Anal. Chem. 1994,66, 3858-3863
Analysis of Double-Stranded Oligonucleotides by Electrospray Mass Spectrometry Ernst Bayer,' Tatjana Bauer, Karl Schmeer, Konrad Blelcher, Martin Maler, and HansJoachlm Gaus Institute of Organic Chemistty, Universitat Tubingen, Auf der Morgenstelle 18, 72076 Tubingen, Germany
Double-stranded oligonucleotides of different lengths and chemical modification have been analyzed by ion spray mass spectrometry. The non-covalent-bonded duplexes can be detected. Therefore, ion spray mass spectrometry is a useful method for investigation of hybridizations of natural and chemically modified oligonucleotides. Since the exact mass of the double strand can be detected, this method can distinguish between specific and nonspecific interaction.
By the techniqueof electrospray ionization (ESI), gas phase ions can be formed directly from solution at atmospheric pressure by protonation or deprotonation and evaporation. In that way created, multiply charged ions allow an accurate molecular mass determination of large biopolymers. Electrospray as well as ion spray (pneumatically assisted electrospray) mass spectrometry also allows the observation of noncovalent complexes of biomolecules as, for example, apoenzyme and prosthetic group' enzyme-product, enzymesubstrate,2 receptor-ligand,3 enzyme-inhibit~r,~proteinGDPS and nucleotide c o m p l e x e ~ . ~The ? ~ recognition of noncovalent base pairing of complementary, single-stranded oligonucleotides, which results in double- and even triplestranded biopolymer is of great interest in the life sciences. Chemically modified oligonucleotides, the so-called antisense oligonucleotides, are of great interest as potential therapeutic agents. Therefore, we investigated the doublestranded oligonucleotideswith natural and chemically modified oligonucleotides. There are no reports about hybridization studies of mixed double strands of natural and chemically modified single strands by mass spectrometric methods. However, this is of great interest because it is this hybridization from an antisenseoligonucleotide which is expected to perform in the target cells to prevent translation and inhibit gene expression.
MATERIAL AND METHODS Synthesis of Oligonucleotides. All unmodified oligonucleotides were synthesized in 1-pmol scale on CPG-Material (MWG-Biotech, Ebersberg, Germany)17J8 by using an AB1 DNA synthesizer, Model 380B (Applied Biosystems, Foster City, CA). The synthesis was carried out using standard (1) Katta, V.; Chait, B. T. J. Am. Chem. SOC.1991, 113, 8534-5. (2) Ganem, B.; Li, Y. T.; Henion, J. D. J. Am. Chem. SOC.1991,113,7818-9. (3) Ganem, B.; Li, Y. T.; Henion, J. D. J. Am. Chem. SOC.1991,113,6294-6. (4) Baca, M., Kent, S.B. H. J. Am. Chem. SOC.1992, 114, 3992-3, ( 5 ) Ganguly, A. K.; Pramanik; B. M., Tsarbopoulos, A.; Covey, T. R.; Huang, E:; Fuhrman, S. A. J . Am. Chem. SOC.1992, 114,655940, (6) Light-Wahl; K. J.; Springer, D. L.; Winger, B. E.; Edmonds, C. G.; Champ, D. G.; Thrall, B. D., Smith, R. D. J. Am. Chem. SOC.1993, 11s. 803-4. (7) Li, Y. T.; Henion, J. D. Tefrahedron Left. 1993, 34, 1145-8.
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Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
phosphoramidite chemistry*-' with the CE 102 cycle from ABI.12 Poly(ethy1ene glycol) (PEG)-modified oligonucleotides,'3-16 well as PEG-modified phosphorothioate oligonucleotides were synthesized on tentacle polymers19(TentaGel Rapp Polymere, Tubingen, Germany), a polystyrene-poly(ethy1ene glycol) copolymer with an average PEG spacer size of 2200-2500 Da and a loading of 0.24 mmol/g. To obtain 3'-PEG-modified oligonucleotides the commonly used succinyl group was selected as an anchor between the PEG spacer of the TentaGel support and hexaethylene glycol.20p21 The first nucleotide was coupled as a phosphoramidite to the remaining hydroxyl group of hexaethylene glycol during automated solid phase synthesis. The preparation of the phosphorothioates was analogous with the exception of the oxidation step, where tetraethylthiuram disulfide was used instead of iodine/water. In all cases, the product was cleaved from the support with a 25% ammonia solution and the removal of the protecting groups was performed in 25% ammonia (12 h, 55 "C). The solution was evaporated and lyophilized several times. Before the single-stranded oligonucleotides were prepared for hybridization, they were purified by membrane filtration (Centricon-3, 3000 Da, Amicon, Beverly, MA). With this method it was easily possible to remove disturbing ions and short failure sequences. A 0.20 mmol sample of each single strand was dissolved in 2 mL of 1 M NH40Ac buffer (pH 7) and concentrated by centrifugation. We always used the Cryofuge 6-6 from Heraeus Christ (Karlsruhe, Germany). This process was repeated four times, and the final 10-4 M solution was taken for hybridization. Hybridization. After the purification of the single strands, 1 mL of equimolar solutions each of the complementary single strands were combined and stirred at 80 "C. After 10 min, (8) Letsinger, R. L.; Ogilvie, K. H; Kelvin, K. J. Am. Chem. Soc. 1967, 89,
4801-3. (9) Letsinger, R. L.; Finnan, J. L.; Heavner, G. A.; Lunsford,W. J. J. Am. Chem. SOC.1975, 97, 3278-9. (10) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lei!. 1981, 22, 1859-62. (1 1) McBride, L. J.; Caruthers, M. H. Terrohedron Left. 1983, 24, 245-8. (12) User Bulletin, Applied Biosystems, 1984. (13) Bonora, G. M.; Bianotto, G.; Moffini, M. Screnin, L. L. Nucleic Acids Res. 1993, 21, 1213-7. (14) Giovannengeli, C.; Montenay-Garestier, T.; Rougct, M.; Chassignol, M; Thuong, N., T.; Helene, C. J. Am. Chem. Soc. 1991, 113, 7775-7. (15) Rumney, S.Kool, E. T. Angew. Chem. 1992, 104, 1686-9. (16) Durand, M.; Chcvrie, K.; Chassignol, K.; Thuong, N. T.; Maurizat, J. C. Nucleic Acids Res. 1990, 18, 6353-9. (17) Stec, W. J.; Zon, G.; Egan, W.;Stec, B. J. J. Am. Chem. Soc. 1984, 106,
6077-9.
(18) (19) (20) (21)
Ott, J.; Eckstcin, F. Biochemistry 1987, 26, 8237-41. Bayer, E. Angew. Chem. 1991, 103, 117-33. Maier, M., submitted for publication in Nucleic Acids Res. Bleicher, K.; Maier, M.; Gaus, H.-J.; Schmeer, K.; Bauer, T. Solid PhaseSynfhesis, Conference Proceedings, Oxford, 3 1 August4 Septembcr 1993; Epton, R., Ed.; SPCC Ltd.: Birmingham, UK, 1994.
0003-2700/94/036&3858$04.50/0
0 1994 Amerlcan Chemlcal Soclety
..
B 5-
a)
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1165
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569.000
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-
. 7.
b)
o,oJ
1000
295.000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
2200
2300
2400
m/z
Figure 1. Influence of purification on the sample (A iB; see Table 1): (a) mass spectrum with a sample that was eight times rebuffered with NH4HCOI; (b) the same sample that was only twice rebuffered.
a mass range of 2400 Da. The oligonucleotide complexes were analyzed by flow injection using a microliter syringe in a medical infusion pump (Harvard Apparatus, Natick, MA). The flow rate ranged from 2 to 5 pL/min. The concentration of the duplex was 10-4M. Calibration was carried out with a NaI solution. All spectra were recorded under the same conditions in the negative mode (dwell time 1 ms, step size 0.5 amu, and 50 scans with the multichannel analyzer). All samples were dissolved in 10 mM NHsHCO3 (pH 8.5), and their concentrations were in all cases 10-4 M. Capillary Electrophoresis. Capillary electrophoresis was carried out with a Grom CE system 100 (Herrenberg, Germany) with an uncoated fused silica capillary (Polymicro Technology, Phoenix, AZ) and on-line detection with a Linear UV/visible detector (Reno, NV) at 260 nm. The buffer for analysis of single strands contained 5 mM borate, 5 mM Tris, 50 mM SDS, and 7 M urea, pH 8.7. Voltage during the separation was 20 kV and the currents ranged from 16 to 18 PA. Duplexes were analyzed with buffer containing 25 mM N H ~ O A CpH , 8.8. Thevoltage and the current were the same as mentioned above. Thermal Denaturation Measurements. Melting points were determined on a Specord M 5000 (Carl Zeiss, Jena, Germany). For the measurement, 300 mL of the solution ( l w mol/L) was taken before rebuffering (1 M NH40Ac) and diluted with the same buffer to a final concentration of lk5mol/L.
-
M ilH
M IOH
U 9H
M 8H
M 7H
M 6H
Charge 01 the DUpIOX
Figure 2. Influence of triethylamine and crown ether on the signal Intensity of the duplex. The duplex signals vanished by addition triethylamine,while addition of crown ether (18-crown-0) improves the signal intensity.
the solution was cooled over a period of 3 h to room temperature and shaken overnight. To prepare the duplex solution for mass spectrometric analysis and capillary electrophoresis, the probe had to be rebuffered to a 10 mM NH4HC03 solution. This was carried out with concentrators in the same manner as described above. Depending on the mass of the duplex, a scutoff" of 3000 or 10 000 Da was chosen. The procedure with 10mM NH4HCO3 buffer was repeated eight times. Each cycle took 20 min in the case of a concentrator of 3000 Da (cutoff) and 10 min in the case of a concentrator of 10000 Da (cutoff) and 4500 rpm. Mass Spectrometry. All mass spectra were recorded on a Sciex API 111 triple-quadrupole mass spectrometer, having an electrospray ion source (Sciex, Toronto, ON, Canada) and
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Analytical Chemistry. Vol. 66, No. 22, November 15, 1994
3859
>-
6 peaks /HyperMass= 12.234[H)S2,47
1.307.250
Dupe71746
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1200
1300
1400
1500
1600
1700
1800
1900
2000
2100
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Flgure 3. Mass spectrum of a 20 mer120 mer duplex Consisting of two mixed 20 mer sequences (strand A*, 5‘CC TTG AGA TTC CCG TGA TCC-3‘; strand B’, 5‘-GGA TCA AGO GAA TCT CAA (303’).
RESULTS AND DISCUSSION The stability of the duplex is determined by different factors. There are interactions between velectron systems of the bases (base stacking). These interactions are supported by London dispersion forces and hydrophobic effects. Further more there are hydrogen bondings in the plane of the bases. Basestacking dominates in aqueous solution where basebase hydrogen bonding is greatly suppressed due to the competition of binding sites by water molecules. Last, electrostatic interactions between cations present in the solvent and the negatively charged phosphate groups have also to be considered.22 Two factors are important for sample preparation: the pH of the solution and cation concentration. The pH value affects the proton equilibrium of the phosphate groups in the range pH 4-1 1 and beyond that range also affects the bases. This means, for the detection of duplexes, on the one hand, free negative charges are necessary, on the other, the compensation of the negative charges by cations is essential for the stability of the duplex. An excess of cations can easily become a problem in measurements of double-stranded oligonucleotides because the formation of cation clusters occurs immediately. Since different clusters are formed, a peak broadening and, consequently, a decrease of the signal intensity is observed. Counterion association leads to a lower mass accuracy, as was reported by Smith et a1.,6 where the difference of measured (22) Saenger, W. Principles of Nucleic Acid Structure, 2nd 4.; Springer: N e w York, 1984; Chapter 6.
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Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
Table 1. Sequences of the Single Strands Used for HybrMlzatbn’ A
5‘-dG GAG GAG 3’-dC CTC CTC C 3’-dCTC CTC CTC CTC D 3‘-dCC E 3’-dC
B
a
AGA TCT TCT TCT TCT
GGG CCC CCC CCC CCC
AGG AAG G-3’ TCC TTC C-5’ TCC TTC CTT CTC-5’ TC-5’ T-5’
The investigated, underivatized duplexes consisted of A + D, and A + E.
C, A
+ B, A +
Table 2. Complementary Strand8 of the Investigated Duplexes Whlch Conrhtod of ModMod Ollgonuckotldes
F 3’-d(S)C CTC CTC TCT CCC TCC TTC C-5’ G PEG-6-O-P(0)2-O-dC CTC CTC TCT CCC TCC TTC C-5’ H PEG-6-O-P(O)2-O-d(S)C CTC CTC TCT CCC TCC TTC C-S‘ ________~
and expected mass was -60 amu and the exact mass of the double strand could not be obtained. In order to avoid cluster formation, a buffer is required whose components will produce volatile compounds during the ion evoporation process. A 10 mM NH4HCO3 buffer meets all those requirements. The mass spectra obtained following two different methods of sample preparation are shown in Figure 1. In spectrum a, the intensity per scan is about the same as in spectrum b. In spectrum b, the final, with three peaks, estimated mass was 12 552.06 amu, having a deviation of 11.32 amu, while in (a) itwas,withsixpeaksestimated, 12 231.82amuwithadeviation
a) 20meri20mer
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2135
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1900
2000
2100
2200
2300
2400
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Flgure 4. (a) Mass spectra of a 20 mer120 mer (A
-+ B) and (b) a 20 mer110 mer (A i- D) duplex with their single strands (see Table 1).
of 2.28 amu. The expected mass was 12 234.0 amu. The sample shown in (b) still contained greater amounts of NH4OAc in spite of rebuffering with NH4HC03. In the sample shown in (a), the buffer was changed eight times and the counterions more completely removed. No results were obtained by measurements in NH40Ac buffer. To avoid these problems, different substances were added to the buffer. Addition of crown ether proved to be a good method for the removal of excess counterions (e.g., Na+, K+, and NH4+) in peptide analysis by fast atom bombardment mass spectrome t r ~ Additon . ~ ~ of small amounts of triethylamine, which generally improves the signal-to-noise ratio of single-stranded oligonucleotides, increased the signal intensity of the single strands a factor of 10, while the duplex signal rested at about the same value. At higher concentrations of triethylamine, duplex signals were no longer detectable. We assume a denaturation of the duplex because very similar behavior was observed in CE experiments. Both derivatized and underivatized duplexes showed the single peak of the duplex in the electropherogram; after addition of triethylamine, two additional peaks, representing the single strands, were observed. All together eight different duplexes were investigated. One of them consisted of two mixed 20 mer sequences (A*, 5'-dGGATCAAGGGAATCT CAA GG-3';B*, 5'-dCCT TGA GAT TCC CGT GAT CC-3'; see Figure 3). Seven other duplexes, that were investigated had always the single
-
(23) Orlando, R. Anal. Chem. 1992, 64, 3 3 2 4 .
20 mer strand in common. At four of those seven duplexes, the length of the complementary strand was varied (see Table l ) , while at the remaining three the complementary purine strand was modified (see Table 2). The spectra of 20 mer/20 mer and 20 mer/l0 mer are shown in Figure 4a,b. The seven signals in Figure 4a belong to the series of the 6-12-fold charged 20 mer/20 mer duplex (A + B). The six signals in Figure 4b, representing the charge series from the 4-9-fold charged 20 mer/l0 mer (A + D) hybrid, can be used for mass determination. This is advantageous for identification of duplexes in comparison to the previous report^,^.^ where only one main signal and an extremely small second signal could be demonstrated. Besides that series, the seven peaks of the single strands-three respectively four peaks for each strand, representing the 3-5fold and the 3-6-fold charged compounds-are also found in the spectrum. We assume that the single strands are formed during the evaporation process or on the way through the nitrogen curtain (in front of the orifice). The extraordinary stability of the duplex is amazing. Even at high orifice voltages the signals of the lower charged duplex remain in the spectrum. At an orifice voltage of 180 V,which is able to fragment dinucleotides almost completely, the signal of the 6-fold charged duplex is the peak of the highest intensity. This is in agreement with the generally observed charge stripping, where with increasing orifice voltage the higher charged species decrease. Analytical Chemistry. Vol. 66, No. 22, November 15, 1994
3861
Table 3. Melting Temperatures of Duplexes Containing an Unmodified Single Strand and Three Modified Single Strands double strands melting temp, O C
thioate/DNA 63 O C
HEG-oligo/DNA
DNA/DNA 77.5 OC
74 "C
HEG-thioate/DNA
62 O
C
HEG-DNAIDNA duplex
single strands
a) -4
Figure 5. Charge distribution of five different duplexes (see Table 1). Highest Intensities are shown by the 20 mer/20 mer and the 20 mer/ 30 mer hybrid (A B and A C).
+
+
T
2500000
2000000
1500000
1000000
500000
I lOmer
8mer
20mer
30mer
Duplex with 20mer
Figure 6. Comparison of signal intensities from single- and doublestranded oligonucleotides, obtained in the mass spectra. All duplexes had one strand in common (A), while the length of the complementary strand (B-E) was varied. Signal intensitiesof the single strand decrease with increasing length of the complementary strand (see Table 1). A maximum stability is found at 20 mer/2O mer strand (A 4- B).
Almost the same behavior is shown by other duplexes (Figure 5 ) . The signal intensity of the duplex increases with the length of the complementary strand. The highest signals are shown by the 20 20 duplex. At the 30 + 20 duplex, the higher charged complex is dominating, which might be due to the deprotonation caused by unpaired parts of the complementary oligonucleotide. The lower intensities, as well as the smaller amount of signals, produced by the duplexes containing 8 and 10 mer strands can be explained by their lower stability. In Figure 6 , the intensity of the signals of the duplex are compared with the intensities of the single strand, which have the four duplexes in common. It can be shown that with increasing length of the complementary strand the signals of single strands decrease while the duplex signals increase up to the same length of the unchanged strand (Figure 6 ) . If the length of the complementary strand increases, the intensity remains of the same order, which can be expected because
+
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Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
I
I
I
0
10
20
-
30
t
[min]
b)
I
I
I
I
0
10
20
30
t[min]
Figure 7. (a) MECC of a HEG2O mer/20 mer duplex; (b) after addition of triethylamine.
there is no increased hybridization possible (A + C in Figure 6). Hybridization of oligonucleotides with single-stranded DNA or RNA plays a key role in the antisense ~ t r a t e g y . ~ ~ ? ~ ~ Specific interaction with target sites occurs as a possibility to control and especially inhibit protein biosynthesis. A large number of oligonucleotide derivates have been introduced either to enhance the duplex stability,26nuclease r e ~ i s t a n c e or , ~ ~cell penetrati~n.~~-~O Nowadays one of the most commonly used modifications is the phosphorothioate oligonucleotides31because of their high nuclease stability. By additional modification of the phosphorothioate oligonucleotides with PEG, a higher import rate into the cell is expected. To investigate the hybridization properties of modified oligonucleotides, usually UV analysis of temperature-dependent stability of the double-stranded fragments is used. Table 3 shows the melting temperatures of the duplexes of the modified 20 mer oligonucleotides (see Table 2) with an unmodified DNA fragment in comparison to an unmodfied double strand of the same sequence. The heteroduplexes were also investigated with electrospray mass spectrometry and capillary electrophoresis. It is possible to partially cleave the bonds between the two single strands of a hexaethylene glycol (HEG)-20 mer/20 mer duplex by addition of triethylamine and to separate single strands and duplex with micellar electrokinetic capillary electrophoresis (MECC) (Figure 7). The mass spectrum of this duplex is shown in Figure 8b. (24) Uhlmann, E.; Peymann, A. Chem. Rev. 1990, 90, 543-84. (25) Milligan, J. F.; Matteucci, M. D.; Martin, J. C. Med. Chem. 1993,14,192337. (26) Pieles, U.; Englisch, U. Nucleic Acids Res. 1989, 17, 285-99. (27) Cohen,J. S. Oligodeoxynucleotides,AntisenseInhibitors of Gene Expression; Macmillan Press Ltd.: London, 1989; Chapter 5. (28) Loke, S. L.; Stein, C. A.; Zhang, H. X.; Mori, K.; Nakanishi, M.; Subasinghe, C.; Cohen, J. S.; Neckers, L. M. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 3474-8. (29) Markus-Sekura,C. J.; Woerner, A. M.; Shinozuka, K.; Zon, G.; Quinnan, G. V. Nucleic Acids Res. 1987, 15, 5749-63. (30) Wickstrom,E. L.; Bacon, T. A.; Gonzales, A.; Freeman, D. L.; Lyman,G. H.; Wickstrom, E. Proc. Natl. Acad. Sci. U S A . 1988, 85, 1028-32. (31) Eckstein, F. Angew. Chem. 1983,95,431-47.
96.667 12 0
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-8 17,3.
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1
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11.5,
5.8.
0.
A
'-
148.000
1068
1000
1100
1200
1300
1400
1500
1600
1700
m/.Z
Figure 8. Mass spectra of (a) the thioate-20 mer120 mer duplex (F), (b) the HEGPO mer / 20 mer duplex (G), and (c) the HEG-thloate-20 mer / 20 mer duplex (H).
The investigation of double strands under denaturating conditions was also carried out during ES-MS analysis with a thioate-20 mer/2O mer duplex. As already mentioned above, the addition of triethylamine to the duplex also leads in this case to complete denaturation. The resulting spectra show exclusively the signal series belonging to the two single strands while no duplex signal is received anymore. The mass spectra of three heteroduplexes are shown in Figure 8.
CONLUSIONS We found a rapid method for preparation of the duplex without the need of an extensive HPLC purification. For analysis of the hybrid, ion spray mass spectrometry proved to be a powerful method. Even smaller impurities such as failure sequences do not disturb the measurement and can even be determined. This is a great advantage in comparison with methods such as CD and UV spectroscopy, where selective
determination of one duplex is not possible, and therefore, contributions to other nonspecific interactions cannot be distinguished. After optimization of the parameters and sample preparation in volatile buffer (10 mM NHdHC03), ion spray mass spectrometry proved to be a fast and powerful method to show specific hybridization of unmodified oligonucleotides, as well as modified oligonucleotides which are essential in the antisense conception. During the investigations it could be shown by mass spectrometric methods, that the stability of the duplex is strictly correlated with the length of its chains. Those results offer new possibilities for the study of hybridization that may be of interest also for other areas of nucleic acid research. The stability of different hybrids can easily be approximated and compared with each other. Received for review May 20, 1994. Accepted August 24, 1994." a Abstract
published in Advance ACS Absfracrs, October 1, 1994.
Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
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