Anal. Chem. 2001, 73, 1567-1571
Cantilever-Based Optical Deflection Assay for Discrimination of DNA Single-Nucleotide Mismatches Karolyn M. Hansen,† Hai-Feng Ji,† Guanghua Wu,‡ Ram Datar,§ Richard Cote,§ Arunava Majumdar,‡ and Thomas Thundat*,†
Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, Department of Mechanical Engineering, University of California, Berkeley, California 94720, and Department of Pathology, University of Southern California, Los Angeles, California 90033
Characterization of single-nucleotide polymorphisms is a major focus of current genomics research. We demonstrate the discrimination of DNA mismatches using an elegantly simple microcantilever-based optical deflection assay, without the need for external labeling. Gold-coated silicon AFM cantilevers were functionalized with thiolated 20- or 25-mer probe DNA oligonucleotides and exposed to target oligonucleotides of varying sequence in static and flow conditions. Hybridization of 10-mer complementary target oligonucleotides resulted in net positive deflection, while hybridization with targets containing one or two internal mismatches resulted in net negative deflection. Mismatched targets produced a stable and measurable signal when only a four-base pair stretch was complementary to the probe sequence. This technique is readily adaptable to a high-throughput array format and provides a distinct positive/negative signal for easy interpretation of oligonucleotide hybridization. A major focus of current genomics research is the detection of single-nucleotide polymorphisms (SNPs) within known gene sequences, as well as in the genome as a whole.1 Point mutations, which can be a deleterious type of SNP, are the hallmarks of several known diseases (e.g., Tay Sachs,2 cystic fibrosis,3 thalassaemia4); efforts to locate and characterize SNPs will aid in the early detection, diagnosis, and perhaps treatment of individuals carrying such mutations. Current methods for detection of SNPs are numerous and include the following: denaturing gradient gel electrophoresis (DGGE),5 oligonucleotide ligation assays (OLA),6,7 * Corresponding author: (e-mail)
[email protected]. † Oak Ridge National Laboratory. ‡ University of California, Berkeley. § University of Southern California, Los Angeles. (1) Schafer, A. J.; Hawkins, J. R. Nat. Biotechnol. 1998, 16, 33-39. (2) Gravel, F. A.; Clarke, J. T. R.; Kaback, M. M.; Mahuran, D.; Sandhoff, K.; Suzuki, K. In The metabolic and molecular basis of inherited disease; Scriver, C. R., Beauder, A. L., Sly, W. S., Valle, D., Eds.; McGraw-Hill: New York, 1995; Vol. 2, pp 2839-2879. (3) Cronin, M. T.; Fucini, R. V.; Kim, S. M.; Masino, R. S.; Wespi, R. M.; Miyada, C. G. Hum. Mutat. 1996, 7, 244-255. (4) Muniz, A.; Martinez, G.; Lavinha, J.; Pacheco, P. Am. J. Hematol. 2000, 64, 7-14. (5) Fischer, S. G.; Lerman, L. S. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 15791583. 10.1021/ac0012748 CCC: $20.00 Published on Web 03/02/2001
© 2001 American Chemical Society
pyrosequencing,8 capillary electorphoresis,9,10 allele-specific oligonuclotide hybridization (ASO),11 chemical cleavage of mismatch (CCM),12 2′-amine acylation,13 single-base chain extension (SBCE),14 carbon nanotube probes,15 and oligonucleotide hybridization detection using QCM.16,17 In this study, we demonstrate the detection of single-base pair mismatches at discrete locations in a 10-mer DNA target oligonucleotide without the use of extrinsic fluorescent or radioactive labeling. Our detection method employs silicon microcantilevers, which are extremely sensitive to specific biomolecular interactions.18,19,20 Microcantilevers with specific functional coatings have been used to determine concentrations of molecules in both gas and liquid phase and exhibit the sensitivity to detect concentrations in the pico- to femtogram range.21 The inherent characteristics of cantilevers that allow such sensitivity are as follows: (1) the ability to bend (deflection) as a function of molecular interactions at the cantilever surface; (2) (6) Iannone, M. A.; Taylor, J. D.; Chen, J. W.; Li, M. S.; Rivers, P.; Slentz-Kesler, K. A.; Weiner, M. P. Cytometry 2000, 39, 131-140. (7) Skobeltsyna, L. M.; Pyshnyi, D. V.; Shishkina, I. G.; Tabatadze, D. R.; Dymshits, G. M.; Zarytova, V. F.; Ivanova, E. M. Mol. Biol. 2000, 34, 321327. (8) Nordstrom, T.; Ronaghi, M.; Forsberg, L.; de Faire, U.; Morgenstern, R.; Nyren, P. Biotechnol. Appl. Biochem. 2000, 31, 107-112. (9) Siles, B. A.; O’Neil, K. A.; Tung, D. L.; Bazar, L.; Collier, G. B.; Lovelace, C. I. P. J. Capillary Electrophor. 1998, 5, 51-58. (10) Gao, Q. F.; Yeung, E. S. Anal. Chem. 2000, 72, 2499-2506. (11) Winichagoon, P.; Saechan, V.; Sripanich, R.; Nopparatana, C.; Kanokpongsakdi, S.; Maggio, A.; Fucharoen, S. Prenat. Diagn. 1999, 19, 428-435. (12) Liu, J. W.; Wu, X. M. Prog. Biochem. Biophys. 1999, 26, 276-280. (13) John, D. M.; Weeks, K. M. Chem. Biol. 2000, 7, 405-410. (14) Chen, J. W.; Iannone, M. A.; Li, M. S.; Taylor, J. D.; Rivers, P.; Nelsen, A. J.; Slentz-Kesler, K. A.; Roses, A.; Weiner, M. P. Genet. Res. 2000, 10, 549557. (15) Woolley, A. T.; Guillemette, C.; Cheung, C. L.; Housman, D. E.; Leiber, C. M. Nat. Biotechnol. 2000, 18, 760-763. (16) Wang, J.; Nielsen, P. E.; Jiang, M.; Cai, X.; Fernandes, J. R.; Grant, D. H.; Ozsoz, M.; Beglieter, A.; Mowat, M. Anal. Chem. 1997, 69, 5200-5202. (17) Okahata, Y.; Kawase, M.; Niikura, K.; Ohtake, F.; Furusawa, H.; Ebara, Y. Anal. Chem. 1998, 70, 1288-1296. (18) Wu, G.; Ji, H.-F.; Hansen, K. M.; Thundat, T.; Datar, R.; Cote, R.; Hagan, M. F.; Chakraborty, A. K.; Majumdar, A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1560-1567. (19) Raiteri, R.; Nelles, G.; Butt, H.-J.; Knoll, W.; Skladal, P. Sens. Actuators, B 1999, 61, 213-217. (20) Fritz, J.; Baller, M. K.; Lang, H. P.; Rothuizen, H.; Vettiger, P.; Meyer, E.; Guntherodt, H.-J.; Gerber, Ch.; Gimzewski, J. K. Science 2000, 288, 316318. (21) Thundat, T.; Oden, P. I.; Warmack, R. J. Microscale Thermophys. Eng. 1997, 1, 185.
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Table 1. DNA Sequences for 20- and 25-Mer Oligonucleotide Experimentsa
a
probe 1: 5′ thiolated, 20 mer probe 2: 5′ thiolated, 20 mer probe 3: 5′ thiolated, 20 mer target: 10 mer comp target: 9 mer comp target: 10-mer MMT target: 10-mer 1MM target: 10-mer 2MM target: 20-mer noncomp
5′-thiol-TT AAG GTC TGG ACT GGC CTG-3′ 5′-thiol-TT AAG GTC TGT CTC GGC CTG-3′ 5′-thiol-TT AAG GTC TGG ACT GGT ACC-3′ 3′-C TGA CCG GAC-5′ 3′-TGA CCG GAC-5′ 3′-G TGA CCG GAC-5′ 3′-C TGA GCG GAC-5′ 3′-C TGA GGG GAC-5′ 3′-TC ATG ACA GAT CTA CTC GTA-5′
probe 4: 5′ thiolated, 25 mer target: 25-mer comp target: 25-mer proximal MMT target: 25-mer distal MMT target: 25-mer 1MM target: 10-mer comp target: 10-mer 1MM target: 10-mer 2MM target: 25-mer noncomp
5′-thiol-T CAT CTG CTA CCA ATC AGT CGC TCG-3′ 3′-A GTA GAC GAT GGT TAG TCA GCG AGC-5′ 3′-T GTA GAC GAT GGT TAG TCA GCG AGC-5′ 3′-A GTA GAC GAT GGT TAG TCA GCG AGT-5′ 3′-A GTA GAC GAT GGC TAG TCA GCG AGC-5′ 3′-GAT GGT TAG T-5′ 3′-GAT GGC TAG T-5′ 3′-GAT GTC TAG T-5′ 3′-A CTT TGA TCC ACG CGT GTA CTT CTC-5′
Noncomplementary bases indicated by italics. MMT, terminal mismatch; 1MM, one internal mismatch; 2MM, two internal mismatches.
the specific resonance frequency of each cantilever which varies due to mass loading.22 The use of deflection and mass loading characteristics eliminates the need for extrinsic labeling (and subsequent label detection) of molecules of interest. In addition, silicon cantilevers can be constructed23,24 and functionalized in microarray format for high-throughput analysis, similar to the array formats currently used for high-throughput DNA analysis.25,26 In this study, we use hybridization-induced cantilever deflection to demonstrate that the number and location of DNA base pair mismatches in a target 10-mer oligonucleotide can be discerned under high-stringency static and flow conditions using gold-coated silicon microcantilevers. Moreover, using the cantilever format, we present evidence demonstrating functional hybridization of probe and target oligonucleotides with a complementary sequence length of only four nucleotides. EXPERIMENTAL PROTOCOL Silicon cantilevers (Contact Ultralevers, Thermomicroscopes, Sunnyvale, CA) were stripped of nonhomogeneous vendor-applied chromium and gold using serial washes of ceric ammonium nitrate and aqua regia solutions, respectively (5 g of ceric ammonium nitrate, 5 mL of H2O, 4 mL of concentrated nitric acid; 1:4 HNO3/ HCl). Chromium (2.5 nm thickness) and gold (20 nm thickness) were sequentially deposited on the upper surface of the cantilevers via vacuum evaporation. Cantilevers were used immediately or stored desiccated; all gold-coated cantilevers were treated with piranha (3 parts H2O2/7 parts H2SO4) just prior to DNA immobilization. Cantilever specifications: triangular, 180 µm length, 1 µm thickness, 0.26 N/m spring constant, 40 kHz resonance frequency in air, and surface area (gold side) ∼10-4 cm2. DNA oligonucleotides were commercially prepared (Synthegen, Houston, TX) and supplied as HPLC-purified preparations (22) Thundat, T.; Chen, G. Y.; Warmack, R. J.; Allison, D. P.; Wachter, E. A. Anal. Chem. 1995, 67, 519-521. (23) Madou, M. J. Fundamentals of microfabrication; CRC Press: Boca Raton, FL, 1997. (24) Perazzo, T.; Mao, M.; Kwon, O.; Majumdar, A.; Varesi, J. B.; Norton, P. Appl. Phys. Lett. 1999, 74, 3567-3569. (25) Marshall, A.; Hodgson, J. Nat. Biotechnol. 1998, 16, 27-31. (26) Ramsay, G. Nat. Biotechnol. 1998, 16, 40-44.
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(Table 1). All probe (surface-immobilized) DNA, 20 and 25 nucleotides in length, were synthesized with C-6 5′-thiol modification for immobilization on the gold side of the cantilever. To verify the robustness of the deflection assay, four thiolated probes were designed, which differed in both sequence and length (Table 1), and two hybridization exposure conditions were employed: flow through (probes 1-3, 20 mer each) and static (probe 4, 25 mer). For the flow-through 20-mer probe experiments, target (fully complementary and mismatched) DNA, 10 nucleotides in length, was complementary to the 3′ end of probe DNA with interaction occurring distal to the gold surface. Three 10-mer mismatch sequences were designed: proximal terminal, one internal, and two internal. For the static 25-mer probe experiments, three 25mer mismatch sequences were designed: distal terminal, proximal terminal, and one internal. For the same 25-mer probe, two 10mer mismatch sequences were also designed: one internal and two internal. Cantilevers were functionalized with freshly deprotected thiolated probe DNA by immersion in solution for 1 h at room temperature (4 cantilevers/mL, 50 µg/mL DNA, 100 mM phosphate buffer (PB), pH 7.0). DNA-coated cantilevers were rinsed several times with PB and stored in PB at 4 °C until use. Cantilever optical deflection assays were conducted using a four-quadrant AFM head with integrated laser and positionsensitive detector (Digital Instruments, Santa Barbara, CA; Figure 1). Probe DNA-coated cantilevers were mounted in the liquid cell (∼250 µL volume) and allowed to equilibrate in PB until a stable baseline was achieved for both static and flow-through experiments (all at 25 °C; 2 mL/h flow rate for flow experiments). Flow was controlled using a syringe pump (IITC, Inc., Woodland Hills, CA) equipped with a low-pressure liquid chromatography injector valve and injection loop (Upchurch Scientific, Oak Harbor, WA). For static experiments, target DNA sequences (20 µg/mL in PB) were injected directly into the fluid cell; for flow experiments, target DNA sequences (20 µg/mL in PB) were injected into the flow stream using a 1-mL injection loop; lag time for the target DNA solution to enter the fluid cell was ∼6 min. Cantilever deflection due to probe-target hybridization was monitored in situ throughout each experiment (measured as voltage change
Figure 1. Schematic diagram of experimental setup showing a fluid cell within which a microcantilever beam was mounted. SEM micrograph on right shows geometry of cantilever beam. Fluid flow was controlled by a syringe pump connected to an in-line injection valve and 1-mL sample loop.
Figure 2. Cantilever deflection for hybridized oligonucleotides. Cantilever surfaces functionalized with thiolated 20-mer probe 1 and challenged with complementary, mismatch, and noncomplementary target oligonucleotides (see Table 1 for oligonucleotide sequences).
using HP Data Logger model 34970A; 1 V ) 100 nm deflection). Prior to these experiments, cantilevers were calibrated (using an AFM) by pressing the tip a known distance and determining the voltage change. In this study, no attempt was made to regenerate hybridized cantilevers; a fresh cantilever was used for each target DNA challenge. Regeneration parameters and reproducibility are being addressed in ongoing research. RESULTS AND DISCUSSION Hybridization of fully complementary 10-mer target DNA to 20-mer immobilized probe DNA resulted in a net upward deflection of the cantilevers (gold upper/silicon lower) (Figure 2). Upward deflection is due to the reduction of compressive forces on the gold side of the cantilever due to dsDNA formation, and as has been pointed out by Wu et al.,18 the origin of this stress relief lies in the drastic reduction in configurational entropy of dsDNA versus ssDNA. Single-strand DNA has a persistence
length of 0.75 nm (equivalent to 2 nucleotides)27 and is therefore coiled and highly flexible, whereas dsDNA has a persistence length of 50 nm (equivalent to 150 base pairs)28 and is present in rodlike configuration. The hybridized 20- and 25-mer probes used in our experiment therefore exist in a rodlike conformation. This steric effect dominates over the increase in electrostatic repulsion due to the additional negative charge in the target ssDNA that hybridizes with the probe. Discrimination of hybridization of 10versus 9-mer oligonucleotides is evident, and the exposure to noncomplementary DNA verified that deflection was indeed due to hybridization (Figure 2). Hybridization of a 10-mer oligonucleotide with a terminal base pair mismatch results in deflection similar to that of the 9-mer oligonucleotide with a difference of ∼1 nm (
