Analysis of Oligonucleotides by On-Column Transient Capillary

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Anal. Chem. 1996, 68, 3907-3911

Analysis of Oligonucleotides by On-Column Transient Capillary Isotachophoresis and Capillary Electrophoresis in Poly(ethylene glycol)-Filled Columns Seppo Auriola,*,† Ilpo Ja 1a 1 skela 1 inen,† Mikko Regina,‡ and Arto Urtti†

Departments of Pharmaceutics and Pharmaceutical Chemistry, University of Kuopio, P.O. Box 1627, SF-70211 Kuopio, Finland

Capillary electrophoresis in poly(ethylene glycol) (PEG) solution-filled uncoated silica capillaries was demonstrated to be suitable for separation of oligonucleotides in 100 mM ammonium formate (pH 4.5) buffers. Reversed polarity (-30 kV at the injector end) was used, because the electroosmotic flow (EOF) at 4.5 was considerably lower than the mobility of the anionic analytes. On-column transient isotachophoresis (ITP) was developed for preconcentration of oligonucleotide samples in PEG solution-filled capillaries. The ITP focusing step allows injection of 20% of column length without loss of peak resolution. The detection limit of the method was 100 ng/mL of 16-mer oligonucleotide by using UV detection at wavelength 254 nm (signal-to-noise ratio 10). Oligonucleotides and DNA restriction fragments have a constant charge-to-size ratio between pH 6 and 8. Therefore, they cannot separated by capillary electrophoresis (CE) in free buffer solution, and some sort of sieving matrix must be used to separate different length fragments.1,2 Base-specific electrophoretic selectivity can be additionally obtained by using low-pH separation buffers, since the negative net charge of each oligonucleotide is different at these conditions.2,3 Polyacrylamide gel-filled capillaries are the most often used CE columns for separation of oligonucleotides and DNA restriction fragments.4,5 However, the rigid gelfilled capillaries have several disadvantages, as their preparation is rather difficult and they are easily destroyed. Also, the number of electrophoretic runs is limited, because the rigid gel-filled capillaries are not washable like buffer-filled CE capillaries. A solution of these problems is to replace the gel with a UVtransparent polymer solution. When the concentration of the polymer exceeds a certain entanglement treshold, the polymer chains become entangled, and they form a transient network of obstacles. The theory of polymer solutions and separation mechanism in polymer solution-filled CE capillaries has been †

Department of Pharmaceutics. Department of Pharmaceutical Chemistry. (1) Heller, C. J. Chromatogr. 1995, 698, 19-31. (2) Keith, G. J. Chromatogr. Libr. 1990, 45A, A103-A141. (3) Cordier, Y.; Roch, O.; Cordier, P.; Bischoff, R. J. Chromatogr. 1994, 680, 479-489. (4) Cohen, A. S.; Narajian, D. R.; Paulus, A.; Guttman, A.; Smith, J. A.; Karger, B. L. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 9660-9663. (5) Li, S. F. Y. J. Chromatogr. Libr. 1992, 52, 155-200; 377-540. ‡

S0003-2700(96)00872-4 CCC: $12.00

© 1996 American Chemical Society

discussed by Heller1 and by Righetti.6 Recently, several groups have succesfully applied CE in viscous polymer solutions for separations of macromolecules. These applications include analysis of low-molecular-mass RNA:s7 or double-stranded DNA using hydroxypropyl methylcellulose polymers8 and analysis of proteins in dextran-filled columns.9 Poly(ethylene glycol) (PEG) polymer with molecular weight 100 000 has been succesfully applied for separation of SDS-protein complexes,10 and the effect of temperature on the sieving properties of PEG-filled CE capillaries has been studied by Guttman et al.11 Chang and Yeung have shown that the resolution of different size DNA restriction fragments can be optimized by varying the poly(ethylene oxide) concentration and molecular weight. The best resolution was obtained by using PEO mixtures containing both mid size (MW 300 000) and large polymers (up to MW 8 000 000).12 In capillary zone electrophoresis, only a small sample volume can be injected to the capillary without a decrease in the separation power. The sensitivity of the system can be increased by developing better detection devices or by using sample preconcentration techniques. Sensitivity of oligonucleotide analysis can be increased by using electrokinetic injection from low-ionicstrength solution. However, in this case, extensive desalting of the sample by dialysis and solid phase extraction is needed before CE analysis.13 Isotachophoresis (ITP) can be used as a sample focusing method either by coupling a separate ITP instrument to CE or by performing the ITP preconcentration step in the CE capillary. In the single-column transient ITP-CE system up to 100-fold higher sample volumes (20-50% of the column volume) can be injected compared to normal CE without loss of peak resolution. In a standard ITP electrolyte system, a leading ion is chosen with an effective mobility higher than those of the sample components, whereas the terminating ion must be lowest in mobility. The theory of ITP-CE and options of different elec(6) Righetti, P. G. J. Chromatogr. 1995, 698, 3-17. (7) Katsivela, E.; Ho ¨hle, M. G. J. Chromatogr. 1995, 700, 125-136. (8) Skeidsvoll, J.; Ueland, P. M. Anal. Biochem. 1995, 231, 359-365. (9) Lausch, R.; Scheper, T.; Reif, O.-W.; Schlo¨sser, J.; Fleischer, J.; Freitag, R. J. Chromatogr. 1993, 654, 190-195. (10) Ganzler, K.; Greve, K. S.; Cohen, A. S.; Karger, B. L.; Guttman, A.; Cooke, N. C. Anal. Chem. 1992, 64, 2665-2671. (11) Guttman, A.; Horvath, J.; Cooke, N. Anal. Chem. 1993, 65, 199-203. (12) Chang, H.-T.; Yeung, E. S. J. Chromatogr. 1995, 669, 113-123. (13) Leeds, J. M.; Graham, M. J.; Truong, L.; Cummins, L. M. Anal. Biochem. 1996, 235, 36-43.

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trolyte systems has been described by Beckers,14 Krivankova et al.,15 and Foret et al.16,17 A special ITP method has been developed for preconcentration of DNA fragments and PCR products in partially polyacrylamide-filled columns. The transition from ITP to CE was achieved by the mobility shift of DNA from free solution to sieving gel buffer. In this system, butyrate ion was chosen for the terminator, because it is slower than DNA in free solution but faster than DNA in the sieving buffer.18 This paper describes an on-column transient isotachophoresisCE analysis method that is performed in a poly(ethylene glycol) solution-filled capillary. Up to 20% of the capillary can be filled with the oligonucleotide sample solution and focused to a narrow zone before the CE separation of the components. The background CE electrolyte (ammonium formate, pH 4.5) was selected so that the formate ion acts as the leading ion in the ITP step. MES was used as the terminating slow anion in the ITP preconcentration. After the ITP preconcentration, the terminator buffer vial was changed to a background buffer vial, and normal CE run was started. The ITP-CE method described in this paper is very easy to set up, because it is based on the use of uncoated silica capillaries and no tedious coating of capillaries, or expensive commercially prepared gel columns, are needed. The method is applicable both for quantitative and qualitative analysis of oligonucleotides. The preconcentration method is also potentially compatible with such sensitive CE detection devices like fluorescence and mass spectrometry. EXPERIMENTAL SECTION Chemicals. Poly(deoxyadenylic acid) pd(A)19-24 was purchased from Pharmacia (Uppsala, Sweden). The 16-mer antisense oligonucleotide (GTG TAT CTC CAT GCA T) used in the migration time reproducibility and detection limit test was synthesized on an automated DNA synthesizer (Gene Assembler Plus, Pharmacia). Poly(ethylene glycol), molecular weight 20 000 (PEG 20000), was obtained from Fluka (Buchs, Switzerland). Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) and 2-(N-morpholino)ethanesulfonic acid (MES) were from Sigma (St. Louis, MO). Other reagents were of analytical grade. Instrumentation. The experiments were performed using Beckman P/ACE System 2100 equipped with a UV absorbance detector (Beckman Instruments, Palo Alto, CA). Fused-silica capillaries, uncoated, 50 µm i.d., were obtained from Polymicro Technologies (Phoenix, AZ). The capillary length was 67 cm (60 cm effective length). After installation, the capillary was washed first with 1 N NaOH (10 min), with water (10 min), with 100 mM ammonium formate (pH 4.5), and with the same buffer containing 4% (w/w) of PEG 20000 (both 30 min). Before and after each run, the capillary was rinsed with the separation buffer. Capillary electrophoresis and ITP were carried out in the reversed polarity mode (-30 kV at the injector end), and the capillary temperature was set to 23 °C. Detection wavelength was 254 nm for oligonucleotides and 214 nm for salicylic acid. Capillary Electrophoresis. (a) Choice of Electrolyte System. The choice to use 100 mM ammonium formate buffer at (14) Beckers, J. L. J. Chromatogr. 1993, 641, 363-373. (15) Krivankova, L.; Foret, F.; Gebauer, P.; Bocek, P. J. Chromatogr. 1987, 390, 3-16. (16) Foret, F.; Szoko, E.; Karger, B. L. J. Chromatogr. 1992, 608, 3-12. (17) Foret, F.; Szoko, E.; Karger, B. L. Electrophoresis 1993, 14, 417-428. (18) van der Schans, M. J.; Beckers, J. L.; Molling, M. C.; Eveaerts, F. M. J. Chromatogr. 1995, 717, 139-147.

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pH 4.5 in these experiments was based on the following facts: (1) At pH close to 4, electroosmotic flow has been reported to be low.19 (2) Ammonium formate buffers are compatible with CEelectrospray mass spectrometry (MS), and the developed CE method can be potentially combined with MS detection.20 (3) At pH 4.5, ammonium formate has buffer capacity (pKa of formate is 3.8). (4) In the transient ITP step, formate ion will act as the leading (fastest) ion (effective mobility of formate at pH 4.5 was calculated to be -49 × 10-9 m2 V-1 s-1). (5) MES was selected to be the terminating ion in the ITP focusing, because it has low effective mobility at pH 4.5 (-0.7 × 10-9 m2 V-1 s-1) at pH 4.5.21 (6) At low pH, the number of negatively charged compounds (potential background peaks) is smaller than at high pH. (b) Measurement of EOF and Effective Mobility of Oligonucleotide Anions. The EOF in the capillary was estimated, by measuring migration time of a standard compound, salicylic acid (pKa 2.9, ion mobility µ ) -35 × 10-9 m2 V-1 s-1). The effective mobility µep, of this standard compound at pH 4.5 was calculated on the basis of equations and data from Pospichal et al.21 to be µep ) -34 × 10-9 m2 V-1 s-1. As the column length (L ) 67 cm), length to the detector (l ) 60 cm), and voltage (V ) 30 kV) were known and the migration time (t ) 590 s for salicylic acid) of the standard was measured, the following equation can be used to calculate the total mobility (µtot) of the ions:

µtot ) Ll/tV

(1)

The equation gives a total mobility of µtot ) -22 × 10-9 m2 V-1 s-1 for salicylic acid. Total mobility, µtot, is the sum of effective ion mobility and EOF. Thus, EOF could be calculated by the equation

EOF ) -µep + µtot

(2)

The resulting value was EOF ) 12 × 10-9 m2 V-1 s-1, which is relatively low.19 The calculation of effective mobility of oligonucleotide anions was based on the same equations. In this case, EOF was known, and migration time was measured in the 100 mM ammonium formate (pH 4.5) buffer. The effective mobility (µep) of pd(A)19-24 was -32 × 10-9 m2 V-1 s-1. (c) CE Separation of Oligonucleotides Using PEG 20000 as a Sieving Polymer. The effect of sieving polymer concentration on the separation of oligonucleotides was studied by adding 0%, 4%, 8%, and 12% (w/w) of PEG 20000 to the 100 mM ammonium formate buffer. A solution of poly(deoxyadenylic acid) (pd(A)19-24, 160 µg/mL, 27 µg/mL each oligomer) was used to examine the separation power of the viscous matrixes. Capillary electrophoresis was carried out in the reversed polarity mode (-30 kV at the injector end), and the capillary temperature was set to 23 °C. Detection wavelength was 254 nm. (d) Transient On-Column Isotachophoresis and Capillary Electrophoresis of Oligonucleotides in PEG-Filled Columns. The capillary was filled with 100 mM ammonium formate buffer (pH 4.5) containing 12% (w/w) of PEG 20000. The oligonucleotide (19) Altria, K. D.; Simpson, C. F. Chromatographia 1987, 24, 527-532. (20) Niessen, W. M. A.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. 1993, 636, 3-19. (21) Pospichal, J.; Gebauer, P.; Bocek, P. Chem. Rev. 1989, 89, 419-430.

samples were dissolved in the buffer without PEG at concentrations of 16, 8, and 1.6 µg/mL (1.3 and 2.7 µg/mL each oligomer, respectively). High-pressure injection was used to fill 10% or 20% of the capillary with the sample (0.8 and 1.6 min, respectively). After injection, the injector end of the column was set to the terminating electrolyte, which was 100 mM MES (titrated to pH 4.5 with 2 N ammonium hydroxide). The time needed for the ITP preconcentration was tested using the following ITP times and voltages: 1.5, 1, 0.5, and 0.25 min (all -30 kV) and 0.25 min (-15 kV). After the ITP step, the injector end of the column was transferred to the background electrolyte vial (ammonium formate), and the CE separation with -30 kV was started. An ITP preconcentration time of 0.5 min was chosen for all following experiments. The suitability of some commonly used biochemical buffers as the sample matrix in ITP preconcentration was tested by analyzing the sample in 100 mM phosphate-buffered saline (PBS, pH 7.4), 100 mM Tris-HCl (pH 7.5), and 50 mM MES-50 mM HEPES-75 mM NaCl (pH 7.4) buffers. The effect of salt concentration on the ITP was tested by analyzing the sample dissolved in 100 mM Tris-HCl (pH 7.5) containing 200, 100, 20, and 0 mM additional NaCl. The reproducibility of migration times, peak areas, and peak heights in the ITP-CE system was tested with an oligonucleotide standard containing 1 µg/mL in 100 mM Tris-HCl buffer (pH 7.5) (n ) 6). The detection limits and suitable concentration range of the method were tested by using standards containing 10, 5, 1, 0.3, and 0.1 µg/mL (n ) 3) of oligonucleotide in same buffer. Twenty percent of the column length was filled with the sample, and a 0.5 min ITP time (-30 kV) was used. RESULTS AND DISCUSSION Separation of Oligonucleotides by CE in PEG-Filled Capillaries. Based on the migration time of salicylic acid in 100 mM ammonium formate buffer at pH 4.5 with no PEG, the EOF can be calculated (eqs 1 and 2) to be relatively low (12 × 10-9 m2 V-1 s-1). When -30 kV reversed polarity was used for CE, the pd(A)19-24 oligonucleotide oligomers sample gives only one peak at 9.27 min, when no PEG was present in the buffer (Figure 1A). This means that all pd(A) oligomers migrated with the same velocity, because their mass-to-charge ratio is constant. The effective mobility of pd(A) oligonucleotides in free buffer was calculated to be -32 × 10-9 m2 V-1 s-1. Addition of PEG 20000 polymer to the buffer resulted in separation of different oligomers (Figure 1B-D). The separation of large analytes can theoretically be achieved when the concentration of the sieving polymer exceeds the critical entanglement threshold, c*, and the polymer forms a transient network of obstacles. The “pore size” of the entangled polymer solution is not dependent on the molecular weight of the polymer as long as threshold c* is exceeded.1 The threshold c* is dependent on the intrinsic viscosity η of the polymer. The value of η can be measured or approximately calculated. For PEG polymers, intrinsic viscosity can be calculated by the equation, η ) 0.02 + (2.4 × 10-4)MW0.73. For PEG 20000, the value of η is 0.35 dL/g.22 An estimate of the entanglement threshold c* can be finally calculated by the equation, c* ≈0.6(η)-1.1 Based on this intristic viscosity value, the entanglement threshold concentration value of PEG 20000 can be calculated to be c* ≈0.6(0.35 dL/g)-1 ) 1.7% (w/w). Figure 1 shows that a PEG 20000 concentration (22) Zeman, L.; Wales, M. Sep. Sci. Technol. 1981, 16, 275-290.

Figure 1. Effect of poly(ethylene glycol) concentration on the CE separation of pd(A)19-24 oligomers. The capillary was filled with 100 mM ammonium formate (pH 4.5) containing the following concentrations of PEG 20000: 0% (A), 4% (B), 8% (C), and 12% (D). Sample was dissolved in water (160 µg/mL), injected by pressure (about 1% of the column length), and electophoresed with -30 kV. Column length was 60 cm to the detector; uncoated silica capillary 50 µm i.d.

of 12% (w/w) must be used before a satisfactory CE separation of oligonucleotides was obtained. At 12% (w/w) concentration entangled solution is theoretically obtained even when using PEG polymers with molecular weight above 1000.22 However, PEG 20000 was used in all CE experiments, because the high viscosity of the buffer was expected to further decrease the EOF and make conditions more favorable for a succesful ITP preconcentration step. Transient On-Column Isotachophoresis and Capillary Electrophoresis of Oligonucleotides in PEG-Filled Columns. Transient on-column preconcentration works best when EOF is slow. This is normally achieved by using coated CE capillaries.17 In this study, slow EOF was achieved by using low pH and by using a highly viscous PEG 20000 solution. The increase in the viscosity was observed by determining the time needed to rinse an injected marker zone from the injector end to the detector using high pressure. The rinse time increased from 0.59 to 7.9 min as the PEG concentration was increased from 0% to 12% (w/w). One of the two basic electrolyte systems for ITP preconcentration described by Foret et al.16 was selected to be used in this study. In this method, the background electrolyte is selected to have an anion (formate) with higher effective mobility than any of the sample ions. ITP focusing is then achieved by using a suitable terminating electrolyte (MES anion) behind the sample zone. After the on-column focusing, the terminating electrolyte vessel is replaced with background electrolyte, and separation in the zone electrophoretic mode begins. Figure 2 shows the effect of the ITP preconcentration time on the focusing of the long sample plug. When 10% of the column was filled with a sample solution containing 16 µg/mL of pd(A)19-24, and the sample was electrophoresed without ITP concentration, the electropherogram showed only a large unresolved hump between 18 and 21 min (Figure 2A). When MES terminator is used for ITP preconcentration, partial focusing can be achieved in 0.25 min using -15 kV voltage (Figure 2B). The peaks appearing in the end of the Analytical Chemistry, Vol. 68, No. 22, November 15, 1996

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Figure 2. Effect of the on-column isotachophoretic preconcentration time on focusing of the sample plug in the capillary. Ten percent of the capillary length was filled with the pd(A)19-24 sample dissolved in ammonium formate buffer (16 µg/mL). MES was used as the terminating ion, and the following ITP times and voltages were used: no ITP (A); 0.25 min, -15 kV (B); 0.5 min, -30 kV (C); 1.5 min, -30 kV (D). Other conditions were as in Figure 1.

unresolved hump clearly indicate that the focusing happens at the injector end of the sample plug. Further increase in the ITP time results in better focusing of the zone (Figure 2C). When the sample zone is completely focused, it begins to move fast to the detector as a narrow zone, and peak separation can be lost. This effect can be seen in Figure 2D, where the migration time of pdA21 has decreased from the original 21.7 to 16.5 min and the peaks are not completely resolved. The suitability of some commonly used biochemical buffers as the sample matrix in ITP preconcentration was tested by analyzing the sample dissolved in 100 mM PBS (pH 7.4, Cl- ion concentration 132 mM, PO4- 9 mM), 100 mM Tris-HCl (Cl- 100 mM, pH 7.5), and 50 mM MES-50 mM HEPES-75mM NaCl (Cl- 75 mM, pH 7.4) buffers. Figure 3A,B shows that samples in buffers containing high-mobility anions (phosphate or chloride) can be focused using this ITP method. The migration times are shorter than when the sample was dissolved in ammonium formate buffer. The sample plug that contains low-mobility MES and HEPES ions is well focused in the ITP, but the different oligomers are not resolved to separate peaks in the following CE 3910 Analytical Chemistry, Vol. 68, No. 22, November 15, 1996

Figure 3. Effect of sample buffer on the ITP preconcentration. The suitability of some commonly used biochemical buffers as the sample matrix in ITP preconcentration was tested by analyzing the sample dissolved in (A) 100 mM phosphate-buffered saline (PBS, pH 7.4, Cl- ion concentration 132 mM, PO4- 9 mM), (B) 100 mM Tris-HCl (Cl- 100 mM, pH 7.5), and (C) 50 mM MES-50 mM HEPES-75 mM NaCl (Cl- 75 mM, pH 7.4) buffers. ITP times was 0.5 min, -30 kV, sample pd(A)19-24, 8 µg/mL, 20% fill, other conditions as in Figure 1.

run (Figure 3C). When the sample was introduced in the buffer system containing MES and HEPES anions, the sample zone is effectively focused, even when no separate ITP step with the MES terminator electrolyte was performed. However, the oligonucleotide oligomers were not electrophoretically resolved, and only one narrow peak with migration time of 12.9 min appears in the electropherogram (data not shown). The effect of salt concentration of the sample on the ITP was tested by analyzing the sample in 100 mM Tris-HCl (Cl- ion concentration, 100 mM, pH 7.5) containing 200, 100, 20, and 0 mM additional NaCl. Figure 4 shows that increasing salt concentration to 300 mM Cl- ion results in an increase of migration time and better resolution of the oligonucleotide isomers. The migration time of the 16-mer antisense oligonucleotide was relatively reproducible (16.83 min (0.15 min SD) when the sample was dissolved in 100 mM Tris-HCl and 0.5 min ITP was used before the CE separation. However, migration times of oligonucleotides are dependent on the salt concentration and quality of ions in the sample. Thus, it would be advisable to include migration time marker compounds in the samples, especially when more complex samples will be analyzed.23

Figure 4. Effect of sample sodium chloride concentration on the ITP preconcentration. (A) Sample in 100 mM Tris-HCl, pH 7.5, total Cl- 120 mM. (B) Sample in 100 mM Tris-HCl, pH 7.5, total Cl- 320 mM. Other conditions as in Figure 3.

The detection limit of the method was 100 ng/mL (signal-tonoise ratio was 10). Below this sample concentration level, the purity of reagents becomes critical as several impurity peaks, which are not dependent on the oligonucleotide concentration, appear in the electropherograms (Figure 5). The quantitative response of the ITP-CE method was tested by measuring the peak areas and peak heights for a set of standards containing 0.1-5 µg/mL of 16-mer (n ) 3). Calibration lines were created using concentration of oligonucleotide (µg/mL) in the x-axis and either peak area or peak height in the y-axis (arbitrary units). The resulting curve equations and correlation coefficients were y ) 0.085x + 0.019, R2 ) 0.987 (peak areas) and y ) 0.001077x + 0.000956, R2 ) 0.949 (peak heights). The peak area measurement appears to be a better method for quantitation of the compounds than peak height measurement, presumably because lowconcentration samples tend to give higher and narrower peaks than more concentrated samples. The reproducibility of peak areas and peak heights was tested using a standard containing 1 µg/mL of 16-mer in Tris-HCl buffer (n ) 6). The relative standard deviations were 13.5% (area) and 9.8% (height).

Figure 5. Electropherogram obtained with an oligonucleotide standard containing 100 ng/mL of 16-mer antisense oligonucleotide in 100 mM Tris-HCl, pH 7.5. Twenty percent of the column was filled with sample, and MES was used as terminator for 0.5 min, -30 kV. Oligonucleotide peak is marked with letter A. Other conditions as in Figure 1.

CONCLUSIONS This study shows that on-column ITP preconcentration and CE separation of oligonucleotides can be performed in uncoated silica capillaries that are filled with viscous PEG ammonium formate buffer solutions. The use of the ITP focusing step makes it possible to increase the injected sample volume by 20 times without loss of peak resolution, which markedly increases the concentration sensitivity of the analysis. The ITP preconcentration is applicable for analyzing oligonucleotide samples in various buffer matrices, and even high salt concentrations are allowed. Use of volatile ammonium formate buffer makes the system potentially compatible with mass spectrometric detection. ACKNOWLEDGMENT This study was supported by Academy of Finland, Emil Aaltonen Foundation, and Ella and George Ehrnrooth Foundation. Received for review August 9, 1996. Accepted August 30, 1996.X AC960872C

(23) Srivatsa, G. S.; Batt, M.; Schuette, J.; Carlson, R. H.; Fitchett, J.; Lee, C.; Cole, D. E. J. Chromatogr. 1994, 680, 469-477.

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Abstract published in Advance ACS Abstracts, October 1, 1996.

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