Oligonucleotide Separation Technology

Transcription

1 Oligonucleotide Separation Technology [ 1 ]

2 Oligonucleotide Separation Technology Waters Oligonucleotide Separation Technology (OST) columns contain second-generation hybrid silica BEH Technology particles functionalized with C 18. The separation of detritylated synthetic oligonucleotide samples is based on the well-established method of ion-pair, reversed-phase chromatography. The availability of 1.7 μm PLC particles and 2.5 μm HPLC particles in various column dimensions meets various lab-scale isolation and analysis needs. Waters OST colums deliver exceptional sample resolution, and superior column life making them the ideal choice for your application. Waters manufacturing and quality-control testing procedures help ensure consistent batch-to-batch and column-to-column performance regardless of application demands. Separation efficiencies equivalent or better than PAGE, CGE, and ion-exchange HPLC methods without the need for desalting Resolution of failure sequences from detritylated full length products Scalable column offerings for lab-scale purification needs Exceptional column life for reduced cost per analysis QC tested with MassPREP OST Standard to ensure performance consistency

3 Separation of Detritylated Oligodeoxythymidine Ladders by Capillary Gel Electrophoresis (CGE) vs. Ion-Pair, Reversed-Phase Chromatography System: CGE Column: Injection: Running: Temp: Capillary Gel Electrophoresis System PEG sieving matrix (BioCap 75 μm x 27.5 (to detector)/ 34.5 cm (total length) 45 injection at 5 kv 15 kv 30 C V 260 nm 22 min LC System: Column: Waters ACQITY PLC System ACQITY OST C 18, 1.7 µm, 2.1 x 50 mm Mobile Phase: A: 15 mm TEA, 400 mm HFIP, ph 7.9 Column Temp: B: 50% A, 50% MeOH 0.4 ml/min 60 C 40 to 48% B in 4 minutes (20-24% MeOH) 260 nm min

4 OLIGONCLEOTIDE SEPARATION TECHNOLOGY EXCEPTIONAL RESOLTION OF OLIGONCLEOTIDE MIXTRES ACQITY PLC OST C 18, 1.7 µm (designed for use with an ACQITY PLC System) and XBridge OST C 18, 2.5 µm columns are well suited for analyzing detritylated oligonucleotides using ion-pair, reversed-phase chromatography. Separations and component resolution are comparable to those obtained by capillary gel electrophoresis (CGE), however, with Waters PLC Technology you can complete the analysis in a fraction of the time. It is also possible to resolve large oligonucleotide sequences (e.g., N from N-1) due to the enhanced resolving power of the sub-3 µm BEH Technology particles. With Waters OST columns, hyphenated Mass Spectrometry methods and MS-friendly eluents, it is possible to characterize the molecular weight of the separated target oligonucleotide product from failure sequences. Separation of Detritylated Oligodeoxythymidine Ladders Separation of a mer Detritylated Oligodeoxythymidine Ladder LC System: Waters ACQITY PLC System with PDA Column: ACQITY PLC OST C 18, 1.7 µm, 2.1 x 50 mm Mobile Phase: A: 15 mm TEA mm HFIP, ph 7.9 B: 50% A and 50% Methanol 0.2 ml/min Column Temp.: 60 C %B in 15 min V: 260 nm, 10 scans per second LC System: Waters ACQITY PLC System with PDA and Q-Tof micro MS Column: ACQITY PLC OST C 18, 1.7 µm, 2.1 x 50 mm Mobile Phase: A: 15 mm TEA mm HFIP, B: Methanol 0.1 ml/min V 260 nm Column Temp: 60 C 19 to % B in 60 minutes V: 260 nm, 10 scans per second MS: 1 scan per second, 0.1 sec interscan V 260 nm 40T 50T 60T MS (TIC) 2 11 min 0 45 min Ion-pair, reversed-phase chromatography of detritylated oligonucleotides is a well accepted method for the analysis of complex synthesis reaction mixtures. Columns containing small particles, such 2.5 µm and 1.7 µm, provide enhanced resolving power comparing to that obtained using more traditional LC columns. While it is routine to accurately measure separated oligonucleotides by V detection, this technique in unable to provide valuable qualitative information. Consequently, mass spectrometry eluents enable V and MS data to be collected in a single run for quantitative and qualitative data collection and post-run analysis. ACQIT Y PLC BEH OST Columns C ertificat e of Application Specific Analysis Each batch of XBridge and ACQITY PLC OST C 18 column material is rigorously tested to ensure consistent performance. The Certificate of Analysis available for each column reports physical, chemical, and chromatographic tests obtained using our MassPREP OST Standard that contains a defined mix of 15, 20, 25, 30, and 35 nucleo-tide (nt) long oligodeoxythymidines. [ 4 ]

5 OLIGONCLEOTIDE SEPARATION TECHNOLOGY FAST, INFORMATION-RICH ACQITY PLC/MS ANALYSES Waters ACQITY PLC systems and column chemistries collectively reduce the time and cost per sample analysis while improving the overall quality and resolution of collected data. A key component that differentiates Waters holistic design is our patented sub-2 μm hybrid particle chemistry. This patented technology outperforms traditional HPLC that uses standard larger particle sizes. These synergistic technologies combine to provide scientists with tools to help better characterize both DNA, RNA, and modified synthetic oligonucleotide sequences. LC/MS Analysis of RNA (21 mer) G C C 20 nt 8.73 min C TEA 5800 m/z 6800 TEA A A G C RNAi 21-mer 1 10 min LC System: TIC MS: Capillary: Sample Cone: Extraction Cone: SYNAPT MS 2000 V 35 V 3 V Desolvation Temp: 200 C Source Temp.: 120 C Cone Gas Flow: Desolvation Gas Flow: Waters ACQITY PLC Column: ACQITY PLC OST C 18, 2.1 x 50 mm, 1.7 µm Column Temp: Sample Injected: Mobile Phase A: Mobile Phase B: 60 C 2.5 µl (100 pmole of 21 nt RNAi) 0.2 ml/min 15 mm TEA. 400 mm HFIP 50% A, 50% methanol 20-40% B in 10 min. PDA, 260 nm V 50 L/hr 600 L/hr The acquisition of the accurate masses from an ACQITY PLC-based separation allowed for an assignment of the peaks of 5 -truncated oligomers as well as some other impurities. The mass of each peak in the MS chromatogram was deconvoluted using MaxEnt 1 software. Nearly the entire sequence of the parent oligonucleotide was elucidated. MS analysis also revealed a presence of an extra uridine mononucleotide added to the target 21-mer RNAi sequence. Manufacturing, and Application Specific Testing for Increased CSTOMER Assurance Waters second-generation hybrid particles are the basis for XBridge and ACQITY PLC OST C 18 columns. sing a proprietary bonding process, we ve developed a robust chemistry for synthetic DNA and RNA separations that are frequently performed at conditions of elevated ph and temperature. In addition, extensive quality-control testing with detritylated oligonucleotide standards help us ensure consistent column-to-column performance. Well characterized, state-of-the-art bonding procedures for short amine-containing ligand Particle structure and bonding chemistry stable at low ph and at elevated temperature Quality-control tested with a relevant labeled OST standard mixture Consistent labeled oligonucleotide separations from batch to batch [ 5 ] Anal. Chem. 2003, 75, ,.S. Patent No. 6,686,035 B2

6 OLIGONCLEOTIDE SEPARATION TECHNOLOGY OTSTANDING COLMN LIFE Waters OST columns, packed with BEH Technology particles, have shown remarkable column longevity under demanding separation conditions, while maintaining outstanding separation performance. By comparison, under these same demanding separation conditions traditional silica-based columns have shorter lifetimes. High ph and Temperature Separation of 5-25 mer Detritylated Oligodeoxythymidine Ladder Analyte: Acenaphthene XBridge C 18 HPLC System: Alliance Bio 2796 with PDA Column: XBridge OST C 18, 2.5 µm (2.1 x 50 mm) Mobile Phase: A: 10% MeOH 90% (385mM HFIP mM TEA) B: 25% MeOH 75% (385mM HFIP mM TEA) Column Temp: 60 C 0-100% B in 30 min (10-25 % MeOH) 1.0 ml/min 260 nm, 5 scans per second % Initial N 5s 70 XTerra MS C 18 Gemini C 18 Luna C18(2) 50 YMC Pro C 18 Zorbax Extend C 18 Injection # hours Hours in 50 mm TEA, ph 10, 50 C Waters OST columns (both our ACQITY PLC OST C 18 and XBridge OST C 18 ) were selected from Waters proprietary base particle material that was designed to tolerate aggressive separation conditions of high temp and ph. It is well known that most if not all 100% silica-based, reversed-phase columns have limited life when used with eluents (e.g., TEA-HFIP, ph 8.3) and at temperatures (e.g., 60 C) frequently employed for ion-pair, reversed-phase chromatographic separations of synthetic oligonucleotide separations. Injection # min Waters XBridge OST C 18 columns show outstanding column lifetime under synthetic oligonucleotide separation conditions of elevated temperature and ph. Compared to use of less stable, 100% silica-based columns, outstanding performance on XBridge, Hybrid Particle Technology columns is maintained even after many sample injections. SCALABLE DNA AND RNA SEPARATIONS WITH GOOD PRODCT RECOVERY XBridge OST C 18 columns are the preferred offering for detritylated oligonucleotide purifications and are available in a range of lengths for varying lab-scale isolation requirements. As indicated in the table below, the scale of the synthesis reaction mixture will determine your choice of XBridge OST C 18 column dimensions and operating flow rates. For researchers involved in gene silencing, it is often necessary to work with high purity RNA. Crude synthetic oligonucleotides used for gene knockout are typically purified. The figure below illustrates a lab-scale purification of 21 mer RNA at various column loads. sing OST columns and the Waters Alliance System, large quantities of crude single-stranded RNA can be successfully purified yielding high purity material, approx 95%, with an estimated yield of 55% based on collected peak area to the total peak area of the sample. In addition, OST columns are well suited for sirna analysis and purification. As shown in the figure below, sirna is well resolved from single-stranded RNA and truncated duplexes. [ 6 ]

7 XBRIDGE OST C 18 COLMN SELECTION GIDE FOR DETRITYLATED OLIGONCLEOTIDE PRIFICATION Scalable DNA and RNAi Separations with Good Product Recovery XBridge OST C 18 columns are the preferred offering for detritylated oligonucleotide purifications due to the availability of column sizes designed to meet lab-scale isolation requirements. As indicated in the table below, the choice of XBridge OST C 18 column dimension and operating flow rate depends rimarily on the scale of the synthesis reaction mixture. Selection of the appropriate column size for the amount of oligonucletide sample loaded is recommended to maximize component resolution and recovery of the target product from non-desired failure sequences. Dimensions Approx Mass Load** mg*** Flow Rate 2.1 x 50 mm 0.04 µmoles 0.2 mg 0.2 ml/min 4.6 x 50 mm 0.20 µmoles 1.0 mg 1.0 ml/min 10 x 50 mm 1.00 µmoles 4.5 mg 4.5 ml/min 19 x 50 mm* 4.00 µmoles 16.0 mg 16.0 ml/min 30 x 50 mm* 9.00 µmoles 40.0 mg 40.0 ml/min 50 x 50 mm* µmoles mg ml/min * OST Custom Column ** Values are only approximates and vary depending on oligonucleotide length, base composition, and heart-cutting fraction collection method used. *** Estimated for average oligonucleotide MW and synthesis yield. Purification of Single Stranded RNA Purification of sirna Duplex from Impurities LC System: Waters Alliance Bio Column: XBridge OST BEH C 18, 2.5 µm, 4.6 x 50 mm Column Temp: 60 C 1.0 ml/min Mobile Phase A: 0.1M TEAA, ph 7.5 Mobile Phase B: 20% Acetonitrile in A % B in 10.0 min (0.15% ACN/min) PDA, 260 nm 140 nmol Purified Oligonucleotide Purity > 95% LC System: Waters Alliance Bio Column: XBridge OST BEH C 18, 2.5 µm, 4.6 x 50 mm Column Temp: 20 C 1.0 ml/min Mobile Phase A: 0.1 M TEAA, ph 7.0 Mobile Phase B: 20% ACN in A 25-75% B in 30.0 min PDA, 260 nm 85 nmol Target sirna duplex N-1 Purity 78% 84 nmol * % PLC purity (area) 15 nmol 28 nmol RNA Impurities sirna Impurities 1.4 nmol 0 18 min 1.5 nmol 0.15 nmol 0 25 min For researchers involved in gene silencing it is often necessary to work with RNA of high purity. This figure illustrates a lab-scale purification of 21 mer RNA at various column loads. sing OST column chemistry, large quantities of crude single stranded RNA can be successfully purified yielding material of high purity with an estimated yield of 55% based on collected peak area to the total peak area of the sample. Waters OST columns are also well suited for purification of sirna duplexes from RNA impurities. As shown in this figure, 85, 15, 1.5 and 0.15 mmoles of sirna duplex material can be successfully separated on XBridge OST BEH C 18, 2.5 μm, 4.6 x 50 mm within 25 min when separated at recommended temperature of 20 C. [ 7 ]

8 oligonucleotide separation technology COLMNS FOR LARGE DNA/RNA SPECIES In general, molecular biology methods for manipulation of DNA rely on restriction enzymes, polymerase-chain reaction (PCR), and sequencing techniques. sing these methods, genomic DNA is typically converted into shorter double stranded (ds)dna sequences, typically base pairs (bp) in length. The shorter dsdna molecules are often analyzed or isolated by methods such as slab gel or capillary electrophoresis. Waters ACQITY BEH300, C 18 reversed-phase or Gen-Pak FAX anion-exchange columns offer alternatives to more traditional electrophoretic methods and are particularly well suited for various analytical and small-scale purifications. Separation of Duplex DNA Fragments: HaeIII and MspI Restriction Enzyme Digests of pbr322 Plasmid LC System: Waters ACQITY PLC Column: ACQITY PLC PST BEH300 C 18, 2.1 x 50 mm, 1.7µm Column Temp: 50 C 0.2 ml/min Mobile Phase A: 0.1M TEAA, ph 7.0 Mobile Phase B: 20% ACN in A % B in 20.0 min PDA, 260 nm The manipulation of plasmid DNA by appropriate restriction enzymes will generate shorter-length, double stranded (ds)dna fragments who purification and analysis is traditional performed using electrophoresis based methods. By comparison, ion-pair, reversed-phase LC of these samples using Waters ACQITY BEH300 C 18, 1.7 µm technology offers an alternative that is particularly well suited for analytical and small scale purification applications min Mas s PREP OST STANDARD The pre-packaged MassPREP OST Standard consist of a carefully defined mixture of 15, 20, 25, 30, and 35 mer synthetic oligodeoxythymidine sequences that are recommended for use with Waters ACQITY ltraperformance LC (PLC) Technology, high-performance liquid chromatographs (HPLC), liquid chromatography/mass spectrometry (LC/MS), or capillary electrophoresis (CE) systems used for synthetic oligonucleotide separation applications. The MassPREP OST Standard increases laboratory productivity by eliminating the need for analysts to prepare and quality control their own standards. Separation of MassPREP OST Standard on ACQITY PLC OST C 18, 1.7 μm Column 15 nt 20 nt 25 nt 30 nt 35 nt Contains a carefully defined mixture of synthesized oligodeoxythymidine fragments Vacuum-sealed in foil pouches to reduce degradation that can occur by excessive exposure to light and air Each batch QC tested and shipped with a certificate of analysis 0 10 min Waters MassPREP OST Standard can be effectively used to quality control various synthetic oligonucleotide analysis applications. Approximate amounts of 15, 20, 25, 30, and 35 nucleo-tide (nt) long oligodeoxythymidines are lyophilized and packaged in 1.5 ml LC vials. The main components are labeled in the shown chromatogram while the peaks eluting between labeled oligonucleotides are N-1, N-2, etc. failure sequences generated during the oligonucleotide syntheses. [ 8 ]

9 oligonucleotide separation technology OLIGONCLEOTIDE DESALTING ON OASIS HLB Desalting of synthetic oligonucleotides is essential for MS analysis (QC, genotyping applications and SNP analysis). Waters new μelution plate is an excellent choice for high throughput analysis with minimal amount of sample. The Oasis μelution plate combines patented plate design, proven Oasis HLB chemistry, and generic protocols enabling elution volumes as low as 25 μl. Now, for the first time, you can perform SPE cleanup and concentration of very small sample volumes. The Oasis HLB sample extraction products incorporate a patented copolymer made from a balanced ratio of two monomers; the lipophilic divinylbenzene and the hydrophilic N-vinylpyrolodone that is ideally suited for this application. Removes salt prior to MS analysis Low elution volumes High sensitivity Sample concentrating High throughput Ordering Information Description Particle Size Pore Size Dimension Part Number ACQITY PLC OST C 18 * 1.7 μm 135Å 2.1 x 50 mm ACQITY PLC OST C 18 * 1.7 μm 135Å 2.1 x 100 mm Custom ACQITY PLC OST C 18 * XBridge OST C μm 135Å 2.1 x 50 mm XBridge OST C μm 135Å 4.6 x 50 mm XBridge OST C μm 135Å 10 x 50 mm Custom XBridge OST C ACQITY PLC BEH300 C µm 2.1 x 50 mm Description Quantity Part Number MassPREP OST Standard 1/pk * For use on Waters ACQITY PLC Systems More information can be found at ACQITY PLC System with XEVO TQ Mass Spectrometer

10 [ ACQITY PLC System Accessories ] Sales Offices Austria and European Export (Central South Eastern Europe, CIS and Middle East) Australia Belgium Brazil Canada x2205 China CIS/Russia / Czech Republic Denmark Finland France Germany Hong Kong The Netherlands Norway Poland Puerto Rico Singapore Spain Sweden Switzerland Taiwan nited Kingdom All other countries: Waters Corporation.S.A Hungary India and India Subcontinent Ireland Italy Japan Korea Mexico Waters Corporation. Waters, The Science of What s Possible, PLC, ACQITY PLC, Oasis, Gen-Pak, ACQITY ltraperformance LC, Alliance, MaxEnt, MassPREP, BEH Technology, Q-Tof micro, SYNAPT, XTerra, XEVO, LCT Premier, and XBridge are trademarks of Waters Corporation. The quality management system of Waters manufacturing facilities in Taunton, Massachusetts and Wexford, Ireland complies with the International Standard ISO 9001:2000 Quality Management and Quality Assurance Standards. Waters quality management system is periodically audited by the registering body to ensure compliance. Gemini and Luna are trademarks of Phenomenex, Inc. Zorbax is a registered trademark of Agilent Technologies. YMC is a trademark of YMC Co., Ltd EN April 2009 SC-AC [ 10 ]