Anal. Chem. 1997, 69, 4349-4353
A Sequencing Method for RNA Oligonucleotides Based on Mass Spectrometry Konrad Faulstich, Karlheinz Wo 1 rner, Hannelore Brill, and Joachim W. Engels*
Institut fu¨ r Organische Chemie, Johann Wolfgang Goethe-Universita¨ t Frankfurt, Marie Curie-Strasse 11, D-60439 Frankfurt am Main, Germany
The increasing use and research in the so-called RNA world of oligoribonucleotides such as ribozymes, hairpins, duplexes, triple helices, and t-, m-, and modified RNAs requires a fast standard sequencing method. Standard procedures rely on twodimensional chromatographic techniques,1-3 Maxam Gilbert sequencing4-7 or reverse transcriptase Sanger sequencing.8,9 Recent developments deal with mass spectrometry, which offers a fast and sensitive method to study the primary structure of nucleic acids.10 There have been many studies in the last few years of the degradation of oligonucleotides such as tandem mass spectrometry using electrospray ionization,11-13 enzymatic reactions using exonucleases10,14 as well as polymerases,15,16and nozzle skimmer dissociation (NS) of ESI generated17,18 ions or spontane-
ous dissociation following matrix-assisted laser desorption/ionization (MALDI) DNA analysis.18-24 We report here a new method using RNA as the target molecule distinguishing very similar molecular weights of uridine (306.17) and cytidine (305.18) using simple instrumentation. There have been some investigations of DNA exonuclease sequencing in combination with HPLC25 with high mass accuracy up to 10 bases. Exonuclease sequencing of an unknown RNA sample has been argued to be impossible by existing methods.26 Here we report a sequencing strategy to distinguish all four nucleotides including U and C using the advantages of fast and accurate mass spectrometric technology. The method could be useful especially for oligoribonucleotides longer than 20 bases since the mass accuracy of some mass analyzers is a limiting factor. The four nucleotides are determined by different masses of fragments produced by 5′ f 3′-exonuclease digestion, U and C are distinguished in particular by different relative abundances of these fragments, resulting in much higher peak intensities for fragments containing 5′ terminal cytidines. This is caused by different rates of phosphodiester bond hydrolysis by the enzyme. The bond to a neighboring cytidine is hydrolized much more slowly than to an uridine. However, the ensemble of fragments produced by this enzymatic degradation procedure contains more fragments with 5′ terminal cytidines. We made the same observation with 5′ terminal adenosine oligoribonucleotides, but these fragments are easily detected by mass differences.. The method is applicable for delayed ion extraction (DE) MALDI27,28 as well as for continuous MALDI measurements. Digestion with 3′ f 5′ exonuclease does not show these differences in peak intensities.
(1) Sanger, F.; Brownlee, G. G.; Barell, B. G. J. Mol. Biol. 1965, 13, 373-398. (2) Brownlee, G. G.; Sanger, F. Eur. J. Biochem. 1969, 11, 395-399. (3) Silberklang, M.; Gillum, A. M.; RajBhandary, U. L. In Methods in Enzymology; Wu, R., Grossmann, L., Eds.; Academic Press Inc.: New York, 1979; Vol. 59, pp 58-109. (4) Maxam, A. M.; Gilbert, W. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 560564. (5) Stahl, D. A.; Krupp, G.; Stackebrandt, E. In Nucleic Acids Sequencing, a Practical Aproach; Howe, C. J., Ward, E. S., Eds.; IRL Press at Oxford University Press: Oxford, UK, 1989; pp 137-184. (6) Waldmann, R.; Gross, H. J.; Krupp, G. Nucleic Acids Res. 1987, 15, 7209. (7) Zhang, Y.; Liu, W.; Feng, Y.; Wang, T. P. Anal. Biochem. 1987, 163, 513516. (8) Sanger, F.; Nicklen, S.; Coulson, A. R. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5463-5467. (9) Hahn, C, S.; Strauss, E., G.; Strauss, J. H. In Methods in Enzymology; Academic Press Inc.: New York, 1989; Vol. 180, pp 121-163. (10) Pieles, U.; Zu ¨ rcher, W.; Scha¨r, M.; Moser, H. E. Nucleic Acids Res. 1993, 21, 3191-3196. (11) McLuckey, S. A.; Habibi-Goudarzi, S. J. Am. Chem. Soc. 1993, 115, 1208512095. (12) Wolter, M. A.; Engels, J. W. Eur. Mass Spectrom. 1995, 1, 583-590. (13) Ni, J.; Pomerantz, S. C.; Rozenski, J.; Zhang, Y.; McCloskey, J. A. Anal. Chem. 1996, 68, 1989-1999.
(14) Limbach, P. A.; McCloskey, J. A.; Crain, P. F. Nucleic Acids Res. Symp. Ser. 1994, 31, 127-128. (15) Roskey, M. T.; Juhasz, P.; Smirnov, I. P.; Takach, E. J.; Martin, S. A.; Haff, L. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4724-4729. (16) Ko ¨ster, H.; Tang, K.; Fu, D.-J.; Braun, A.; van den Boom, D.; Smith, C. L.; Cotter, R. J.; Cantor, C. R. Nat. Biotechnol. 1996, 14, 1123-1128. (17) Loo, J. A.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1988, 2, 207-210. (18) Little, D. P.; Chorush, R. A.; Speir, J. P.; Senko, M. W.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1994, 116, 4893-4897. (19) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (20) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (21) Nordhoff, E.; Karas, M.; Cramer, R.; Hahner, S.; Hillenkamp, F.; Kirpekar, F.; Lezius, A.; Muth, J.; Meier, C.; Engels, J. W. J. Mass Spectrom. 1995, 30, 99-112. (22) Little, D. P.; McLafferty, F. W. J. Am. Chem. Soc. 1995, 117, 6783-6784. (23) Wu, K. J.; Shaler, T. A.; Becker, H. Anal. Chem. 1994, 66, 1637-1645. (24) Nordhoff, E.; Cramer, R.; Karas, M.; Hillenkamp, F.; Kirpekar, F.; Kristiansen, K.; Roepstorff, P. Nucleic Acids Res. 1993, 21, 3347-3357. (25) Glover, R. P.; Sweetman, G. M. A.; Farmer, P. B.; Roberts, G. C. K. Rapid Commun. Mass Spectrom. 1995, 9, 897-901. (26) Kirpekar, F.; Nordhoff, E.; Kristiansen, K.; Roepstorff, P.; Lezius, A.; Hahner, S.; Karas, M.; Hillenkamp, F. Nucleic Acids Res. 1994, 22, 3866-3870.
Synthetic oligoribonucleotides up to 22 bases have been sequenced by observing different kinetics in exonucleaseinduced phosphodiester bond hydrolysis and detecting the fragments by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Common mass spectrometric sequencing methods have disadvantages concerning read length, cost, and specialist instrumentation using RNA as the target molecule because uridine and cytidine have similar masses. Now we are in the position to distinguish U and C by different peak intensities. The method proposed has been verified using specific endonucleases and 13C-labeled nucleotides. This new nongel-based and nonlabeling sequencing strategy offers first RNA sequencing data using the advantages of fast and accurate MALDI-TOF-MS. Preparation steps and measurements are performed in less than 1 h.
S0003-2700(96)01186-9 CCC: $14.00
© 1997 American Chemical Society
Analytical Chemistry, Vol. 69, No. 21, November 1, 1997 4349
Table 1. Protocols for Enzymatic Digestion of Oligoribonucleotides oligoribonucleotide 1 digestion with 5′ f 3′ bovine spleen phosphodiesterase digestion with 3′ f 5′ Crotalus durissus phosphodiesterase digestion with RNase CL3 from chicken liver
oligoribonucleotide 2
oligoribonucleotide 3
1290 pmol of RNA in 9 µL of water, 530 pmol of RNA in 9 µL of water, 740 pmol of RNA in 15.2 µL of water, 24 mU (6 µL) of enzyme, 22 °C 24 mU (6 µL) of enzyme, 22 °C 24 mU (6 µL) of enzyme, 22 °C 530 pmol of RNA in 9 µL of water, 740 pmol of RNA in 15.2 µL of water, 0.6 mU (3 µL) of enzyme in 0.1 M 0.6 mU (3 µL) of enzyme in 0.1 M ammonium citrate, pH 5.5, 40 °C ammonium citrate, pH 5.5, 40 °C 1290 pmol of RNA in 9 µL of water, 400 mU of enzyme in 8 µL of 8 M urea, 50 °C
Table 2. Masses of Nucleotide Leaving Groups during Enzymatic Degradation nucleotide leaving group
mass (Da)
G
A
C
U
345.20
329.20
305.18
306.17
Table 3. Masses and Sequences (5′ f 3′ Direction) of Oligoribonucleotide 1 Fragments Produced by Enzymatic Digestion (Corresponding to Figure 1) peak 1 2 3 4 5 6 7 2 a
sequence
mass calcd (Da) mass (Da) mass diffa (Da)
(a) With 5′ f 3′ Phosphodiesterase CAUGUGAC 2505.5 2503.7b AUGUGAC 2200.3 2199.1 UGUGAC 1871.1 1871.1 GUGAC 1565.0 1565.9 UGAC 1220.0 1221.3 GAC 913.6 915.3 AC 568.4 571.1 AUGUGAC
(b) With RNase CL3 2200.3 2199.1
304.6 328.0 305.2 344.6 306.0 344.2 /
Peak x - peak (x + 1). b Calculated: m/z ) 2504.5 (z ) -1).
MATERIALS AND METHODS Linear continuous MALDI-time of flight (TOF) mass spectrometry was performed using a Fisons VG TOF spec mass spectrometer (for an 8 mer and a 22 mer) and a PerSeptive Biosystems Voyager RP-DE mass spectrometer (for a 16 mer) containing a UV nitrogen laser emitting at 337 nm. The laser pulse width is 4 ns. Source voltage for experiments was between 25 000 and 27 000V. Mass accuracy is
