[ ]1

Oligonucleotide Separation Technology

Waters Oligonucleotide Separation Technology (OST) columns contain second-generation hybrid silica BEH Technology

particles functionalized with C18. 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 UPLC® 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

Oligonucleotide Separation Technology

Separation of Detritylated Oligodeoxythymidine Ladders by Capillary Gel Electrophoresis (CGE) vs. Ion-Pair, Reversed-Phase Chromatography

0 4 min

20

25

30

35

12 22 min

UV 260 nm

10

30

15

0 4 min

20

25

30

35

12 22 min

UV 260 nm

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30

15

System: Capillary Gel Electrophoresis SystemCGE Column: PEG sieving matrix (BioCap 75 μm x 27.5 (to detector)/ 34.5 cm (total length)Injection: 45 injection at 5 kVRunning: 15 kVTemp: 30 ˚C

LC System: Waters ACQUITY UPLC® System Column: ACQUITY OST C18, 1.7 µm, 2.1 x 50 mmMobile Phase: A: 15 mM TEA, 400 mM HFIP, pH 7.9 B: 50% A, 50% MeOHFlow Rate: 0.4 mL/minColumn Temp: 60 ˚CGradient: 40 to 48% B in 4 minutes (20-24% MeOH)Detection: 260 nm

OLIGONUCLEOTIDE SEPARATION TECHNOLOGY

EXC EPT IONAL RESOLUT ION OF OLIGONUCLEOT IDE MIXTURES ACQUITY UPLC OST C18, 1.7 µm (designed for use with an ACQUITY UPLC System) and XBridge™ OST C18, 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 UPLC 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.

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40T

2 11 min

50T

60T

UV

260

nm

Separation of Detritylated Oligodeoxythymidine LaddersSeparation of a 15-60 mer Detritylated Oligodeoxythymidine Ladder

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 UV detection, this technique in unable to provide valuable qualitative information. Consequently, mass spectrometry eluents enable UV and MS data to be collected in a single run for quantitative and qualitative data collection and post-run analysis.

0 45 min

UV 260 nm

MS (TIC)

LC System: Waters ACQUITY UPLC System with PDA and Q-Tof micro™ MSColumn: ACQUITY UPLC OST C18, 1.7 µm, 2.1 x 50 mmMobile Phase: A: 15 mM TEA - 400 mM HFIP, B: MethanolFlow Rate: 0.1 mL/min

Column Temp: 60 ˚CGradient: 19 to 26.5.5% B in 60 minutesDetection: UV: 260 nm, 10 scans per second MS: 1 scan per second, 0.1 sec interscan

Each batch of XBridge and ACQUITY UPLC OST C18 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.

ACQUIT Y UPLC BEH OST COLUMNS C ERT IF ICAT E OF “AP PLICAT ION SPECIF IC” ANALYSIS

LC System: Waters ACQUITY UPLC System with PDAColumn: ACQUITY UPLC OST C18, 1.7 µm, 2.1 x 50 mmMobile Phase: A: 15 mM TEA - 400 mM HFIP, pH 7.9 B: 50% A and 50% MethanolFlow Rate: 0.2 mL/minColumn Temp.: 60 °CGradient: 45-49.5 %B in 15 minDetection: UV: 260 nm, 10 scans per second

OLIGONUCLEOTIDE SEPARATION TECHNOLOGY

[ ]5

FAST, INFORMAT ION-RICH ACQUIT Y UPLC /MS ANALYSES

Waters ACQUITY UPLC 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)

U

U

UG

UCCU

U

UCUG

AA

CUU

21-mer

1 10 min

RNAi

20 nt8.73 min

TEATE

A

m/z5800 6800

TIC MS: SYNAPT™ MSCapillary: 2000 VSample Cone: 35 VExtraction Cone: 3 VDesolvation Temp: 200 °CSource Temp.: 120 °CCone Gas Flow: 50 L/hrDesolvation Gas Flow: 600 L/hr

LC System: Waters ACQUITY UPLCColumn: ACQUITY UPLC OST C18, 2.1 x 50 mm, 1.7 µmColumn Temp: 60 ˚CSample Injected: 2.5 µL (100 pmole of 21 nt RNAi)Flow Rate: 0.2 mL/minMobile Phase A: 15 mM TEA. 400 mM HFIPMobile Phase B: 50% A, 50% methanolGradient: 20-40% B in 10 min.Detection: PDA, 260 nm UV

The acquisition of the accurate masses from an ACQUITY UPLC-based separation allowed for an assignment of the peaks of 5’-truncated oligomers as well as some other impurities. T he 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 AP PLICAT ION SPECIF IC T EST ING FOR INC REASED CUSTOMER ASSURANC E

Waters second-generation hybrid particles are the basis for XBridge and ACQUITY UPLC OST C18 columns. Using 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.

Anal. Chem. 2003, 75, 6781-6788, U.S. Patent No. 6,686,035 B2

■ 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

OLIGONUCLEOTIDE SEPARATION TECHNOLOGY

[ ]6

OUTSTANDING COLUMN 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

Waters OST columns (both our ACQUITY UPLC OST C18 and XBridge OST C18) 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.

Waters XBridge OST C18 columns show outstanding column lifetime under synthetic oligo-nucleotide 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 SEPARAT IONS WIT H GOOD P RODUCT RECOV ERY

XBridge OST C18 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 C18 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. Using 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.

Analyte: Acenaphthene

030

50

70

90

110

50 100 150 200 hours

Hours in 50 mM TEA, pH 10, 50° C

% In

itial

N5s

XBridge™ C18

XTerra® MS C18

Gemini™ C18

Luna® C18(2)

YMC™ Pro C18

Zorbax® Extend C18

Injection #4

0 3 6 9 12 15 18 21 24 27 30 min

Injection #1001

HPLC System: Alliance ® Bio 2796 with PDAColumn: XBridge OST C18, 2.5 µm (2.1 x 50 mm)Mobile Phase: A: 10% MeOH 90% (385mM HFIP + 14.3mM TEA) B: 25% MeOH 75% (385mM HFIP + 14.3mM TEA)

Column Temp: 60 ˚CGradient: 0-100% B in 30 min (10-25 % MeOH)Flow Rate: 1.0 mL/minDetection: 260 nm, 5 scans per second

0 6

Purified Oligonucleotide

Purity > 95%

*

N-1

140 nmol

84 nmol

28 nmol

1.4 nmol

0 18 min

97.8% UPLC purity (area)Purity ≈ 78%

XBRIDGE OST C18 COLUMN SELECT ION GUIDE FOR DET RIT YLAT ED OLIGONUCLEOT IDE PURIF ICAT ION

Scalable DNA and RNAi Separations with Good Product Recovery

XBridge OST C18 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 C18 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* 25.00 µmoles 110.0 mg 110.0 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

For researchers involved in gene silencing it is often necessary to work with RNA of high purity. T his figure illustrates a lab-scale purification of 21 mer RNA at various column loads. Using 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.

LC System: Waters Alliance BioColumn: XBridge OST BEH C18, 2.5 µm, 4.6 x 50 mmColumn Temp: 60 °CFlow Rate: 1.0 mL/minMobile Phase A: 0.1M TEAA, pH 7.5Mobile Phase B: 20% Acetonitrile in AGradient: 30 – 52.5% B in 10.0 min (0.15% ACN/min) Detection: PDA, 260 nm

Purification of siRNA Duplex from Impurities

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 C18, 2.5 µm, 4.6 x 50 mm within 25 min when separated at recommended temperature of 20 °C.

[ ]7

0 25 min

1.5 nmol

0.15 nmol

RNAImpurities

siRNAImpurities

Target siRNAduplex85 nmol

15 nmol

LC System: Waters Alliance Bio™ Column: XBridge OST BEH C18 , 2.5 µm, 4.6 x 50 mmColumn Temp: 20 °CFlow Rate: 1.0 mL/minMobile Phase A: 0.1 M TEAA, pH 7.0Mobile Phase B: 20% ACN in AGradient: 25 - 75% B in 30.0 minDetection: PDA, 260 nm

COLUMNS 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. Using these methods, genomic DNA is typically converted into shorter double stranded (ds)DNA sequences, typically 100-1000 base pairs (bp) in length. The shorter dsDNA molecules are often analyzed or isolated by methods such as slab gel or capillary electrophoresis. Waters ACQUITY BEH300, C18 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

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 electro-

phoresis based methods. By comparison, ion-pair,

reversed-phase LC of these samples using Waters

ACQUITY BEH300 C18, 1.7 µm technology offers

an alternative that is particularly well suited for

analytical and small scale purification applications.

MassP REP 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 ACQUITY UltraPerfor-mance LC® (UPLC) 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.

1 20 min

LC System: Waters ACQUITY UPLCColumn: ACQUITY UPLC PST BEH300 C18 , 2.1 x 50 mm, 1.7µmColumn Temp: 50 °CFlow Rate: 0.2 mL/minMobile Phase A: 0.1M TEAA, pH 7.0Mobile Phase B: 20% ACN in AGradient: 57.5 – 84.5 % B in 20.0 minDetection: PDA, 260 nm

OLIGONUCLEOTIDE SEPARATION TECHNOLOGY

Separation of MassPREP OST Standard on ACQUITY UPLC OST C18, 1.7 μm Column

0 10 min

15 nt

20 nt25 nt

30 nt35 nt

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. T he 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

■ Contains a carefully defined mixture of synthesized oligo- deoxythymidine 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

[ ]10

OLIGONUCLEOTIDE SEPARATION TECHNOLOGY

Description Particle Size Pore Size Dimension Part Number

ACQUITY UPLC OST C18* 1.7 μm 135Å 2.1 x 50 mm 186003949

ACQUITY UPLC OST C18* 1.7 μm 135Å 2.1 x 100 mm 186003950

Custom ACQUITY UPLC OST C18* — — — 186003951

XBridge OST C18 2.5 μm 135Å 2.1 x 50 mm 186003952

XBridge OST C18 2.5 μm 135Å 4.6 x 50 mm 186003953

XBridge OST C18 2.5 μm 135Å 10 x 50 mm 186003954

Custom XBridge OST C18 — — — 186003955

ACQUITY UPLC BEH300 C18 1.7 µm — 2.1 x 50 mm 186003685

Description Quantity Part Number

MassPREP OST Standard 1/pk 186004135

OLIGONUCLEOT IDE DESALT ING 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 INFORMAT ION

* For use on Waters ACQUITY UPLC Systems

ACQUITY UPLC System with XEVO TQ Mass Spectrometer

More information can be found at www.waters.com/biosep

[ ACQUITY UPLC SYSTEm ACCESSORIES ]

[ ]10

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■ Sample concentrating

■ High throughput