants simultaneously that might be expressed at low levels. In this study, we report a nonfluorescent DNA array platform on a gold-coated glass slide to detect multiple DNA targets simultaneously on a single array spot through a sandwich structure utilizing DNA-AuP-RTag probes for BRCA1 alternative (17) Lin, C.; Liu, Y.; Yan, H. Nano Lett. 2007, 7, 507-512. (18) Ni, J.; Lipert, R. J.; Dawson, G. B.; Porter, M. D. Anal. Chem. 1999, 71, 4903-4908. (19) Mulvaney, S. P.; Musick, M. D.; Keating, C. D.; Natan, M. J. Langmuir 2003, 19, 4784-4790. (20) Isola, N. R.; Stokes, D. L.; Vo-Dinh, T. Anal. Chem. 1998, 70, 1352-1356. (21) Zhang, C. Y.; Johnson, L. W. J. Am. Chem. Soc. 2006, 128, 5324-5325. (22) Grubisha, D. S.; Lipert, R. J.; Park, H. Y.; Driskell, J.; Porter, M. D. Anal. Chem. 2003, 75, 5936-5943. (23) Docherty, F. T.; Clark, M.; McNay, G.; Graham, D.; Smith, W. E. Faraday Discuss. 2004, 126, 281-288. (24) Koo, T.-W.; Chan, S.; Sun, L.; Su, X.; Zhang, J.; Berlin, A. A. Appl. Spectrosc. 2004, 58, 1401-1407. (25) Nie, S.; Emory, S. R. Science 1997, 275, 1102-1106. (26) Graham, D.; Mallinder, B. J.; Whitcombe, D.; Watson, N. D.; Smith, W. E. Anal. Chem. 2002, 74, 1069-1074. (27) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536-1540. (28) Graham, D.; Smith, W. E.; Linacre, A. M. T.; Munro, C. H.; Watson, N. D.; White, P. C. Anal. Chem. 1997, 69, 4703-4707. (29) Faulds, K.; Smith, W. E.; Graham, D. Anal. Chem. 2004, 76, 412-417. (30) Wabuyele, M. B.; Vo-Dinh, T. Anal. Chem. 2005, 77, 7810-7815. (31) Sun, L.; Yu, C.; Irudayaraj, J. Anal. Chem. 2007, 79, 3981-3988.
splicing explorations. As a proof of concept, four model BRCA1 alternative splice variants were detected using DNA targets featuring sequences of exon/exon junctions along with a detailed set of experiments to evaluate specificity. Detection sensitivity of this platform was evaluated by determining a limit of detection (LOD) for each of the probes prescribed. It should be noted that, with the high-throughput array format, a large number of tests could be performed on a single slide with a one-time sampling process. EXPERIMENTAL SECTION Materials. DNA oligonucleotides were purchased from IDT (Coralville, IA). The Reductacryl was obtained from EMD Biosciences (San Diego, CA). Silver enhancement solution was ordered from Ted Pella (Redding, CA). Gold(III) chloride trihydrate, trisodium citrate dehydrate, Tween 20, 4-mercaptopyridine, 2-thiazoline-2-thiol, 4,6-dimethyl-2-pyrimidinethiol, and 2-thiouracil were purchased from Sigma-Aldrich (St. Louis, MO). Preparation of DNA-AuP-RTag Probes. Gold colloid (40 nm) was synthesized by citrate reduction of HAuCl4.32 DNAAuP-RTag probes were fabricated using the procedures described in Sun et al. with slight modification.31 Thiolated DNA oligonucleotides in a disulfide form were first reduced using dithiothreitol (DTT) immobilized onto acrylamide resin at a ratio of 1 mg oligo to 50 mg resin. Thiolated DNA oligonucleotides were added to the red oily precipitate obtained from 10 mL of gold colloid centrifuged for 15 min at 5800 rpm (Clinical 100, VWR, West Chester, PA) to result in a 1 mL solution with a final oligo concentration of 1 µM. After 24 h, the solution was buffered at pH 7.5 (10 mM phosphate buffer with 0.01% Tween 20). After 30 min, salting was initiated slowly with 4 M NaCl until the desired salt concentration (0.3 M) was obtained. The solution was then allowed to “age” under these conditions for an additional 40 h, and the excess reagents were removed by centrifugation for 20 min at 8000 rpm (Microfuge 18 centrifuge, Beckman Coulter Inc., Fullerton, CA). Following the removal of the supernatant, the red oily precipitate was washed twice with 0.3 M NaCl, 0.01% Tween 20, 10 mM phosphate buffer (pH 7.5) (0.3 M PBS) by successive centrifugation and redispersion. The attachment of oligo was confirmed by measuring the difference in absorbance at 260 nm between the original DNA solution and the supernatant collected through centrifugation using a UV-vis spectrometer (V-570, Jasco, Japan). 4-Mercaptopyridine, 2-thiazoline-2-thiol, 4,6-dimethyl-2pyrimidinethiol, and 2-thiouracil were chosen as RTag-1-4, respectively. For each DNA-AuP-RTag probe, 1 mL of the corresponding RTag solution (1 mM) was added to the red oily precipitate of DNA-functionalized gold colloid. The mixture was gently stirred for 24 h and then centrifuged for 20 min at 8000 rpm to remove the supernatant. After washing three times with 0.3 M PBS (pH 7.5), the final red oily precipitate of DNA-AuPRTag was redispersed in the same buffer. Attachment of RTag was confirmed by the fingerprint Raman spectrum of the respective RTags. Array-Format Sandwich Assay. The gold-coated glass slide was first treated with piranha solution (3:1 concentrated sulfuric acid to hydrogen peroxide solution) for 1 h to clean off the organic residue and to produce a hydrophilic surface. The slide was then (32) Frens, G. Nature (London), Phys. Sci. 1973, 241, 20-22.
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Figure 1. Structures of the BRCA1 alternative splice variants and the corresponding sandwich assays. Numbers denote different exons, and the missing exons are shown by connecting lines in each variant. Exons are not drawn to scale, although, as indicated, exon 11 is much longer than the others. Note that exons 1a and 1b are shown with the full-length mRNA species only, because it is currently unknown whether the splicing profiles of the further 3′ exons are different for these two mutually exclusive exons (ref 43). 11q, the last 3309 nt are missing from exon 11; 5q, the last 22 nt are missing from exon 5. PS, probing strand; TS, target strand; CS, capturing strand.
covered with a silicone mask with an array of holes (3 mm in diameter) to avoid cross-talk between neighboring spots. An amount of 10 µL of 1 µM single or multiple thiolated capturing strands (CS) in TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.5) with 1 M NaCl was spotted on the slide and incubated for 6 h in a humidity chamber to be immobilized onto the designated spots through S-Au interaction. After a wash with Nanopure water, the slide was spotted with 10 µL of 1 mM 6-mercapto-1hexanol and incubated overnight in a humidity chamber to reduce nonspecific binding. The slide was washed again with Nanopure water and spotted with 10 µL of single or multiple target strands (TS) with various concentrations in 0.3 M PBS (pH 7.5) and incubated in a humidity chamber at 37 °C to induce hybridization. After 4 h, the slide was washed with Nanopure water. Then 10 µL of different DNA-AuP-RTag probes or mixtures of probes were added to the designated spots and incubated in a humidity chamber at 37 °C for an additional 4 h to facilitate hybridization with the overhanging region of the TS. After the formation of the sandwich structure, the slide was washed with 0.3 M PBS solution to remove nonspecifically bound probes and then washed with 0.6 M NaNO3 PBS solution (pH 7.5) to remove chloride ions, which could form insoluble precipitates with silver ions in later steps. After washing steps, the slide was immediately treated with a silver enhancement solution27 for 10 min, rinsed with Nanopure 3344
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water, and dried in nitrogen, and the spotted slide was ready for Raman measurement. SERS Measurement and Postprocessing of the Spectral Data. In this study, all Raman spectra were acquired using the SENTERRA confocal Raman system (Bruker Optics Inc., Billerica, MA) with a 50× air objective (N.A. 0.7, infinity and flat field corrected). A 785 nm diode laser with 10 mW power at the laser source was used for excitation. A 50 µm pinhole was used for confocal purpose. The integration time was 20 s, and the resolution was 3 to ∼5 cm-1. OPUS software was used to chop the spectra to reveal Raman bands in the 400-1800 cm-1 range, and baseline correction was done by the rubber-band method using a “rubberband-like” string icon stretched between the endpoints of the spectrum to provide the spectrum minima. RESULTS AND DISCUSSION Design of DNA Oligos and Array-Format Detection. The DNA sequences chosen in this study are specific to BRCA1 alternative splice variants ∆(11q), ∆(9, 10), ∆(5), and ∆(5q, 6) as described in Figure 1. Among these variants, ∆(11q) and ∆(9, 10) are two of the four predominant splice variants expressed in a variety of tissues.3,33,34 ∆(5) and ∆(5q, 6) are the other two well(33) Lu, M.; Conzen, S. D.; Cole, C. N.; Arrick, B. A. Cancer Res. 1996, 56, 4578-4581.
Figure 2. DNA detection with a sandwich complex for BRCA1 alternative splice variants. (1) Immobilization of capturing strands (CS); (2) immobilization of 6-mercapto-1-hexanol to reduce nonspecific binding; (3) hybridize target strands (TS) to CS; (4) hybridize DNA-AuP-RTag probes to the overhanging region of TS; (5) silver enhancement. (a), (b), and (c) represent multiplex detection using DNA sequences specific to BRCA1 alternative splice variants.
studied variants.35,36 Specific sequences of these alternative splice variants were obtained from NIH maintained GenBank. TS for detection was single-stranded DNA (ssDNA) specific to the exon/ exon junction. The probing strand (PS) used in the DNA-AuPRTag probe was ssDNA complementary to the downstream half of the TS with thiol modification at the 5′ end. When designing the sequences of PS, multiple C or G residues were avoided at the 3′ end extending off the gold nanoparticle to avoid particle aggregation.37 CS is ssDNA complementary to the upstream half of the TS with thiol modification at the 3′ end. T10 was used as a linker for PS and CS in order to reduce nonspecific binding and improve hybridization efficiency because thymine has the lowest affinity to gold compared to other nucleotides.38 To detect TS, a sandwich complex was formed on the gold-coated glass slides as illustrated in Figure 2. In the expanded view, two-plex, threeplex, and four-plex detection is depicted for the detection of multiple DNA targets specific to the four model alternative splice variants. Multiplex Detection of Alternative Splice Variants. Strong multiplexing capability of the DNA-AuP-RTag probes is one of the most important advantages that this approach offers for (34) Xu, C.-F.; Chambers, J. A.; Nicolai, H.; Brown, M. A.; Hujeirat, Y.; Mohammed, S.; Hodgson, S.; Kelsell, D. P.; Spurr, N. K.; Bishop, D. T.; Solomon, E. Genes, Chromosomes Cancer 1997, 18, 102-110. (35) Claes, K.; Vandesompele, J.; Poppe, B.; Dahan, k.; Coene, I.; Paepe, A. D.; Messiaen, L. Oncogene 2002, 21, 4171-4175. (36) Munnes, M.; Zuther, I.; Schmitz, B.; Doerfler, W. Gene Funct. Dis. 2000, 1, 38-47. (37) Hill, H. D.; Mirkin, C. A. Nat. Protoc. 2006, 1, 324-336. (38) Kimura-Suda, H.; Petrovykh, D. Y.; Tarlov, M. J.; Whitman, L. J. J. Am. Chem. Soc. 2003, 125, 9014-9015.
possible identification of alternative splice variants. Characteristic peaks corresponding to multiple DNA-AuP-RTag probes can be differentiated by simple visual observation. Before detecting multiple DNA targets specific to the four alternative splice variants, the feasibility of multiplexing in a sandwich structure on a slide was first verified using the CS, TS, and PS sequences specific to the ∆(11q) variant and probes synthesized with the same PS but four different RTags, one type of RTag denoting one specific probe. Two-plex (∆(11q)-AuP-RTag-1 and -2), three-plex (∆(11q)AuP-RTag-1-3), and four-plex (∆(11q)-AuP-RTag-1-4) detection were demonstrated using mixtures of respective probes to hybridize with the overhanging region of TS, which hybridized to its appropriate complement presented by the sequence of the CS immobilized to the gold-coated slide. Raman signals from DNA-AuP-RTag-1 and -2 were strong, and the Raman signal from DNA-AuP-RTag-3 was weaker due to the intrinsic Raman cross section of the respective RTags. When the Raman spectra using different concentrations and combination of the probes were compared, the optimal ratio was determined to be 1:1:2:1 among ∆(11q)-AuP-RTag-1-4. The resulting spectra of two-plex, threeplex, and four-plex detection are shown in Figure 3. The peaks are labeled in different colors depicting the origin of different DNA-AuP-RTag probes even though the variant sequence chosen is the same. Characteristic peaks for these chosen Raman tags provided in the preliminary work by Sun et al.31 were observed, ensuring that multiplex detection of splice junctions using a sandwich construct is possible. Raman bands observed for detection using sequences specific to ∆(11q) and individual ∆(11q)-AuP-RTag probes and mixtures of probes are listed in Analytical Chemistry, Vol. 80, No. 9, May 1, 2008
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Figure 3. Multiplex detection with DNA sequences specific to ∆(11q) variant and mixtures of ∆(11q)-AuP-RTag probes: two-plex, ∆(11q)AuP-RTag-1 and -2; three-plex, ∆(11q)-AuP-RTag-1-3; four-plex, ∆(11q)-AuP-RTag-1-4. Table 1. Raman Bands Observed for Detection Using Different ∆(11q)-AuP-RTag Probes probe name
obsd Raman peaks (cm-1)
∆(11q)-AuP-RTag-1 ∆(11q)-AuP-RTag-2
422, 487, 999, 1058, 1091, 1198, 1574, 1600 420, 447, 491, 645, 759, 863, 1024, 1142, 1294, 1349 565, 874, 984, 1239, 1382 436,565, 717, 922, 1158, 1372
∆(11q)-AuP-RTag-3 ∆(11q)-AuP-RTag-4
Table 2. Raman Bands Observed for Multiplex Detection Using DNA Sequences Specific to ∆(11q) Variant sample name
mixture components
obsd Raman peaks (cm-1)
two-plex
∆(11q)-AuP-RTag-1 ∆(11q)-AuP-RTag-2
1002, 1060, 1090, 1209, 1572, 1609 446, 755, 1149, 1294, 1349
three-plex
∆(11q)-AuP-RTag-1 ∆(11q)-AuP-RTag-2 ∆(11q)-AuP-RTag-3
1007, 1062, 1096, 1198, 1577, 1610 452, 765, 1139, 1356 573, 874, 1246
four-plex
∆(11q)-AuP-RTag-1 ∆(11q)-AuP-RTag-2 ∆(11q)-AuP-RTag-3 ∆(11q)-AuP-RTag-4
1004, 1059, 1092, 1207, 1577, 1606 449, 1299, 1351 872, 1240 922, 1162
Tables 1 and 2. The fingerprints revealed for each probe in different tests were consistent. To demonstrate multiplex detection of alternative splice variants and evaluate the specificity of the proposed system, five cases were examined including two two-plex cases (two-plex (A), target 3346
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∆(11q) and ∆(9, 10); two-plex (B), target ∆(9, 10) and ∆(5)), two three-plex cases (three-plex (A), target ∆(11q), ∆(9, 10), and ∆(5); three-plex (B), target ∆(9, 10), ∆(5), and ∆(5q, 6)), one fourplex case (target, all four alternative splice variants) with appropriate control (PBS buffer added instead of target samples) as described in Figure 4. All the targets have equal molar ratio. For each of the experiments, a mixture of four probes featuring different alternative splice variants were used to simulate a realistic scenario of monitoring for unknown targets for a reliable assessment of the detection specificity of the proposed scheme. As discussed in the last section, DNA-AuP-RTag-1 and -2 revealed much higher Raman intensity than the other two probes. Intensity of Raman signals should be balanced among various DNA-AuPRTag probes for best multiplexing of Raman signals. There are two possible ways to optimize Raman signals: first, adjusting the RTag concentration at the probe synthesis stage; second, optimizing the concentration ratios of the DNA-AuP-RTag probes synthesized with equal RTag concentration in the probe mixtures as performed in the last section. Because the second method leads to different concentration of probing strands for different alternative splice variants, it is more difficult to quantify alternative splice variants based on signal intensity. Therefore, we chose the first method. The concentration of RTag-1 and -2 was reduced to 50 µM during probe synthesis based on the concentration study of RTag discussed in Sun et al.31 in order to reduce their Raman signals by half to be compatible with the optimal concentration ratio discussed in last section. It should be noted that based on
Figure 4. Multiplex detection using DNA sequences specific to alternative splice variants of ∆(11q), ∆(9, 10), ∆(5), and ∆(5q, 6).
several tests there was no need to reduce the concentration of RTag-4 in probe synthesis although an optical concentration ratio of 1:1:2:1 was discussed in the last section. This can be explained by the minor difference in signal intensity between RTags-3 and -4 and the strength of final signals depending on not only the difference in intrinsic Raman cross sections of RTags but also the difference in immobilization and hybridization efficiency for different sequences. The concentration of RTag-3 and -4 were kept at 1 mM for probe synthesis. In addition, the percentage of formamide was determined to be 15% to reduce the nonspecific interaction among oligonucleotides.37 The resulting spectra depicting the different levels of multiplexing are shown in Figure 4. For clarity, the sample spectrum was intentionally separated from the control for each section of Figure 4 with no change in relative Raman intensity. Fingerprint Raman bands were labeled in different colors encoding origins from different DNA-AuP-RTag probes in Figure 4 and summarized in Table 3. Two to four DNA targets in the corresponding target mixtures could be easily identified even by visual observation. Since the identification of each target was based on multiple characteristic Raman bands in most instances, the accuracy of detection is guaranteed. Furthermore, in each multiplex case the presence of specific target DNA is accurately correlated to the fingerprints of the corresponding DNA-AuP-RTag probe, i.e., no nonspecific peaks that could rise to false positives were noted, which indicates good specificity of the multiplex system. Characteristic peaks revealed for each probe
Table 3. Raman Bands Observed for Multiplex Detection of DNA Sequences Specific to Alternative Splice Variants of ∆(11q), ∆(9, 10), ∆(5), and ∆(5q, 6) target types
obsd Raman peaks (cm-1)
two-plex (A)
∆(11q) ∆(9, 10)
1001, 1060, 1092, 1200, 1574, 1607 1295, 1344
two-plex (B)
∆(9, 10) ∆(5)
455, 759, 1292, 1355 563, 1234
three-plex (A)
∆(11q) ∆(9, 10) ∆(5)
1005, 1060, 1093, 1578, 1608 450, 862, 1142, 1296, 1355 563, 1249
three-plex (B)
∆(9, 10) ∆(5) ∆(5q, 6)
452, 1291, 1354 882, 1243 720, 1158
four-plex
∆(11q) ∆(9, 10) ∆(5) ∆(5q, 6)
1005, 1060, 1094, 1573 1355 872, 1244 924, 1160
case name
in different cases were consistent. It should be noted that shifts of Raman peaks were observed between the measured spectra after the sandwich structure was formed on the slide and the standard, which was more significant for multiplex detection. However, the shift was expected because the sandwich structure and the local environment of the spot may affect the orientation, hence, the molecular vibration, of RTags on the slide. In addition, multiplex detection added complexity to the system. Therefore, Analytical Chemistry, Vol. 80, No. 9, May 1, 2008
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Figure 5. Sensitivity study of the array platform for DNA detection using DNA target specific to ∆(11q) variant and ∆(11q)-AuP-RTag-1-4.
a further shift was expected. In spite of the shift (∼10 cm-1), individual DNA-AuP-RTag probes could still be clearly differentiated since the characteristic peaks of the chosen tags are well separated from each other. Current results verified the feasibility of array-format multiplex detection for alternative splice variants identification. In fact the multiplexing level has good potential to be improved, based on the eight nonfluorescent probes as previously demonstrated.31 And with other choices of RTags, the broader spectral range (beyond 400-1800 cm-1) to be utilized and the aid of multivariate analysis, a further increase in the level of multiplexing is an expected hypothesis to be tested. Then an array format combined with highly multiplexed assay on each array spot could lead to a workstation with much higher throughput providing alternative splicing patterns across species, cell types, disease states, and stages of development simultaneously to be adapted for other nucleic acid detection based applications. Detection of BRCA1 alternative splice variants in the mRNA pool extracted from different cell lines are in progress in the authors’ lab. Here, the interrogated ssDNA targets obtained from isolated mRNA through S1 nuclease assays and alkaline hydrolysis can be directly probed for several alternative splice variants from cells, which will lead to possible answers to fundamental issues related to tumorigenesis. 3348
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Evaluation of Detection Sensitivity. With the multiplexing capability demonstrated, it is important to understand the sensitivity of the detection system before developing it into a reliable workstation. To evaluate detection sensitivity of this array platform, target strands for ∆(11q) variant with decreasing concentration ranging from 1 mM to 1 fM in steps of 10× were detected using ∆(11q)-AuP-RTag-1, -2, -3, and -4, respectively. Control spots using PBS buffer instead of TS were also prepared for each of the probes for comparison. Spectra for target concentration of 1 µM, 1 pM, 1 nM, and 1 fM using individual DNA-AuP-RTag probes are shown in different sections of Figure 5. The top spectra for individual sections are the standard spectra of the corresponding DNA-AuP-RTag probes. Characteristic peaks were labeled and characterized corresponding to the different probes. Some bands other than characteristic Raman peaks of the DNA-AuPRTag probes were also observed, which could arise from the silver enhancement solution or other solutions since these bands were noted in almost all the spectra including control. The purpose of this sensitivity study was to determine the lower LOD for this array-format detection. From the spectra we can see that the major peaks were differentiable under a target concentration of 1 fM for all the probes used so that a supersensitive assay could be developed based on this study. To provide further evidence of
stretch of the bonds between the nitrogen at position 3 and the neighboring carbon atoms.42 The peak intensity obtained by integrating specific peaks relative to the local baseline clearly shows that the Raman intensity corresponding to a concentration of 1 fM target is three standard deviations above the control as indicated by the dotted line (Figure 6). Very high sensitivity was obtained due to the intrinsic design of the DNA-AuP-RTag probe, by which a single hybridization event was amplified in the Raman signal through the attachment of a vast number of RTag molecules to a single gold nanoparticle. This highly sensitive (up to 1 fM) assay will lead to the possible detection of low-abundance alternative splice variants.
Figure 6. Raman intensity of detection with 1 fM TS specific to ∆(11q) variant and controls using different ∆(11q)-AuP-RTag probes. Dotted lines indicate three standard deviations above controls.
sensitivity and to assess the LOD, spectra of target concentration of 1 fM were compared with controls by integrating one of the major peaks for detection using each DNA-AuP-RTag probe as shown in Figure 6. For RTag-1, peak 1091 cm-1 was chosen, which is possibly from the in-plane C-H deformation;39 for RTag-2, peak 1349 cm-1 was chosen, which could come from vibration of thioamide;40 for RTag-3, peak 1239 cm-1 was chosen, which might be due to C-H bending on the pyridine ring;41 for RTag-4, peak 922 cm-1 was chosen, which may correspond to the in-phase (39) Wang, Z.; Rothberg, L. J. J. Phys. Chem. B 2005, 109, 3387-3391. (40) Joy, V. T.; Srinivasan, T. K. K. J. Raman Spectrosc. 2001, 32, 785-793. (41) Yang, W.-h.; Hulteen, J.; Schatz, G. C.; Van Duyne, R. P. J. Chem. Phys. 1996, 104, 4313. (42) Aguiar, H. B.; Ana, A. C. S.; Temperini, M. L. A.; Corio, P.; Cunha, F. Vib. Spectrosc. 2006, 40, 127-132. (43) Orban, T. I.; Olah, E. Mol. Pathol. 2003, 56, 191-197.
CONCLUSION We have demonstrated the use of nonfluorescent Raman tags for multiplex DNA detection. This is also the first proof-of-concept study to apply a SERS-based detection system to investigate alternative gene splicing. A four-plex detection scheme was successfully tested without the need for complex deconvolution methods for data analysis with a potential to further extend the multiplexing level to eight or more. Sensitivity of the probes was shown to be 1 fM. On the basis of this work, a microarray platform could be developed for alternative splicing profiling of BRCA1 (or other genes) in order to address the fundamental questions related to tumorigenesis. We envision that such an approach could be extended to other nucleic acid based diagnostics where low-level expressions need to be assessed. ACKNOWLEDGMENT Funding from NIH-NCI (5 RO3 CA 121347), Oncological Sciences Center, and Center for Food Safety Engineering at Purdue University is acknowledged. Work was done at the Physiological Sensing Facility in the Bindley Bioscience Center. Received for review December 16, 2007. Accepted February 13, 2008. AC702542N
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