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Sequence Confirmation of Modified Oligonucleotides Using Chemical Degradation, Electrospray Ionization, Time-of-Flight, and Tandem Mass Spectrometry Julie Farand* and Michael Beverly Department of Process Research, Merck Research Laboratories, 2950 Wilderness Place, Boulder, Colorado 80301 We report the sequencing of highly modified oligonucleotides containing a mixture of 2′-deoxy, 2′-fluoro, 2′-Omethyl, abasic, and ribonucleotides. The passenger and guide strands each containing 48% and 86% of modified nucleotides, respectively, are representative sequences of synthetic short interfering RNAs (siRNAs). We describe herein the sequence confirmation of both strands using a series of robust chemical reactions, followed by analysis via ESI-TOF and ion trap mass spectrometry (ITMS). The following method enables the rapid sequence confirmation of highly modified oligonucleotides. Synthetic oligonucleotides are finding increased applications as novel therapeutics, diagnostic agents, and biological research tools.1,2 The incorporation of unnatural nucleotides into oligonucleotides has led to significant improvements in their physical properties.3-5 Yet, characterization and structure elucidation of oligonucleotides still remains a challenge. The confirmation of the primary sequence of oligonucleotides is essential regardless of their intended function or application. Highly modified oligonucleotides often resist common methods of sequencing, such as enzymatic digestion, used for DNA and RNA. As a result, pressure for the development of a sequence confirmation methodology exists among emerging RNA-based process research, manufacturing quality control, and regulatory departments. A variety of chemical, biochemical, and analytical techniques have been employed to determine the sequence of DNA and RNA.6-8 However, most methods have been limited to unmodified oligonucleotides. Chemical methods have been used for the digestion of DNA and RNA, followed by analysis using polyacrylamide gel electrophoresis (PAGE). The success of these methods relies on the following conditions: only the desired bases are * To whom correspondence should be addressed. E-mail: julie_farand@ merck.com. (1) Opalinska, J. B.; Gewirtz, A. M. Nat. Rev. Drug Discovery 2002, 1, 503– 514. (2) Goodchild, J. Curr. Opin. Mol. Ther. 2004, 6, 120–128. (3) Morrissey, D. V.; Zinnen, S. P.; Dickinson, B. A.; Jensen, K.; McSwiggen, J. A.; Vargeese, C.; Polisky, B. Pharm. Discovery 2005, 5, 16–20. (4) Verma, S.; Eckstein, F. Annu. Rev. Biochem. 1998, 67, 99–134. (5) Manoharan, M. Curr. Opin. Chem. Biol. 2004, 8, 570–579. (6) Limbach, P. A. Mass Spectrom. Rev. 1996, 15, 297–336. (7) Maxam, A. M.;.; Gilbert, W. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 560– 564. (8) Peattie, D. A. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 1760–1764.
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chemically modified, cleavage of the phosphate backbone must occur at the modified site, and PAGE must clearly separate the resulting fragments according to their size in order to create a complete sequence ladder. PAGE is labor-intensive and requires fluorescent dye labeling or radiolabeling. Moreover, the presence of secondary structures and modified bases has shown to affect the electrophoretic mobility of the fragments during PAGE analysis (e.g., band compression).9,10 The Sanger chain-termination method is an additional sequencing technique, whereby DNA polymerase (or reverse transcriptase for RNA) is utilized to transcribe primary DNA/RNA into cDNA in the presence of a radiolabeled primer, dNTPs, and ddNTPs.11,12 Since ddNTPs lack the 3′ terminal hydroxyl, the transcription is terminated and fragments of various lengths are generated, thus creating a sequence ladder using PAGE. Because a primer is required, the Sanger method is generally difficult when using short oligonucleotides (∼10-20 mer).13 More importantly, this method relies on building a cDNA strand which captures the sequence information of the unknown DNA/RNA. This method will not accurately sequence abasic nucleotides or distinguish nucleotides bearing the same heterocycle with different 2′ modifications (e.g., 2′-OMeU vs 2′-fluU) (Table 1). Recognition and digestion of modified oligonucleotides using enzymatic approaches, such as endo- and exonucleases, is problematic. Because of the incorporation of foreign nucleotides and modified phosphodiesters, only partial sequencing can be obtained using these techniques.14-16 As part of the drug development process, the nature of the modifications is constantly evolving and enzymatic activity toward such modifications is likely to vary. Therefore, direct sequencing of oligonucleotides by mass (9) Frank, R.; Ko ¨ster, H. Nucleic Acids Res. 1979, 6, 2069–2087. (10) Li, S.; Haces, A.; Stupar, L.; Gebeyehu, G.; Pless, R. C. Nucleic Acids Res. 1993, 21, 2709–2714. (11) Sanger, F.; Nicklen, S.; Coulson, A. R. Proc. Natl. Acad. U.S.A. 1977, 74, 5463–5467. (12) Shahn, C.; Strauss, E. G.; Strauss, J. H. Methods Enzymol. 1989, 180, 121– 130. (13) Tang, J.; Roskey, A. M.; Agrawal, S. Anal. Biochem. 1993, 212, 134–137. (14) Shaw, J.-P.; Kent, K.; Bird, J.; Fishback, J.; Froehler, B. Nucleic Acids Res. 1991, 19, 747–750. (15) Kawasaki, A.; Casper, M. D.; Freier, S. M.; Lesnik, E. A.; Zounes, M. C.; Cummins, L. L.; Gonzalez, C.; Cook, P. D. J. Med. Chem. 1993, 36, 831– 841. (16) Egli, M.; Minasov, G.; Tereshko, V.; Pallan, P. S.; Teplova, M.; Inamati, G. B.; Lesnik, E. A.; Owens, S. R.; Ross, B. S.; Prakash, T. P.; Manoharan, M. Biochemistry 2005, 44, 9045–9057. 10.1021/ac8011158 CCC: $40.75 2008 American Chemical Society Published on Web 08/27/2008
Table 1. Natural and Chemically Modified Nucleotides Utilized in the Passenger and Guide Strands
spectrometry is increasingly becoming the preferred alternative to enzymatic sequencing due to its efficiency, sensitivity, and accuracy. Matrix-assisted laser desorption/ionization (MALDI) has been widely used for DNA and RNA analysis.17 Although most mass spectrometry based sequencing studies have been conducted on unmodified oligonucleotides, it has been shown that RNA and 2′fluoro modifications dramatically reduce the fragmentation of oligonucleotides in MALDI and would therefore limit sequencing information of highly modified oligonucleotides.18 In addition, few applications of MS/MS using modified oligonucleotides have been reported.19-21 According to the nature of the modification, fragmentation can be difficult and may differ from patterns observed with DNA and RNA.22,23 The use of ITMS for sequence elucidation of oligonucleotides g15 nucleotides (nt) in length has proven to be a challenging task due to limited mass accuracy and resolution of the analyzer.24 Also, spectral interpretation can be difficult and time-consuming for large oligonucleotides, and as a result, tandem mass spectrometry has mainly been applied toward shorter oligonucleotides. The limitations of the techniques mentioned above warrant the development of novel sequence confirmation and analysis methods for highly modified oligonucleotides. As part of our research efforts on RNA-based therapeutics, we sought to develop a broadly applicable, robust method for the sequence confirmation of highly modified oligonucleotides. Our strategy involves the chemical degradation of intact oligonucleotides to generate all fragments containing the 3′ terminal hydroxyl. Using mass spectrometry analysis, a sequence ladder is created by sorting the fragment masses in order (N to N-x, where x ) (n - 1) and n is the number (17) Nordhoff, E.; Kirpekar, F.; Roepstorff, P. Mass Spectrom. Rev. 1996, 15, 67–138. (18) Tang, W.; Zhu, L.; Smith, L. M. Anal. Chem. 1997, 69, 302–312. (19) Barry, J. P.; Vouros, P.; Schepdael, A. V.; Law, S.-J. J. Mass Spectrom. 1995, 30, 993–1006. (20) McLuckey, S. A.; Habibi-Goudarzi, S. J. Am. Soc. Mass Spectrom. 1994, 5, 740–747. (21) Monn, S. T. M.; Tromp, J. M.; Schu ¨ rch, S. Chimia 2005, 59, 822–825. (22) McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1992, 3, 60–70. (23) Wu, J.; McLuckey, S. A. Int. J. Mass Spectrom. 2004, 237, 197–241. (24) Ho ¨lzl, G.; Oberacher, H.; Pitsch, S.; Stutz, A.; Huber, C. G. Anal. Chem. 2005, 77, 673–680.
of nucleotides in the strand) (Figure 1). Since the mass difference between each sequential fragment affords the identity of the cleaved nucleotide, the sequence of the intact oligonucleotide is established and confirmed. Herein, we demonstrate the application of this methodology on both the passenger and guide strands of a chemically modified synthetic siRNA. We selected ESI with time-of-flight (TOF) or ion trap mass spectrometry (ITMS) to analyze the chemical digestion products from highly modified oligonucleotides.25-27 A TOF mass analyzer can distinguish the 1 u mass difference between nucleotides uridine and cytosine and provides excellent mass accuracy. However, during accurate mass analysis, short fragments (1-3 nt in length) containing the last nucleotides of the strand are poorly retained by LC-MS and elute with the solvent front. Therefore, instead of using ESI-TOF, we use tandem mass spectrometry to confirm the sequence of the final two or three nucleotides (5000 u were deconvoluted in order to rapidly obtain the average masses (See Experimental Section). In many cases, different chemical reactions yielded the same fragment. Therefore, we have specified the reactions which have generated the observed masses listed in Tables 5 and 6. Total Ion Chromatogram. The extent of digestion using diethyl pyrocarbonate/piperidine with strands 1 and 2 is illustrated in Figures 4 and 5. Unlabeled peaks correspond to masses of unknown structures which may be the result of decomposition or unknown reaction pathways. The direction of these chemical digestions (5′ f 3′ or 3′ f 5′) deserves mention. Unlike enzymes, whereby exonucleases can be selected according to their direction of DNA or RNA digestion, the direction and mechanism of a chemical digestion has not been extensively studied. The application of this Analytical Chemistry, Vol. 80, No. 19, October 1, 2008
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Figure 4. Total ion chromatogram (TIC) of 1 after DEPC/piperidine digestion. / represents fragments containing the 5′ terminal hydroxyl in the presence or absence of a 3′ terminal phosphate.
Figure 5. Total ion chromatogram (TIC) of 2 after DEPC/piperidine digestion.
methodology generated all fragments N to N-20 for 1 and N to N-18 for 2, containing the 3′-end terminal hydroxyl in the presence or absence of the 5′-end terminal phosphate. The propensity of these 3′ hydroxyl-terminated fragments has also been observed with MALDI-TOF.24 Interestingly, the detection of fragments containing the 5′ terminal hydroxyl has been limited to strand 1 under the following conditions: NH2OH, piperidine, aniline, or sodium hydroxide. Furthermore, the observed fragments were the result of a modification/cleavage adjacent to deoxyribonucleotides and 2′-fluorouridine only. The scarcity of fragments containing the 5′ terminal hydroxyl does, however, simplify the analysis without jeopardizing valuable sequence information. We are currently investigating the mechanism of these reactions in order to elucidate this 7420
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interesting observation. Nonetheless, fragments containing the 3′-end hydroxyl have allowed us to confirm the sequences of 1 and 2 via ESI-TOF and ITMS analysis (Table 5-7). Ion Trap Mass Spectrometry. Fragments with masses
