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Rapid and Direct Sequencing of Double-Stranded DNA Using Exonuclease III and MALDI-TOF MS Uraiwan Puapaiboon,† Jaran Jai-nhuknan,‡ and J. A. Cowan*,†
Evans Laboratory of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, and Bruker Daltonics, Inc., 47697 Westinghouse Drive, Fremont, California 94539
Application of MALDI-TOF MS to direct sequencing of dsDNA substrates is demonstrated using a strategy that employs exonuclease III digestion of a target sequence. Experimental conditions for exonuclease III have been optimized for this application, including addition of essential divalent metal ion cofactors. A short cationexchange column was designed to provide efficient sample cleanup and overcome major problems arising from salt interference. Sequence determination and characterization of oligonucleotides is a significant problem in biological and medical science. In recent years, much effort has been dedicated to developing methods for screening and sequencing of oligonucleotides by utilizing mass spectrometric techniques, particularly MALDI MS,1-6 to replace traditional time-consuming and labor-intensive gel electrophoretic methods. Single-stranded DNA (ssDNA, 1035 nt in length) has previously been sequenced by MALDI MS using either an in-source fragmentation approach7-9 or nucleolytic digestion strategies.10,11 Few reports have described sequencing of double-stranded DNA (dsDNA) by MALDI MS. Taranenko et al. have utilized a cycle sequencing and chain termination method to sequence a single-stranded and double-stranded 50-base-pair template.12 However, this method relies on appropriate commercial †
The Ohio State University. Bruker Daltonics, Inc. (1) Owens, D. R.; Bothner, B.; Phung, Q.; Harris, K.; Siuzdak, G. Bioorg. Med. Chem. 1998, 6, 1547-1554. (2) Nordhoff, E.; Ingendoh, A.; Cramer, R.; Overberg, A.; Stohl, B.; Karas, M.; Hillenkamp, F.; Crain, P. F. Rapid Commun. Mass Spectrom. 1993, 6, 771. (3) Chait, B. T.; Wang, R.; Beavis, R. C.; Kent, S. B. H. Science 1993, 262, 89. (4) Taranenko, N. I.; Tang, K.; Allman, S. L.; Chang, L. Y.; Chen, C. H. Rapid Commun. Mass Spectrom. 1994, 8, 1001-1006. (5) Tang, K.; Taranenko, S. L.; Allman, S. L.; Chang, L. Y.; Cheng, C. H. Rapid Commun. Mass Spectrom. 1994, 8, 727-730. (6) Tang, K.; Taranenko, S. L.; Allman, S. L.; Chen, C. H.; Chong, L. Y.; Jacobson, K. B. Rapid Commun. Mass Spectrom. 1994, 8, 673-677. (7) Parr, G. R.; Fitzgerald, M. C.; Smith, L. M. Rapid Commun. Mass Spectrom. 1992, 6, 369-372. (8) Zhu, L.; Parr, G. R.; Fitzgerald, M. C.; Nelson, C. M.; Smith, L. M. J. Am. Chem. Soc. 1995, 117, 6048-6056. (9) Zhu, Y. F.; Taranenko, N. I.; Allman, S. L.; Taranenko, N. V.; Martin, S. A.; Hoff, L. A.; Chen, C. H. Rapid Commun. Mass Spectrom. 1997, 11, 897903. (10) Schuette, J. M.; Pieles, U.; Maleknia, S. D.; Srivatsa, G. S.; Cole, D. L.; Moser, H. E.; Afeyan, N. B. J. Pharm. Biomed. Anal. 1995, 13, 1195-1203. (11) Pieles, U.; Zercher, W.; Schar, M.; Moser, H. E. Nucleic Acids Res. 1993, 21, 3191-3196. (12) Taranenko, N. I.; Chung, C. N.; Zhu, Y. F.; Allman, S. L.; Golovlev, V. V.; Isola, N. R.; Martin, S. A.; Hoff, L. A.; Chen, C. H. Rapid Commun. Mass. Spectrom. 1997, 11, 386-392. ‡
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protocols that are used to generate large quantities of DNA ladders; otherwise, MALDI-TOF MS with higher detection sensitivity and resolution would be required. Complications in obtaining short single-stranded oligonucleotides from the corresponding double-stranded oligonucleotide for either chemical degradation or chain termination sequencing are known.13 In this paper, we report a novel strategy using exonuclease III for direct sequencing of double-stranded DNA by MALDI MS, and discuss possible applications in biomedical research. Escherichia coli exonuclease III is a multifunctional enzyme that is doublestrand specific.14,15 The enzyme possesses four activities; namely, 3′-5′ exonuclease, RNase H, 3′-phosphatase, and AP endonuclease activities. For exonuclease activity, the enzyme consecutively degrades the phosphodiester bond from the 3′-end of doublestranded DNA releasing 5′-mononucleotides. This characteristic of exonuclease III affords new opportunities for use of the enzyme, in concert with MALDI-TOF MS, to directly determine the sequence of dsDNA. The enzyme has an absolute requirement for divalent metal ion cofactors to promote activity, and so the choice of an appropriate added divalent cation is also a significant consideration to achieve optimal working conditions. A major problem in the application of both MALDI analysis and other mass spectrometric techniques is interference by salts or added divalent cations. Previous applications of MALDI MS to study the activity of enzymes have been carried out in aqueous solutions lacking mineral salt to avoid such problems.10,11 Smirnov et al. have used an ammonium citrate solution to eliminate the desalting step,16 where the pH could be adjusted by titration with ammonium hydroxide to achieve optimal solution conditions for reaction of snake venom phosphodiesterase (pH 9.4). However, phosphodiesterase does not utilize divalent metal cofactors, while the conditions required for reaction of exonuclease III with dsDNA, and used in this present study, are more stringent,17 requiring a high ionic strength (50 mM KCl, 20 mM Tris, pH 7.5, and 8 mM divalent cation) in the reaction mixture. To achieve the optimum activity of exonuclease III, we have devised a modified sample preparation procedure for MALDI analysis by employing a more efficient sample cleanup technique. (13) Brown, T. A. DNA Sequencing: The Basics; Oxford University Press: New York, 1994; pp 22-24 and 83-84. (14) Jo ¨rg, D. H. Anal. Biochem. 1993, 209, 238-246. (15) Weiss, B. Methods Enzymol. 1980, 65, 203-231. (16) Smirnov, I. P.; Roskey, M. T.; Juhasz, P.; Takach, E. J.; Martin, S. A.; Haff, L. A. Anal. Biochem. 1996, 238, 19-25. (17) Black, C. B.; Cowan, J. A. Eur. J. Biochem. 1997, 243, 684-689. 10.1021/ac000181n CCC: $19.00
© 2000 American Chemical Society Published on Web 06/10/2000
EXPERIMENTAL SECTION 30-Mer (5′-CTA GCG TAA TCA TAG TCC CCA TAG TAA CCT-3′), 30-mer (5′-AGG TTA CTA TGG GGA CTA TGA TTA CGC TAG-3′), and 34-mer (5′-AGG TTA CTA TGG GGA CTA TGA TTA CGC TAG TGC A-3′) oligonucleotides were purchased from Integrated DNA Technologies, Inc. and used without further purification. 3-Hydroxypicolinic acid (3-HPA) and ammonium citrate were purchased from Aldrich. Cation-exchange resin, 50 W-X8, mesh size 200-400 µm, was purchased from Bio-Rad. Exonuclease III was prepared and purified by standard procedures.18 Bruker’s Proflex III (Bruker, Billerica, MA) was used for MALDI-TOF data collection. The instrument is equipped with a 150-200-µJ, 3-ns, N2 laser (Laser Science, Inc.), a delay-extraction two-stage gridless source, and a two-stage gridless reflectron with a total flight path of 120 cm. The vacuum chamber was held at pressures below 6.0 × 10-6 Torr with a 70 L/s turbo pump. Ions were detected using microchannel plate (Galileo Corp., Sturbridge, MA), and data were recorded on a 1-GHz analog-to-digital converter (LeCroy, Chestnut Ridge, NY) controlled by a Sun workstation (Sun Microsystems, Mountain View, CA). A ds-oligonucleotide substrate was prepared by annealing equimolar concentrations of ss-30-mer oligonucleotides (5′-CTA GCG TAA TCA TAG TCC CCA TAG TAA CCT-3′) with 30-mer (5′-AGG TTA CTA TGG GGA CTA TGA TTA CGC TAG-3′) (for sequence 1) and ss-30-mer (5′-CTA GCG TAA TCA TAG TCC CCA TAG TAA CCT-3′) with 34-mer (5′-AGG TTA CTA TGG GGA CTA TGA TTA CGC TAG TGC A-3′) (for sequence 2). Annealing was carried out by heating at 90 °C for 10 min in 20 mM Tris-HCl, pH 7.5 and 50 mM KCl, followed by slowly cooling to room temperature. The pelleted dsDNA was obtained by ethanol precipitation and allowed to air-dry. Enzymatic degradation was carried out at room temperature by mixing 7.5 µL of 0.32 µM exonuclease III solution (0.11 units) and 7.5 µL of a 15 µM solution of annealed substrate. The solution buffer consisted of 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM, 2-mercaptoethanol, and 8 mM MnCl2. The reaction was stopped at the indicated time points by mixing 2-µL aliquots of the assay with 1 µL of the 3-HPA)/ammonium citrate solution defined below. The resulting mixture was loaded onto a short cation-exchange column, containing the NH4+-form cation-exchange resin specified earlier, and eluted by centrifugation at 4000 rpm for 1 min. The cation-exchange column was prepared by simply adding approximately 50-70 µL an aqueous suspension of cations-exchange beads into the pipet tip containing a piece of glass wool at the end to prevent leaking of the beads. For MALDI analysis, 0.5 µL of the matrix solution, containing 0.3 M hydroxypicolinic acid and 30 mM ammonium citrate in 30% acetonitrile/H2O, was deposited onto the sample target disk and air-dried. The purified enzymatic degradation products (0.5 µL) were applied onto the top of the matrix crystal and allowed to air-dry prior to analysis by MALDI MS. RESULTS AND DISCUSSION A critical contribution to the success of these experiments has been the use of both a cation-exchange resin for purification and an ammonium citrate additive to reduce formation of alkali salt (18) Kuo, C. F.; McKee, D. E.; Cunningham, R. P.; Tainer, J. A. J. Mol. Biol. 1993, 229, 239-242.
adducts. The use of both cation-exchange resins and ammonium citrate has previously been reported.2,11,16 However, we have found that an on-probe cleanup does not provide effective removal of metal cofactors from the buffer solution required by exonuclease III, which includes added divalent cation and 50 mM KCl for optimal activity. Accordingly, we have devised a strategy that uses a cation-exchange column to clean up the products from the reaction mixture. By use of this simple and unique modification, dramatic changes (improvement in signal intensity and higher resolution) in the results were obtained that allowed us to directly sequence dsDNA by use of exonuclease III and MALDI-TOF MS. Exonuclease III catalyzes the hydrolysis of terminal nucleotides from dsDNA in a 3′-5′ orientation.15 However, a complementary oligonucleotide is required, and DNA duplexes with 3′-overhangs are nonsubstrates. Typically the rate of reaction from each end will be distinct, and so two strategies can be adopted. First, complete sequence information can be obtained for blunt-end dsDNA (Figure 1), assuming the enzyme achieves at least 50% cleavage on each strand. For sequence 1 (Figure 1), exonuclease III degrades the dsDNA substrate slightly faster from the 3′-end of strand B. In cases where the strands are nonpalindromic, the mass spectrum will exhibit two series of sequence information. One series indicates the sequence of bases from 3′-5′ degradation of strand A, while the other corresponds to 3′-5′ degradation of strand B. The complete sequence is obtained by complementing the data obtained from strands A and B. This method is demonstrated by the data shown in Figure 1. Enzyme digestion products were generated within 2 min. The peak at m/z 9111 and 9302 in the MALDI spectrum represent the intact 30-mer oligonucleotides: one for the sense strand (strand A) and the other for the antisense strand (strand B). Consistent with previous findings, only single-stranded oligonucleotides were observed,19-22 as a result of both the acidic pH of the matrix and the laser desorption/ionization in MALDI. The relationship between mass differences of reaction products to the base sequence derived from the digestion reaction of exonuclease III with the blunt-end doublestranded oligonucleotide substrate is summarized in Figure 1. An advantage of employing blunt-end DNA is that one can obtain complete sequence information of dsDNA, since the enzyme operates on both ends and extends to a region of overlap. The strategy is also suitable for direct sequencing of samples that are amplified by PCR. An alternative approach is to create a double-stranded sequence with one blunt end and one 3′-overhang (Figure 2). Restriction enzymes that possess appropriate recognition sequences can be employed to produce the desired substrate.14 In doing so, the dsDNA substrate will be unidirectionally degraded from the blunt end, and the 3′-overhang is protected from exonuclease III digestion. By selection of appropriate restriction endonucleases to produce a desirable substrate for enzymatic digestion, virtually full sequence information can be obtained. To imitate the 3′overhang fragment generated by the restriction endonuclease, Pst I, sequence 2 (Figure 2) was designed. Again, the peaks at m/z (19) Benner, W. H.; Horn, D.; Katz, J.; Jaklevic, J. Rapid Commun. Mass. Spectrom. 1995, 9, 537. (20) Liu, Y. H.; Lubman, D. M.; Venta, P. J. Anal. Chem. 1995, 67, 3482. (21) Siegert, C. W.; Jacob, A.; Ko ¨ster, H. Anal. Biochem. 1996, 243, 55. (22) Doktycz, M. J.; Hurst, G. B.; Habibi-Goudazi, S.; McLuckey, S. A.; Tong, K.; Chen, C. H.; Vziel, M.; Jacobson, K. B.; Woychik, R. P.; Buchanan, M. V. Anal. Biochem. 1995, 230, 205.
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Figure 1. Mass spectral data obtained on sequence 1 following digestion with exonuclease III for 2 min. The top strand is A, and the bottom strand is B. Peaks from strand A are indicated by *.
Figure 2. Mass spectral data obtained on sequence 2 following digestion with exonuclease III for 4 (a) and 12 min (b). Peaks from strand A are indicated by *.
9111 and 10537 represent the intact 30-mer (strand A) and 34mer (strand B) oligonucleotide, respectively. Our results indicate that exonuclease III degrades only the 30-mer oligonucleotide from the blunt 3′-end. Even after 12 min of digestion, the peak at m/z 10537 remains while the peak at m/z 9111 has disappeared (data not shown). Direct measurement of the mass difference of the digestion products from aliquots removed after 4 and 12 min (Figure 2) unambiguously identify almost the entire sequence of the desired 30-mer oligonucleotide. 3340
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CONCLUDING REMARKS Our results demonstrate the utility of MALDI-TOF MS, combined with exonuclease III digestion, for rapid and direct determination of DNA sequence. A combination of rapid nuclease cleavage, with the speed and sensitivity of MALDI-TOF MS, makes the approach especially attractive. Our use of a short cationexchange column provided a quick and efficient technique to clean sample mixtures containing divalent cation species, which are essential cofactors for the enzyme-mediated reactions. Our success
makes this new approach to dsDNA sequencing possible. In addition, this modified strategy, which is simple and inexpensive, can be utilized in studying other enzymatic reactions that require divalent cations to promote activity. Furthermore, as a result of the simplicity of the methods, and advances in both liquid handling techniques and MALDI-TOF MS, sequence determination of DNA samples may be done automatically. The ability to directly sequence short dsDNA, without the need to prepare ssDNA PCR products, would provide an invaluable diagnostic tool for application to biological and medical research, and these techniques should be applicable to large genomic sequencing projects and the diagnosis of genetic defects. For example, sequencing of short ds oligonucleotides (∼30 nt) by combined use of exonuclease III digestion and MALDI-TOF MS is convenient and could be used for direct characterization of mutation sites underlying genetic diseases such as sickle cell anemia, β-thalassemia, phenylketoneuria, etc.23 Moreover, the facile degradation of dsDNA by exonuclease III (∼60 bases/min
at 22 °C, and 200 bases/min at 30 °C)24 affords other opportunities to sequence larger fragments. By controlling the rate of digestion of exonuclease III and removing aliquots from restriction digests obtained at a series of time points, a representative array of fragments is obtained that represents the entire DNA to be sequenced.24 By arranging the digested fragments on a DNA chip, their masses from many regions of the template could be directly measured in parallel by MALDI-TOF MS. By combining the strategy described above with our MS sequencing technique, direct sequencing of large genomic fragments is feasible.
(23) Sutherland, G. R.; Mulley, J. C. In Nucleic Acid Probes; Symons, R. H., Ed.; CRC Pres, Inc.: Boca Raton, FL, 1989; pp 159-203.
(24) Sorge, J. A.; Blinderman, L. A. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 92089212.
ACKNOWLEDGMENT This work was supported by the National Science Foundation, Grant CHE-9706904. J.A.C. was a Camille Dreyfus TeacherScholar (1994-1999). Received for review February 14, 2000. Accepted May 2, 2000. AC000181N
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