16th Intern. Symp. on Column Liquid Chrom. - June, 1992 Poster # 404

Transcription

1 ".. ssentials in " Poster Presentation 16th Intern. Symp. on Column Liquid Chrom. - June, 1992 Poster # 404 Analysis of Synthetic Deoxyoligonucleotides by Anion xchange HPLC and Capillary Gel lectrophoresis William J. Warren, Jeanne DiGiorgio and George Vella Millipore Corporation, Waters Chromatography Division 34 Maple Street, Milford, MA U.S.A. MILLIPOR Waters 34 Maple Chromatography Street Division / Milford, MA J

2 m s Introduction Syntheticdeoxyoligonucleotides are becoming increasingly important in a wide variety of applications ranging from use as hybridization probes, primers for DNA sequencing and the polymerase chain reaction, to utilization as potential therapeutics in antisense and related technology investigations. Although improved chemistries and automated instrumentation have significantly simplified the synthesis of these molecules, the actual amount of desired full length product varies depending upon coupling efficiencies as well as on the length of the desired DNA product. The analysis of DNA synthesis reaction mixtures is therefore often desired to determine the quality of synthesized material prior to use or to ascertain the degree of product homogeneity resulting from post-synthesis purification. This poster will describe the principles and utilization of capillary gel electrophoresis (CG) and high resolution, anion exchange HPLC for the analysis of synthetic deoxyoligonucleotides. Synthetic DNA of various chain length and sequence will be used to demonstrate the relative resolving power of each analytical technique. In addition, the migration / retention time as well as the quantitation reproducibility of each methodology will be shown. Finally, the comparative strengths / weaknesses of each technique will also be described. 2

3 Principles of Separation Techniques Capillary Gel lectrophoresis: Capillary gel electrophoresis (CG) has been cited as an efficient technique for the analysis of synthetic deoxyoligonucleotides (1,2 and 3). The basis of the separation involves the molecular sieving of the various size DNA species as they are electrophoretically driven through the gel matrix (e.g. cross-linked polyacrylamide). A variety of oligonucleotide structural characteristics (e.g. length, charge to mass ratio and Stokes radius) influence the gers ability to separate the DNA molecules. Compared to slab gels, the small internal diameter (e.g. 0.75tim ) of gel filled device enables excellent heat dissipation from the gel matrix during the separation. Therefore, CG separations can be performed at higher field strengths (250 Volts/cm) than are possible using conventional slab gels. This results in very efficient separations in relatively short run times. In addition, current instrument design uses on-line UV detection for real-time monitoring of the separated DNA species as they migrate through the capillary. The actual detector flow cell is comprised of a discrete section of the capillary in which the protective polyamide coating has been removed. This design allows for the detection of nanogram to picogram quantities oligonucleotides.

4 Anion xchange HPLC: Anion exchange HPLC has been described as an effective technique for the analysis of synthetic oligonucleotides (4, 5). Separations rely primarily upon the interaction of the negatively charged phosphate groups on the DNA backbone with the positively charged cations contained on the column packing. lution of the bound deoxyoligonucleotides, in order of increasing chain length, occurs using a gradient of increasing ionic strength. The degree of component resolution depends upon the HPLC column packing and conditions used well as the overall length of the oligonucleotides analyzed. Use of an appropriate high performance anion exchange column, can provide N from N-1 chain length resolution particularly for samples less than 30 bases in length. Compared to conventional slab gel electrophoretic techniques, HPLC instrument design enables real-time, on-line monitoring of the DNA species as they elute from the column and pass through the UV detector. Staining or post-run visualization techniques are not required for the detection of nanogram amounts of sample. 4 Q

5 Sample Preparation: Materials and Methods.,, Phosphodeoxythymidine [pd(t) 18-24] and phosphodeoxyadenine [pd(a) and pd(a) 40-60] standards were purchased from Pharmacia LKB Biotechnology. Deoxyoligonucleotides (i.e. 3mer, 12mer, 13mer, 14mer, 15mer, 16mer) were synthesized at the 200 nm scale using either a Millipore Cyclone Plus or 8750 DNA instrument with 13-cyanoethylphosphoramidite chemistry. When required, final sample detritylation was performed on the instrument. Product cleavage from the controlled pore glass support and phosphate / exocyclic nitrogen deprotection were performed off-line by incubation overnight at room temperature with 30% ammonium hydroxide. A detritylated 76mer deoxyoligonucleotide was obtained from Ms. JoAnn Iwasa, Scios, Inc. Mountain View, CA. Prior to CG or HPLC, the samples were filtered through a Millipore 0.45 l.tm Millex-HV membrane. The concentration of the deoxyoligonucleotides was d6termined by measuring the optical density (OD) at 260nm for each sample using a Milton Roy Spectronic 1001 Split- Beam Spectrophotometer. An OD of 1.0 corresponds to 33 l.tg/ml of deoxyoligonucleotide.

6 Instrumentation: The following were used for the anion exchange HPLC investigations: - Waters Gen-PakTM FAX Anion xchanger, 2.5#m, Non-porous (4.6 x 100mm) - Waters 600 Gradient Solvent Delivery System - Waters 717 Autosampler - Waters Temperature Control System - Waters 486 Tunable Absorbance Detector - Waters xpert asetm Chromatography Workstation The following were used for the gel filled capillary investigations: - J&W Scientific #PAG-5TMGelFilled Capillary (5%T/5%C) 75#mx60cm - Waters QuantaTM 4000 Capillary lectrophoresis System with Negative Power Supply - Waters xpert asetm Chromatography Workstation 6

7 ffect of Sample Load on Resolution (Figures 1A and 1B)._-, Results: - The degree of deoxyoligonucleotide component resolution in CG and anion exchange HPLC is affected by the amount of sample used in the analysis. - A greater range of oligonucleotide sample loads can be satisfactorily analyzed via anion exchange HPLC than CG.

8 Figure 1A CG Separations C LO O4 [: Injection: 5 sec at -5 kv c LO _ I 1 1 I I I I I I Injection: 2 sec at -5 kv 21_ I I I I I I 1 I 1 I c Injection: 10 sec at -5 kv 19/ ' 18 ' 20 ' 22 ' 2"4 26 ' 28 ' 30 ' 32' 34 ' Minutes Sample: pd(t) (660 ng/lal) Capillary: IJPAG-5gel filled lectrolyte: 100mM Tris-Borate, ph 8.3 with 7M Urea Injection: lectrokinetic Run: -13 kv Temp: Ambient 8

9 Figure 1B HPLC Separations - - Injection: 40 ng O LO eq I I 1 1 I I I t- O (.(3 Od Injection: 140 ng 21/ ,,,,,,,,,, Injection: 300 ng /24 " 19 ocd ] I T "r t t t r "-I Minutes Sample: pd(t) (20 ng/ij.i) Gradient: 25-60%B in 30 min Column: Gen-Pak FAX Flow: 0.75 ml/min Buffer A: 25mM Tris-HCI, ph 8.0 with 10% Acetonitrile Temp: 30 C Buffer B: A + 1.0M NaCI

10 Analysis of Self-Complementary Oligonucleotide Sequences (Figures 2A and 2B) Results: - Although CG separations are performed in electrolyte containing denaturant (i.e. 7M urea), these conditions could not completely resolve a mixture of self-complementary oligonucleotides. - Because of accelerated gel matrix degradation at elevated temperatures, CG separations were only performed at ambient temperature. - Anion exchange HPLC, when performed at denaturing temperatures (e.g. 80 C), results in baseline resolution of the same mixture of self-complementary DNA. - The anion exchange HPLC column tested is stable when used at oligonucleotide denaturing temperatures. 1 0

11 Figure 2A o.oo4_- Sample not heated prior to analysis CG Separations I I I I I Sample heated at 80 C for 30 min prior to analysis t' f! I I I I Minutes Sample: pd(t) and pd(a) at 330 ng/ul Capillary: I_PAG-5gel filled lectrolyte: 100mM Tris-Borate, ph 8.3 with 7M Urea Injection: 5 sec at -5 kv Run: -13 kv Temp: Ambient 11

12 Separation at 30 C Figure 2B pd(t) and pd(a) complex HPLC Separations t- O.D pd(t) and pd(a) monomers I I I I I I I I I I pd(t) 24 Separation at 80 C pd(t) 19 pd(a) 25 3d(A) 30 0 cxl I" T T 1 T T T 3" T T Minutes Sample: pd(t) and pcl(a)25-30 (66 ng/pl) Column: Gen-Pak FAX BufferA: 25raM Tris-HCI, ph 8.0 with 10% Acetonitrile Buffer B: A + 1.0M NaCL Injection: 13.2 pg Gradient: 25-60%B in 30 min Flow: 0.75 ml/min I2 i,g

13 Factors Affecting Resolution (Figures 3A and 3B).k Results: - Applied voltage during CG is one factor which influences oligonucleotide separations. Lower running voltages result in marginal improvements in component resolution while they significantly increase the total analysis time. - Gradient conditions used during anion exchange HPLC of oligonucleotides is a significant factor influencing their separation. Shallow gradients dramatically improve component resolution while only slightly increasing analysis times.

14 pd(a) Figure 3A Separation performed at -13 kv CG Separations c to e,,i _ ),,, pd(a) Separation performed at -6.5 kv C to C,I A_,_1 _ r 3" 1" T I Minutes Sample: pd(a) (660 ng/ul) Capillary: IJPAG-5gel filled lectrolyte: 100mM Tris-Borate, ph 8.3 with 7M Urea Injection: 5 sec at -5 kv Temp: Ambient 14 f

15 Figure 3B HPLC Separations t- O CO O4 Gradient: %B in 30 rain L 1 I I I I I 1 I I Gradient: 10-50% B in 0.01 min. Then, 50-60%B in 30 min c O " I I I I I I I I I I Sample: pd(a) (660 ng/pl) Column: Gen-Pak FAX Minutes Injection: 3.3 _g Flow: 0.75 ml/min Buffer A: 25mM Tris-HCI, ph 8.0 with 10% Acetonitrile Temp: 80 C Buffer B: A + 1.0M NaCI 15

16 Ruggedness and Reproducibility of Separations Results: - The gel filled capillary tested performed well up to analyses before significant reduction in component resolution was observed. - The anion exchange HPLC column tested provided high resolution separations for hundreds of analyses. - Table1 summarizes the reproducibility of each technique. Table 1: Reproducibility of CG and Anion xchange HPLC Analysis of a pd(a) Standard Mixture(N=6) Technique Migration/ Retention Time Quantitation %RSD %RSD ppag-5 CG* 0.79% 2.0% Gen-Pak FAX HPLC 0.07% 0.9% * Changes in electrolyte composition occur during each CG separation. In this study, fresh sample electrolyte was used for each analysis in order to obtain maximum migration time. reproducibility. t6

17 ffect of Charge to Mass Ratio on Separations (Figure 4) Results: - Compared to anion exchange HPLC, the separation of oligonucleotides in CG is significantly affected by their charge to mass ratio. - Because oligonucleotides of lower charge to mass ratio migrate slower in CG than samples possessing higher ratios, disproportionate sample loading occurs. In addition, oligonucleotides of larger size and charge to mass ratio may actually migrate through the gel filled capillary faster than some smaller sequences. 17

18 Figure 4 pd(a) Sample Charge to Mass Ratio pd(a) 25 d(a)3 [5'-OH] 2/1073 = I d(a) 3 CG pd(a)25 [5'-PO4] 25 / 9577 = _II _"q m _ Total corrected Total corrected 04 pd(a)30 [5'-PO4] 30 / = area = 87% area = 13% l I I I I I I 0 d(a) _ pd(a) 30 HPLC pd(a) 25 t- O tj3 04 _ Total area = 28% --._--Totalarea = 72% " " " I I I I I I I Minutes Top: I.LPAG-5CG separation Bottom: Gen-Pak FAX HPLC separation Sample: d(a) 3 and pd(a) (330 ng/iji) Sample: d(a) 3 and pd(a) (66 ng/iji) lectrolyte: 100ram Tris-Borate, ph 8.0 with 7M Urea Buffer A: 25raM Tris-HCl, ph 8.0 with 10% ACN Injection: 5 sec at -5 kv Buffer B: A + 1.0M NaCI Run: -13 kv Injection: 660 ng. Flow: 0.75 ml/min. Temp: 80 C Temp: Ambient Gradient: 0-60%B in 30 rain. Then hold at 60%B for 10 min ]_8

19 Result: Influence of 5' Dimethoxytrityl (DMT) Group (Figure 5) - The presence of 5' DMT groups results in longer migration / elution times of oligonucleotide samples compared to detritylated species. The analyses of detritylated oligonucleotide samples is thus recommended for both CG and anion exchange HPLC.. 19

20 Figure 5 CG mer (DMT-off) 16mer: (5' CAT-GTG-CCA-GAA-CTG-T 3') 16mer (DMT-on) LO I I i I ] I i I i I I mer (DMT-off) HPLC 16mer DMT-on) '- O ( Failure sequences o,oooo-_ k_.._..,, _,_...,.. "1" r 1 T T T 1 T 1 -r Minutes Top: ppag-5 CG separation Bottom: Gen-Pak FAX HPLC separation Sample: Mixture of 16mer samples with and Sample: Same as for CG separation without DMT group at 1.5 pg/ul Buffer A: 25mM Tris-HCI, ph 8.0 with 10% ACN lectrolyte: 100mM Tris-Borate, ph 8.0 with 7M Urea Buffer B: A+ 1.0M NaCI Injection: 5 sec at -5 kv Injection: 15 pg. Flow: 0.75 mvmin. Temp: 80 C. Run: -13 kv Gradient: 10-60%B in 30 min Temp: Ambient 2O

21 4_ N From N-1 Resolution (F,gures 1, 3 and 6).. Results: - CG and anion exchange HPLC can provide N from N-1 degrees of component resolution of various size oligonucleotides (See Figures 1 and 3). - Co-elution of different size oligonucleotides can occur in CG if the separation, influenced by the gel matrix, is offset by differences in migration velocities due to charge to mass ratio differences (e.g. co-elution of 12 and 13mer in Figure 6). 21

22 Figure 6 12 & 13 / CG Sample Charge to Mass Ratio 12mer: 5' GCA-ACT-GGT-GAT 3' mer: 5' A-GCA-ACT-GGT-GAT 3' t- 14mer: 5' TA-GCA-ACT-GGT-GAT 3' '<" 15mer: 5' TTA-GCA-ACT-GGT-GAT 3' _' ^ I I I I I I 0 30 O HPLC t- O (D 04 IA L _[_L''JL_A ^ _.J _ L.,, _, I T 7" T T Minutes Top: lapag-5 CG separation Bottem: Gen-Pak FAX HPLC separation Sample: 12,13,14 and 15mer (DMT-off) at 110 ng/ul Sample: Same as for CG separation lectrolyte: 100mM Tris-Borate, ph 8.0 with 7M Urea Buffer A: 25mM Tris-HCI, ph 8.0 with 10% ACN Injection: 5 sec at -5 kv Buffer B: A + 1.0M NaCI Run: -13 kv Injection: 550 ng. Flow: 0.75 ml/min. Temp: 80 C Temp: Ambient Gradient: 25-60%B in 30 min -. 22,,Ib

23 Results: Analysis of Deoxyoligonucleotides of qual Length but Different Sequence.. (Figure 7) - Neither CG or anion exchange HPLC can be used to determine the absolute length of an unknown oligonucleotide as shown in this example. 2 3

24 I Figure 7 B&C Sample ;_ CG 16mer A: 5' GGG-AGT-TCG-AGG-AAC-G 3' 16mer B: 5' TGT-CAA-GAC-CGT-GTA-C 3' c 16mer C: 5' CAT-GTG-CCA-GAA-CTG-T 3' to 04 u.uuuu- w,, j,, 0 B 30 o.a210- _ c HPLC f/ - O _o (N o.oooo-... " I I I I I I Minutes Top: I_PAG-5 CG separation Bottom: Gen-Pak FAX HPLC Separation Sample: (3) 16mers (DMT-off) at 550 ng/ul Sample: Same as for CG separation lectrolyte: 100mM Tris-Borate, ph 8.0 with 7M Urea Buffer A: 25mM Tris-HCI, ph 8.0 with 10% ACN Injection: 5 sec at -5 kv Buffer B: A + 1.0M NaCI " Run: -13 kv Injection:19 Ilg Flow: 0.75 ml/min. Temp: 80 C Temp: Ambient Gradient: 10-60%B in 30 min 24

25 Analysis of "Large" Deoxyoligonucleotide Sequences (Figure 8) - Results: - The gel filled capillary tested provides better resolution of large oligonucleotides (e.g. 76mer) than the anion exchange HPLC column evaluated in this study. 25

26 Figure 8 76mer 76mer: 5' CTT-TCT-ATT-CTC-ACT-CCG-CTG-AAC-CAC-GTA-AAA-CTT- CCT-CCG-GAA-GTG-CAG-AAG-ATC-CGC-CTC-CAC-CTC-CAC-C 3' c U3 (XI Failure sequences ,30 Minutes _,5 76mer HPLC o Failures sequences x.- o I'5 :_o is _o 3s Minutes Top: NPAG-5 CG separation Bottom: Gen-Pak FAX HPLC separation Sample: 76mer (DMT-off) at 330 ng/ul Sample: Same as for CG separation lectrolyte: 100mM Tris-Borate, ph 8.0 with 7M Urea Buffer A: 25mM Tris-HCI, ph 8.0 with 10% ACN Injection: 20 sec at -5 kv Buffer B: A + 1.0M NaCI : Run: -13 kv Injection: 3.3 p.g. Flow: 0.75 ml/min. Temp: 80 C Temp: Ambient Gradient: %B in 30 min,r 26 J.,

27 Conclusions This study directly compared the analyses of several deoxyoligonucleotides using CG (J&W Scientific's p.pag-5gel filled capillary) and high performance, anion exchange HPLC (Waters' Gen-Pak FAX column). Although improvements in both gel filled capillary devices as well as HPLC columns are anticipated, the present state of the art favors HPLC for the analyses of most deoxyoligonucleotide samples. Table 2 summarizes the results obtained in this study. 27

28 Table 2: Characteristics of _PAG-5 and Gen-Pak FAX HPLC for the Analysis of DNA Attribute Characteristicsof Characteristicsof ppag-5 Capillary Gen-Pak FAX Column - Life of stored device. 3 to 4 months > 1 year - # of analyses per device Run to run time. Less than 45 min. Less than 60 min. - # of samples analyzed 1 1 per run. - Technique requires No Yes methods development. - Resolution effected Yes Yes by sample load. - Ability to resolve Good xcellent self-complementary DNA. - Reproducibility using device. Good xcellent - Disproportionate loading due to Yes No differing DNA charge to mass ratios. - Capable of confirming length No No of oligonucleotide unknown. - Ability to resolve "large" xcellent Good -' (e.g. 76mer) sequences. 28

29 =rf References 1) Cohen, A.S., D.R. Najarian, A. Paulus, A. Gutterman, J.A. Smith and B.L. Karger Rapid separation and Purificationof Oligonucleotides by High-Performance CapillaryGel lectrophoresis. Proc. Natl. Acad. Sci. U.S.A. 85: ) Guttman, A. and N. Cooke Capillary Gel Affinity lectrophoresis of DNA Fragments. Anal. Chem. 63: ) Turner, K New Dimensions in Capillary lectrophoresis Columns. LC/GC. 9: ) Drager, R.R. and F.. Regnier High-Performance Anion-xchange Chromatography of Oligonucleotides. Anal. Biochem. 145: ) Kato, Y. T. Kitamura, A. Mitsui, Y. Yamasaki, T. Hashimoto, T. Murotsu, S. Fukushige and K. Matsubara Separation of Oligonucleotides by High-Performance Ion-xchange Chromatography on a Non-Porous Ion xchanger. J. Chrom. 447: