Anal. Chem. 1996, 68, 3405-3412
Detailed Characterization of Antisense DNA Oligonucleotides T. Keough,* J. D. Shaffer, and M. P. Lacey
The Procter and Gamble Company, Miami Valley Laboratories, P.O. Box 538707, Cincinnati, Ohio 45253-8707 T. A. Riley, W. B. Marvin, M. A. Scurria, J. A. Hasselfield, and E. P. Hesselberth
JBL Scientific, Inc., 277 Granada Drive, San Luis Obisbo, California 93401-5809
We have developed methods for verification of the structures of novel, chemically synthesized oligonucleotides having alternating methylphosphonate/phosphodiester internucleotide linkages. Matrix-assisted laser desorption ionization mass spectrometry was used to measure the molecular masses of full-length oligonucleotides, failure synthesis products, and degradation products formed by enzymatic and chemical means. These measurements provide detailed structural information, including molecular mass, length, base composition, complete nucleotide sequence, and confirmation of the sugar moieties and internucleotide linkages. Antisense oligonucleotides are a relatively new class of molecules that are potentially effective as therapies for viral infections, cancers, and other diseases. Oligonucleotides function as antisense agents by binding to complementary strands of mRNA. This impedes the translation of specific disease-related proteins, and certain classes of bound antisense oligonucleotides stimulate the action of Ribonuclease H, an enzyme that selectively cleaves and destroys the bound mRNA.1 Natural phosphodiester oligonucleotides have proven to be ineffective against genetic targets in living systems because of difficulties in delivering those molecules to cells and because of rapid digestion by nucleases. Considerable effort has, therefore, been directed toward the production of modified oligonucleotides, such as phosphorothioates, methylphosphonates, carbamates, and phosphoroamidates. Modified oligonucleotides are nuclease resistant, have enhanced delivery to target cells, and have shown improved specificities for their targeted mRNA.1,2 Crooke3 and Kambhampati and co-workers4 have recently discussed some of the regulatory issues associated with antisense oligonucleotides. Key issues include chemistry, pharmacology, adsorption, distribution, metabolism, excretion, and toxicology. Identification of these new drug substances is of fundamental importance. Verification of identity requires determination of the molecular mass and length of the oligonucleotide as well as its base composition, its sequence, and the identity of the sugar moieties and internucleotide linkages. The methods used for (1) Cohen, J. S.; Hogan, M. E. Sci. Am. 1994, 76-82. (2) Fathi, R.; Haung, Q.; Coppola, G.; Delaney, W.; Teasdale, R.; Kreig, A. M.; Cook, A. F. Nucl. Acids Res. 1994, 22, 5416-5424. (3) Crooke, S. T. Antisense Res. Dev. 1993, 3, 301-306. (4) Kambhapati, R. V. B.; Chiu, Y. Y.; Chen, C. W.; Blumenstein, J. J. Antisense Res. Dev. 1993, 3, 405-410. S0003-2700(96)00298-3 CCC: $12.00
© 1996 American Chemical Society
proof of structure should be specific, allowing identification of modified bases, addition or deletion products, and depurination products. Where possible, determination of the base sequence must be complete, and it must be shown that any chemical or enzymatic manipulations do not adversely affect either the bases or the backbone. Characterization of modified oligonucleotides is particularly challenging if they are nuclease stable. Many of the methods previously developed for detailed characterization of phosphodiester oligonucleotides simply cannot be applied to enzyme-resistant antisense DNA oligonucleotides. In this paper, we demonstrate the use of time-of-flight matrixassisted laser desorption ionization (MALDI) mass spectrometry for verification of the structures of two novel oligonucleotides having alternating methylphosphonate/phosphodiester (MP/ PDE) internucleotide linkages. The basic approach, which is also applicable to other classes of antisense DNA oligonucleotides, first involves analyses of purified full-length materials and the corresponding failure synthesis products.5 Failure synthesis analysis provides easy confirmation of oligonucleotide length, partial base composition, and partial sequence information from the 5′-end of the molecule. The remaining base composition and sequence information is obtained by MALDI analyses of degradation products, formed by various enzymatic and chemical means. Molecular mass measurements, which reflect subtle changes in structure, also allow verification that the bases, sugar moieties, and internucleotide linkages have not been affected by the enzymatic and chemical procedures used for sequencing. The method is sensitive, requiring only low picomoles of material for analysis, and no labeling of either the 5′- or the 3′-end is required. The failure synthesis method should be adaptable to other classes of antisense oligonucleotides, as already demonstrated with methylphosphonates.5 In fact, failure or termination synthesis should prove generally useful for the characterization of biomolecules synthesized in a stepwise fashion on solid supports. We recently employed intentional termination synthesis for the rapid identification of biologically active peptides isolated from supportbound combinatorial libraries.6,7 (5) Keough, T.; Baker, T. R.; Dobson, R. L. M.; Lacey, M. P.; Riley, T. A.; Hasselfield, J. A.; Hesselberth, P. E. Rapid Commun. Mass Spectrom. 1993, 7, 195-200. (6) Youngquist, R. S.; Fuentes, G. R.; Lacey, M. P.; Keough, T. Rapid Commun. Mass Spectrom. 1994, 8, 77-81. (7) Youngquist, R. S.; Fuentes, G. R.; Lacey, M. P.; Keough, T. J. Am. Chem. Soc. 1995, 117, 3900-3906.
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EXPERIMENTAL SECTION Oligonucleotide Synthesis and Purification. Chirally pure deoxymethylphosphonate dimers were prepared by coupling the appropriate methylphosphonamidite monomer to the corresponding 3′-silyl-protected 5′-alcohol. The racemic phosphonite intermediate was oxidized with a peroxide to prevent further skewing of the R/S ratio. The diastereomers were separated on normalphase silica with good recoveries. The silyl groups were removed with fluoride, and the corresponding 3′-alcohol was derivatized with diisopropyl β-cyanoethylphosphorochlorodite to form the dimer amidite synthon. The oligonucleotides were synthesized from the 3′- to 5′-end, beginning with a methylphosphonamidite monomer, to form a racemic methylphosphonate linkage between the penultimate and the ultimate bases on the 3′-end. The rest of the oligomer was synthesized with chirally pure Rp methylphosphonate dimer βcyanoethyl amidite synthons. This synthetic strategy results in alternating methylphosphonate/phosphodiester linkages in the oligomer. The last trityl group was removed on the synthesizer. The oligomers were cleaved from the solid support and deprotected using ethylenediamine.8 Crude samples containing failure sequences were desalted with reverse-phase cartridges and dried using a speed-vac. Purification to obtain pure full-length materials was carried out using normal-phase conditions on Cyclobond columns.9 MALDI Mass Spectrometry. All MALDI mass spectra were obtained on a Vestec VT 2000 linear time-of-flight mass spectrometer (PerSeptive Biosystems, Vestec Division, Houston, TX) previously described.5 Spectra were produced from 50-100 laser pulses, which were accumulated in a transient digitizer, TR8828D (LeCroy, Chestnut Ridge, NY), using LeCroy-supplied software. Spectra were imported into a Compaq 386/33 (Houston, TX) and processed using a commercially available software program (Galactic Industries, Salem, NH). The molecular masses of the full-length products were determined using bovine insulin as an internal mass standard, and they were confirmed by independent ion spray measurements (MH+ 12-mer ) 3535.9 Da; MH+ 18mer ) 5398.9 Da) on a PE Sciex API III triple-quadrupole mass spectrometer (Thornhill, Ontario, Canada). Calibration of the nuclease and chemical digestion mixtures was accomplished using the calculated chemically averaged masses of the full-length oligonucleotides (12-mer ) 3536.3 Da, 18-mer ) 5399.9 Da) and the known mass of the UV-absorbing matrix (ferulic acid MH+ ) 195.2 Da) as internal mass standards. HPLC. All HPLC experiments were carried out on a Waters 600 MS HPLC equipped with a 490E programmable multiplewavelength detector operated at 260 nm (Waters Corp., Milford, MA). Normal-phase separations were carried out on a 4.6 mm × 250 mm Cyclobond I 2000 β-cyclodextrin column, 5 µm particle size, made by Advanced Separation Technologies, Inc. (Whippany, NJ, part no. 20024). Separations were carried out isocratically with the solvent system consisting of 0.1 M NH4Ac (pH ) 6)/ acetonitrile (45:55). Reversed-phase separations were carried out on a 2 mm × 150 mm Delta-Pak C18 column, 300 Å pore size, 5 µm particles. The column was purchased from Waters Corp. (part no. WAT023650). The gradient was 0-50% B over 30 min, where solvent A is 0.1 M NH4Ac (pH ) 7) and B is acetonitrile. The column flow rate was 200 µL/min. (8) Hogrefe, R. I.; Reynolds, M. A.; Vaghefi, M. M.; Young, K. M.; Riley, T. A.; Klem, R. E.; Arnold, L. J., Jr. Methods Mol. Biol. 1993, 20, 143-164. (9) Snyder, L.; Riley, T. A.; Reynolds, M. A.; Klem, R. E., unpublished.
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Sample Preparation for MALDI. Full-length oligonucleotides and the various digest mixtures were dissolved in deionized water to a concentration of about 10 pM/µL. Small volumes of the analyte solutions (typically 2-5 µL) were mixed with equal volumes of a ferulic acid solution, which was prepared fresh daily at a concentration of 10 g/L in aqueous TFA (0.1%)/acetonitrile (70:30). One microliter aliquots of the resulting solutions were loaded onto the end of a stainless-steel solids probe and allowed to dry at ambient temperature. The probe tip was thoroughly washed with cold deionized water prior to analysis. Snake Venom Phosphodiesterase Digestion (3′-5′ Sequencing). The snake venom phosphodiesterase (SVP) digests of the full-length oligonucleotides were carried out in Tris buffer (0.11 M Tris‚HCl with 0.2 mM MgCl2) at a pH of 8.9 (adjusted with 1 N NaOH). SVP (Worthington Biochemical Corp., Freehold, NJ, lot no. 32N288L) was first dissolved in the Tris buffer at a concentration of 0.003 unit/µL (0.3 mg/2 mL). The oligonucleotides were dissolved in separate Tris buffer solutions at a concentration of 0.1 mg/100 µL. Five microliters of the SVP solution was mixed with 25 µL of the oligonucleotide solutions, and the reactions were carried out at 37 °C for 15 min. Reactions were quenched by freezing after addition of 35 µL of acetonitrile. SVP digests were carried out with 0.1-0.3 mg of full-length oligonucleotides when HPLC isolation of individual digestion products was required. The HPLC isolates were dried and then reconstituted in 250 µL of deionized water prior to use. Smallscale SVP subdigests of various HPLC isolates were carried out for 15 min at 37 °C by mixing 10 µL of the reconstituted products with SVP in Tris buffer. Calf Spleen Phosphodiesterase Digestion (5′-3′ Sequencing). Attempted calf spleen phosphodiesterase (CSP) digests of oligonucleotides were carried out in 0.1 M NH4Ac, with the pH adjusted to 5.5 using glacial acetic acid. The NH4Ac buffer was used because it can be easily removed on a speed-vac after the digestion has been carried out. CSP (Boehringer Mannheim, Indianapolis, IN, lot no. 14091120-21/April 95) was supplied in solution at an activity of 2 units/mL. The oligonucleotides were dissolved in the buffer at a concentration of 0.1 mg/100 µL. Three microliters of the CSP solution (0.006 unit) was added to 100 µL solutions of the oligonucleotides, which were heated at 37 °C for variable time periods (15 min to overnight). Mung Bean Nuclease I Digestion. The mung bean nuclease I digestions of oligonucleotides were carried out in a buffer consisting of 30 mM NH4Ac, 50 mM NH4Cl and 1 mM zinc acetate, pH ) 4.6. Mung bean nuclease I (Boehringer Mannheim, lot no. GHA122/July 95) was supplied in a storage buffer having an activity of 45 units/µL. One microliter of that solution was diluted into 100 µL of the digestion buffer (A) prior to use with the 12mer. The original enzyme solution was diluted 10-fold (B) prior to use with the 18-mer. The oligonucleotides were dissolved in separate buffer solutions (0.1 mg/100 µL). The mung bean nuclease solution (20 µL of A) was added to 50 µL of the 12-mer solution, while 10 µL of mung bean nuclease solution (B) was added to 50 µL of the 18-mer solution. The reactions proceeded 15 min at 37 °C. Endonuclease S1 Digestion. The endonuclease S1 digestion of the 12-mer was carried out in a buffer consisting of 33 mM NH4Ac, 50 mM NH4Cl, and 0.03 mM ZnSO4, pH adjusted to 4.5 with glacial acetic acid. Endonuclease S1 (Boehringer Mannheim, lot no. 13504525-44/May 96) was supplied in a storage buffer
Figure 1. MALDI mass spectra of the 12-mer: (a) the purified oligonucleotide and (b) the crude synthetic products.
having an activity of 400 units/µL. The enzyme solution (1 µL) was diluted 40-fold in buffer prior to use. The oligonucleotide was dissolved in a separate buffer solution at a concentration of 0.1 mg/100 µL. The diluted endonuclease S1 solution (10 µL) was mixed with 10 µL of the 12-mer solution, and the reaction proceeded at 37 °C for 60 min. Base-Catalyzed Hydrolysis. Large-scale base-catalyzed hydrolyses of the full-length oligonucleotides were carried out by dissolving about 150 nM (0.5 mg) of oligonucleotides in 1.5 mL of 3% NH4OH. Reactions were typically carried out at 75 °C for 1.5 h. The digestion products were desalted on C18 reversed-phase SEP-PAK cartridges (Millipore Corp., Milford, MA) as previously described.5 Small-scale hydrolyses of HPLC isolates were carried out by mixing 8% of the isolates (20 µL of the components that had been reconstituted in 250 µL of deionized water) with 20 µL of 6% NH4OH. These reactions were carried out at 75 °C for 1.5-2 h. The products were dried on a speed-vac to remove excess base and were reconstituted in 20 µL of H2O prior to analysis by MALDI. RESULTS AND DISCUSSION MALDI Mass Spectra of Oligonucleotides Having Alternating Methylphosphonate/Phosphodiester Internucleotide Linkages. The positive ion MALDI mass spectrum obtained from about 5 pmol of a purified 12-mer, d(CA-TC-TT-CC-TC-GT), where - indicates phosphodiester linkages, is given in Figure 1a. The spectrum is dominated by an intense protonated molecule (MH+)
designated (12-1)+, where the 3′-end of the oligonucleotide is defined as nucleotide 1 and the 5′-end is nucleotide 12. The measured chemically averaged molecular mass (3536.3 Da) is in close agreement with the calculated value of 3536.3 Da. The mass measurement error is 2500 Da) because the mass resolution of the linear time-of-flight analyzer is too low. This problem is illustrated in the MALDI mass spectrum of the mixture of hydrolysis products from the full-length 12-mer, Figure 3. The 3′- and 5′-nucleotides cannot be accurately assigned (both indicated with an X) because the MH+ molecular masses of (12-2) and (11-1) overlap. This problem also affects assignment of the next phosphodiester dinucleotides from each end of the molecule, even though the MH+ molecular ions of (124) and (9-1) are partially resolved. This results because the mass
Figure 4. Normal-phase HPLC separation of the SVP digestion products of the 12-mer.
Figure 5. MALDI mass spectrum of the products formed by hydrolysis of (12-5). The unlabeled doublets correspond to the products that contain a free methylphosphonate group on either the 3′- or 5′-end.
differences between [(11-1) and (9-1)] as well as [(12-2) and (124)] cannot be accurately determined. In spite of this limitation, half of the overlapping dinucleotide sequence, residues (9-4), can be verified from this spectrum, b. The mass resolution (R) needed to separate ions differing in mass by ∆M decreases as mass (M) decreases (R ) M/∆M). Therefore, it should be possible to obtain complete sequence information from both ends of the molecule by hydrolysis of appropriate lower-mass nuclease digestion products. SVP digestion of the 12-mer produced a number of lower-mass components, including (12-3), (12-5), (12-7), (12-9), (p6-1), and (p4-1), as shown in Figure 2. HPLC was used to separate those components, and the products identified by mass are indicated on the chromatogram shown in Figure 4. Obviously, (12-5) and (p4-1) span the entire sequence of the 12-mer, and hydrolysis of those two components should allow completion of the overlapping dinucleotide sequence b. The two components were isolated for base subdigestion, and the MALDI mass spectrum of the hydrolysis products from (12-5) is given in Figure 5. The expected quartet of ions resulting from hydrolysis of the terminal nucleotides collapsed to a doublet, because C is present at both ends of the molecule. The hydrolysis products (12-6) and (11-5) have the same molecular masses, as do the products (p11-5) and (12-6p). The other quartets of ions are clearly resolved, and verification of the complete dinucleotide sequence of (12-5) was achieved, b.
Completion of the sequence at the 3′-end of the molecule was more difficult than that for the 5′-end. Sequence confirmation was not obtained by hydrolysis of (p4-1), even though it was hydrolyzed for 3.5 h at 75 °C in 3% NH4OH. Expected products (p4-2) and (3-1) were not observed. Increasing the concentration of base to 30% had no apparent effect. A number of common MALDI matrices were evaluated with the hopes of improving detection of the low-mass hydrolysis products. Again, they were not detected. Detection of very-low-mass hydrolysis products, trimers and lower, has been a consistent problem throughout this study. Completion of the overlapping sequence at the 3′-end of the oligonucleotide required additional nuclease digests of the fulllength material to produce a higher-mass fragment containing that end of the molecule. CSP, endonuclease S1, and mung bean nuclease were evaluated for this purpose. CSP yielded no reaction, while mung bean nuclease and endonuclease S1 did react. The mixture of products from mung bean nuclease digestion corresponded to a series of oligonucleotides and 5′-phosphoryl oligonucleotides, (12-3), (12-5), (12-7), and (12-9) and (p10-1), (p8-1), (p6-1) and (p4-1), in agreement with the known specificity of the enzyme.35,36 This digest provided complete confirmation of the structure given in a. However, the mixture was complex. The MALDI spectrum of the endonuclease S1 mixture was quite different, and it showed two dominant components, (12-7) and (p6-1), in addition to the full-length 12-mer. Initial attempts to isolate (p6-1) from the endonuclease S1 digest were complicated because (p6-1) and (12-1) were not chromatographically resolved on our normal-phase HPLC system. However, two approaches proved successful for isolation of the component containing the 3′-end. The simplest involved rechromatographing the fraction containing (12-1) and (p6-1) on a C18 reversed-phase column. Nearly baseline resolution was obtained between these two components. Alternatively, that fraction was treated with alkaline phosphatase, an enzyme that exhibits nonspecific phosphomonoesterase activity.37 The resulting mixture was rechromatographed on the β-cyclodextrin column, and (6-1) and (12-1) were easily resolved. Both (p6-1) and (6-1) were subjected to subsequent base hydrolysis. The MALDI spectra from (p6-1) and (6-1) showed the highermass hydrolysis products (5-1) and (p6-2) or (5-1) and (6-2). Both experiments confirmed the pC-5′ (or C-5′) and the T-3′ residues, b. Repeated attempts to detect (3-1), (p6-4), or (6-4) failed, and it was not possible to directly confirm (3-2) as (GC) using this approach. However, this last dinucleotide can be established by consideration of the available base composition information. The complete base composition of the 12-mer was determined above as (C5T5AG). The combined set of hydrolysis experiments (b) establishes the composition of 10 of the nucleotides, (12-4) + 1 ) C4T5A. The composition of (3-2) can be determined as (GC) by difference. Only 32 sequences are consistent with the structure given in b. Final sequence verification of the 12-mer was achieved by comparing the overlapping sequences from a and b. The 5′-nucleotide (residue 12) was confirmed as C from base hydrolysis of (12-5), b. The dinucleotide (12-11) is (CA) according to a. Since residue 12 is C, residue 11 must be A. The phosphodiester dinucleotide (11-10) is (AT) according to b. Since 11 is A, 10 (35) Mikulski, A. J.; Laskowski, M., Jr. J. Biol. Chem. 1970, 245, 5026-5031. (36) Martin, S. A.; Ulrich, R. C.; Meyer, W. L. Biochim. Biophys. Acta 1986, 867, 76-80. (37) Garen, A.; Levinthal, C. Biochim. Biophys. Acta 1960, 38, 470-483.
Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
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must be T. The methylphosphonate dinucleotide (10-9) is (TC) according to a. Since 10 is T, 9 must be C. This process is carried out to completion, giving the final result shown in c. 11
9
7
5
3
1
5′ CA-TC-TT-CC-TC-GT 3′
(c)
These data confirm the one unique sequence of the 12-mer and exclude the other 16 777 215 theoretical possibilities for an oligonucleotide of that length. This sequence confirms the synthetic procedure. Sequence Verification of the 18-mer. The same basic strategy was used to verify the sequence of the 18-Mer. First, the methylphosphonate dinucleotide sequence was established. Seven of the nine methylphosphonate dinucleotides were obtained directly from the failure synthesis analysis. The last two dinucleotides at the 3′-end were verified by MALDI analysis of the products formed by SVP digestion of (p10-1). The overlapping phosphodiester dinucleotide sequence was established by MALDI analyses of the base hydrolysis products of (18-1), (18-11), and (p10-1). Then, the one unique sequence of the 18-mer was verified by combining the MP dinucleotide sequence and the PDE dinucleotide sequence, as was discussed in detail for the 12-mer. This procedure excludes the more than 1010 other sequences that are at least theoretically possible for oligonucleotide 18-mers. Base Modifications, Sugar Moieties, and Internucleotide Linkages. Subtle chemical modifications to either the bases or sugar moieties in antisense DNA oligonucleotides would, in general, be reflected by modifications of the molecular masses of the full-length materials or their various chemical and enzymatic degradation products. During the course of these studies, we determined the molecular masses of the full-length 12- and 18mers and the masses of various truncated forms produced by failure synthesis or SVP digestion. In the case of the 12-mer, the measurements were used to deduce the masses of the complete set of methylphosphonate dinucleotides comprising the molecule, (12-11), (10-9), (8-7), (6-5), (4-3), and (2-1). The masses of all of those dinucleotides agreed with the expected MP dinucleotide masses (with a root-mean-square error of only 0.9 Da). The molecular masses of various hydrolysis products were also established. From those measurements, it was shown that the overlapping phosphodiester dinucleotides (11-10), (9-8), (7-6), (54), (3-2), and the 5′- and 3′-nucleotides also had masses that agreed with the expected values (root-mean-square error, (0.5 Da). Similar mass measurement accuracy was observed in the analyses of the 18-mer and its degradation products. These results indicate that no chemical modifications occurred to any bases or sugars during the various digestions and analyses. The results also suggest that the current method is accurate enough to detect chemically stable modifications that result in mass changes of only a few daltons.
The identities of the various internucleotide linkages can be confirmed chemically for alternating MP/PDE oligonucleotides. This results because the PDE linkages are labile to nucleases and stable to base, while the MP linkages are labile to base and stable to nucleases. The internucleotide linkages of the 12-mer and the 18-mer were exposed to nucleases and base during the sequencing part of the study. All of the digestion patterns were consistent with the alternating MP/PDE structure. CONCLUSIONS A time-of-flight MALDI method has been developed for confirmation of the structures of novel, chemically synthesized antisense DNA oligonucleotides having alternating MP/PDE internucleotide linkages. Length, base composition, and complete nucleotide sequence have been obtained for a 12-mer and an 18mer. The method allows detection of modified bases and sugars, if the modifications result in molecular mass changes of more than a few daltons. The alternating MP/PDE backbone was verified by nuclease and chemical digestion. The experiments demonstrate the utility of failure synthesis molecular ions in two ways. First, failure synthesis ions provide sequence information at the 5′-ends of these oligonucleotides. This is important because CSP digestion, which is typically used for 5′-3′ sequencing, did not work for this class of materials. Second, failure synthesis ions code for length, base composition, and nucleotide sequence, which makes them an especially valuable link between the difficult-tocharacterize high-mass oligonucleotides (>7-mers) and the easyto-characterize low-mass oligonucleotides (
