2

Solid-phase Synthesis and Probe Labeling Processes

Purification and Diafiltration Processes 

for Nucleic Acid Diagnostic Testing (NAT) Products

Vũ Mạnh HuỳnhTiến Sĩ Hóa Học

Solid-phase Synthesis & Labeling Processes for Nucleic Acid Diagnostic Testings (NAT) 

Phophoramidite chemistry for oligo synthesis:1. Potential for automated synthesis.2. High coupling efficiency: 99.8 – 99.9%3. Ease of site-specific:

– Non-nucleosidal base.– Linker incorporation.

4. Ready scalability: – AKTA OligoPilot 10/100: 1 - 50µmole (1 gram of

20 mer), 50µmole - 9 mmole (15 grams of 20 mer).– OligoPilot 400: 4 - 30mmole Scale (150 grams of

20 mer).– OligoProcess: 50mmole-1mole (5 kilograms of 20

mer).

DNA/RNA Synthesizers from GE Healthcare Biosciences

Nucleoside Phosphoramidites for DNA Synthesis

Non-Nucleosidal Phosphoramidites using for Automated DNA Synthesis

HN

NH

O

O

O

OP O

O

NOMe

CN

H3CO

OOO

OMe

H3CO OP ON

CN

OOR O R

O O

O

O

HN

O

O

P

N

OCN

ON N

O

OO

NO

PN

O

CN

O OHN

O

HN CPG

O

OO

NN N(CH3)2

OMe

H3CO

Linker Phosphoramidite

Spacer 9 Linker Phosphoramidite

6-TAMRA Phosphoramidite

Dabcyl CPG Fluorescein Phosphoramidite (6-FAM)

Automated Solid-Phased Synthesis

1. Oligonucleotides can be rapidly assembled by repeatedly addition of monomers using solid-phase methods provided coupling efficiencies are consistently high.

2. Eliminate isolation & purification after each cycle3. Syntheses are continuing with four synthesis steps:

• Step 1: Detritylation• Step 2: Base Condensation• Step 3: Oxidation• Step 4: Capping

Post Synthesis: Cleavage/Deprotection1. Standard Condition: Conc. Ammonium Hydroxide, at 56 -

60° C, 16 hours.

2. Mild Condition: t-Butylamine : Me-OH : H2O; 1:1:2, at 56 - 60° C, 16 hours.

Automated DNA/RNA Synthesis Using for Manufacturing

Step 2: Base Condensation

Step 3: Oxidation Reaction

In the coupling step, the base was added to the previous base:

1. Form unstable phosphite triester linkage.

2. Iodine in water is used as oxidation solution.

3. Unstable phosphite triester linkage is oxidized to form a more stable phosphate triester linkage.

4. Cyanoethyl protecting group is still on the linkage.

The oxidation process of the unstable phosphite linkage to a more stable phosphate linkage.

Step 4: CAP Reaction

After oxidation steps, 5’-hydroxyl groups of the previous base that do not react with tetrazolides are capped with Acetic Anhydride. If this group is left uncapped: coupling failure N-minus pieces to form a complex mixture of oligonucleotides.

Complete Cycle in Automated DNA Synthesis

Post Synthesis: Cleavage/Deprotection

After all bases have been added the oligonucleotide• It is cleaved from the solid support • It is deprotected before it can be purified by HPLC.

Analytical HPLC of Crude DNA/RNA Oligo 51mer

Minutes

AU

Oligo synthesized on the AKTA

On PAC-dA-PS, 43ml/min, 46%

On TAC-dA-CPG, 100ml/min, 59%

Vu, H. “Improved Large Scale Synthesis of Oligonucleotide-Containing Blocks of Poly dA, 2’-Deoxyadenosines.” Presented at poster session at TIDES 2004, Oligonucleotide and Peptide Technology Conferences, April 25-29, 2004, Las Vegas, NV.

Manufacturing of Oligonucleotide containing linker

After deprotection, crude oligo is collected by filtration to remove solid support.

1. Desired oligo

2. N-Plus pieces

3. N-Minus pieces

4. Protecting groups

The crude is purified by RP-HPLC or IEX-HPLC.

The pure oligo is subjected to the next step.

Molecular Beacon Torch Manufacturing

1. Labeling process uses modified cycle.

2. Use fresh dye-reagent.

3. 4 minutes coupling.

4. No CAP Step.

5. Deprotect under mild condition.

6. Purify by IEX-HPLC.

7. Unstable in NaOH.

8. Stable in Sodium Chloride.

9. Process can be scaled up to MFG full-scale.

Mechanism of Molecular Beacon

Reporter Dye Quencher

Stem Structure

Molecular Beacon

Target Nucleic Acid

Molecular BeaconProbeNon-fluorescent

Probe-Target HybridFluorescent

+

Mechanism of action of molecular beacons in the presence of a complementary target nucleic acid.

Dyes using in probe synthesis

Dyes Ex (nm) Em (nm) Company 1. FAM 494 518 Molecular Probes*2. Oregon Green 488 496 524 Molecular Probes*3. Oregon Green 514 511 530 Molecular Probes*4. Rhodamine Green 502 527 Molecular Probes*5. 6-JOE 520 548 Molecular Probes*6. Carboxyrhodamine 6G 525 555 Molecular Probes*7. 6-TET 521 536 Molecular Probes*8. HEX 535 556 Molecular Probes*9. TAMRA 555 580 Molecular Probes*10. Lissamine Rhodamine B 570 590 Molecular Probes*11. Rhodamine Red-X 580 590 Molecular Probes*12. ROX 580 605 Molecular Probes*13. Texas Red 595 615 Molecular Probes*14. Naphthofluorescein 602 672 Molecular Probes*15. BODIPY Dyes var var Molecular Probes*16. Alexa Fluor Dyes var var Molecular Probes17. CyDyes var var Amersham

Biosciences18. Oyster Dyes var var Denovo Biolabels19. IRDyes var var LI-COR20. Epoch Pharm dyes var var Epoch

Pharmaceuticals*Dye available from this company; not necessarily the patent owner

Design & Synthesis of Quencher

1. Cyclic structure, that has molecular model on a plane, such as Benzene ring. This factor will allow quencher to be in a close proximity to the dye.

2. At least one six-member ring.3. The wave-length, which is closed to that of the dye.4. Diazo linkages, quinones, carbonyl group.., those type of

compounds would emit higher wave-length.5. Pyrene (Pyrene-CPG support is commercially available for other

purpose in oligonucleotide chemistry). λEx 345 ; λEm 3766. Acridine (Acridine-CPG support is commercially available as

intercalating agent in oligonucleotide chemistry). λEx 480 to 4907. Napthalene8. 1,8-Naphalic anhydride, 9. Anthracene10. Xanthene11. Fluorene12. Gallocyanine, λEx 610 nm13. Coumarine, λEx 450 ; λEm 505 nm

OHO

H2N OHOH

OCH3

OCH3

OHN O

OH2

1 4290-87 4290-884290-89

OCH3

OCH3

O NH

O

OHO

O

O

HN OHO

OH

OCH3

OCH3

O NH

O

ONH O

O

4290-90

CPG

i ii iii

iv

v H2N OHO

6

HNO

4290-93

OCH3

OCH3NH

O

ONHO

OCPG

4290-108

i) EDAC, HOBT, DMF anh. ii) DCM anh., DIEA, DMT.Cl iii) Succinic anh., Pyr. anh., DMAP iv) TBTU, HOBT, N-Ethyl morpholine v) DCC, HOBT, DMF anh.

H. Vu et al., Bioconjugate Chem. 1995, 6, 599-607.R. Pon et al., Bioconjugate Chem. 1999, 10, 1051-1057.M. Komiyama et al., Bioconjugate Chem. 2002, 13, 365-369.

HNO

iv

4290-94ii, iii, iv

OH

O

NH

OHOH

O HNO

O

Synthesis of anthracene-CPGs containing short and long linkers

Reverse-Phased and Ion Exchanged HPLC Purification Processes Using for MFG.

On-Column Detritylation 1. Crude oligos with DMT group, is firstly separated

from Non-DMT.

2. N-minus pieces, eluted from the column.

3. Oligo with DMT is detritylated by trifluoro acetic acid (TFA) in the column(OCD).

4. the “DMT-OFF” mixture, is then purified by the same HPLC gradient system.

Reversed-Phase HPLC Purification Processes

On-Column Detritylation 1. Crude oligos with hydrophobic DMT group, is firstly

separated from Non-DMT.

2. N-minus pieces, eluted from the column, separate from “DMT-ON” Oligos

3. Oligos with DMT is detritylated by trifluoro acetic acid (TFA, 3–10%) in the column(OCD, 3-10 min.)

4. “DMT-OFF” mixture, is then purified by the same HPLC gradient system.

5. The oligo product with purity >90%, is collected by fraction collector.

Reversed-Phase Purification Method

On Column Detritylation (OCD)On Column Detritylation (OCD)

DMT+Product DMT-off failures

AcidDMT

• Load and wash: Retain DMT-on oligos and wash off DMT-off failures

Detritylation: Remove DMT group from oligo by an acid pulse

DMT-off product

Elution and Recovery : Remove acid and elute product oligo

AcidDMT

Reversed-Phase HPLC Purification Processfor 1 – 10 grams Scale

On-Column Detritylation Process1. Biotage HPLC MFG System.2. Amberchrome CG300 support3. Column 7.5cmx15cm. 4. Radial compression with ethanol operating

pressures 120psi. 5. Trifluoroacetic acid (TFA). 6. Triethylamine acetate (TEAA), Acetonitrile.7. Flow-rate: 264mL/min.8. Detection: 260nm and 298nm. 9. Recovery: 45%. Purity >90%

Ion Exchange Purification Method

M i x e d M o d e o n C o l u m n D e t r i t y l a t i o nM i x e d M o d e o n C o l u m n D e t r i t y l a t i o n

M i n u t e s

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0

mA

U

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

%

0

2 0

4 0

6 0

8 0

1 0 0D e t 1 6 8 - 5 - 2 6 0 n mD N A 1 9 6 , 4 2 1 3 1 1 2 a

P u m p G r a d i e n t CD N A 1 9 6 , 4 2 1 3 1 1 2 a

L o a d ( H y d r o p h o b i c m o d e ) : R e t a i n D M T - o n ( T r + ) o l i g o s

W a s h w / s a l t ( I o n e x c h a n g e m o d e ) : E l u t e D M T - o ff ( T r - ) f a i l u r e sD M T + P r o d u c t D M T - o f f f a i l u r e s

D M T - o f f f a i l u r e sD M T + P r o d u c t

D e t r i t y l a t i o n : R e m o v e D M T g r o u p f r o m o l i g o s b y a n a c i d p u l s e ( T F A )

A c i dD M T - o f f D M T E q u i l i b r a t e c o l u m n w / b a s e : R e m o v e a c i d & e q u i l i b r a t e c o l u m n f o r S A X

D M T - o f f p r o d u c t & i m p u r i t i e s A c i d

E l u t i o n w / s a l t ( I o n E x c h a n g e M o d e ) : E l u t e n , n - 1 , n + 1

S a l t N N + 1N + 1N - 1

E q u i l i b r a t e c o l u m n w / b a s e : R e m o v e a c i d & e q u i l i b r a t e c o l u m n f o r S A X E q u i l i b r a t e c o l u m n w / b a s e : R e m o v e a c i d & e q u i l i b r a t e c o l u m n

f o r S A X

Ion Exchange HPLC Purification Processfor 1 – 5 grams Scale

1. Varian SD-1 Prep HPLC System.

2. UV-VIS Detector. pH meter. Conductivity meter

3. Two pumpheads A, B with flowrate: 10–800 ml/min.

4. A: 1 mM NaOH, B: 1.5 M NaCl/10mM NaOH.

5. Methods for short oligos: 30% B 10 min., 40% B 10 min., 40-70% B 40 min.

6. Method for long oligos: 40% B 10 min., 50% B 10 min., 50-75 %B 30 min.

7. Method MMOCD: A: 1 mM NaOH, B: 1.5 M NaCl/10mM NaOH, C: 0.4% TFA.

8. Flowrate: 100 – 200 ml/min.

9. Recovery Yield ~44% (Purity: >90%), ~20% (> 97%).

Minutes

AU

Oligo synthesized on the AKTA

On PAC-dA-PS, 43ml/min, 46%

On TAC-dA-CPG, 100ml/min, 59%

Analytical HPLC of Crude DNA/RNAOligo 51mer

Large Scale HPLC Purification Process on SOURCE 30Q, MMOCD Method, Purity: >90%

-0.020

0.000

0.020

0.040

0.060

0.080

0.100

0.120

0.140

0.160

0.180

12.0 13.8 15.0 16.3 17.5 18.8 20.0 21.3 22.5 23.8 25.0 26.3 27.5 28.8 30.0 32.0

AU

min

1 - 06252001 #4 na0174 mmbiotF1 UV_VIS_12 - 06252001 #6 na0174 mmbiotF2 UV_VIS_13 - 06252001 #8 na0174 mmbiotF3 UV_VIS_14 - 06252001 #10 na0174 mmbiotF4 UV_VIS_15 - 06252001 #12 na0174 mmbiotF5 UV_VIS_16 - 06252001 #14 [modified by Administrator] na0174 mmbiotF6 UV_VIS_17 - 06252001 #16 [modified by Administrator] na0174 mmbiotF7 UV_VIS_1

7

6

5

4

3

2

1

No. Collection(AU)

Conc.(OD/ml)

V (ml) OD OligoRecovery

(%)

Purity(%)

Full-lengthOD

Full-lengthRecovery

(%)1 0.05-0.18 0.7 220 154 2.88 14.30 22.02 0.742 0.18-0.28 1.62 110 178.2 3.33 46.10 82.15 2.763 0.28-0.56 2.7 110 297 5.55 65.20 193.64 6.51

4 0.56-1.22 6.8 110 748 13.98 90.10 673.95 22.675 1.22-0.89 7.88 110 866.8 16.20 91.50 793.12 26.68

6 0.89-0.24 3.26 110 358.6 6.70 79.40 284.73 9.587 0.24-0.12 1 110 110 2.06 36.10 39.71 1.34

Total 0.05-0.12 880 2712.6 50.68 77.02 2089.32 70.29

No. 4~5 0.56-0.89 220 1614.8 30.17 90.85 1467.07 49.35No. 3-6 0.28-0.24 440 2270.4 42.42 85.69 1945.44 65.45

Large Scale HPLC Purification Process on SOURCE 30Q, MMOCD Method, Purity: >96%

-0.010

0.020

0.040

0.060

0.080

0.100

0.120

0.140

0.160

0.180

0.200

0.220

0.240

12.0 13.8 15.0 16.3 17.5 18.8 20.0 21.3 22.5 23.8 25.0 26.3 27.5 28.8 30.0 32.0

AU

min

1 - 06282001 #2 [modified by Administrator] na0174 r3 F1 UV_VIS_12 - 06282001 #4 [modified by Administrator] na0174 R3 F2 UV_VIS_13 - 06282001 #6 [modified by Administrator] na0174 R3 F3 UV_VIS_14 - 06282001 #8 [modified by Administrator] na0174 r3 f4 UV_VIS_15 - 06282001 #10 [modified by Administrator] na0174 r3 f5 UV_VIS_1

5

4

3

2

1

No. Collection(AU)

Conc.(OD/ml)

V (ml) OD OligoRecovery

(%)

Purity(%)

Full-lengthOD

Full-lengthRecovery

(%)1 0.1-0.38 1.76 110 193.6 7.23 95.00 183.92 12.372 0.38-0.377 2.7 110 297 11.10 97.33 289.07 19.453 0.377-0.27 2.06 110 226.6 8.47 96.24 218.08 14.674 0.27-0.148 1.26 110 138.6 5.18 76.45 105.96 7.135 0.148-? 0.48 110 52.8 1.97 72.00 38.02 2.56

Total 0.1 ~ <0.15 550 908.6 33.95 91.90 835.05 56.18

No. 1-3 0.1-0.27 330 717.2 26.80 96.36 691.07 46.50

No. 1-4 0.1-0.15 440 855.8 31.98 93.13 797.03 53.63

Small Scale Purification Process on Reverse Phase Media (~ 40 mg)

• Column: Amberchrome Profile XT20; 20µ; 10mm x 200 mm

• Flow rate: ~5.0 ml/min• Mobile Phase: A: 3.0% DCA; B: Acetonitrile; C: 0.1M

TEAA.• Gradient: A 100% 15 –18 min; 0% rest of the run• B 0–15 min 15%; 15–18 min 0%; 18–25 min 5%; 25–35

min 10%; 30–45 min 12.5%; 45-55 min 85%; 55–65 min 15%

• Monitoring nms: 260 & 298 nm

Small Scale Purification Process on Ion Exchange Media (~ 10 mg)

• Column: PL-SAX Semi-prep 1000 A; 8 µ; 150 x 7.5 mm• Load: ~ 10 mg• Flow rate: 3.5 ml/min• Mobile Phase: A: 0.1 M NaOH; B: 0.5 M NaClO4; C:

Purified Water• Gradient: A 10% throughout run• B 0 – 10 min 27.5%; 10 – 20 min 32.5 %; 20 - 30 min

32.5 – 45%; 30 – 35 min 45%; 35 – 45 min 90%• Monitoring nms: 260 and 495 nm

Diafiltration/Desalting Process

1. Ethanol Precipitation using Sodium Acetate.2. Ultra-filtration: Membrane with MW Cut-Off

• A polymer coated membrane• Sodium salt passes through Membrane• Scale: ~ 0.5 liter per membrane• Problems: Pinholes, Defects

3. Ultra-filtration: Hollow Fiber Cartridges• Similar to Straws• Sodium salt passes through• Avoid problem of membrane• Scale-up to 10 liters• Faster & cheaper• Concentrate to 40 ml

DNA Aplification, PCR• Phương pháp sinh sản nhanh chóng và chính xác những đoạn

DNA đặc thù cần thiết cho việc khảo cứu về gia phả liên hệ gia đình, điều tra phạm tội.

In Vitro DNA, RNA Amplification (Phương Pháp PCR, TMA)

• Phương pháp amplification được dùng để xác nhận sự tồn tại của virus, tế bào vi sinh vật, động vật, và thực vật

• Phương pháp này có thể sản xuất một triệu - một tỷ copies cua di truyền

• Amplification đã được đánh giá cao trong khoa học hiện đại, vì phản ứng giữa enzyme và DNA/RNA có thể sinh sản nhanh chóng những DNA rất đặc thù , và được xác nhận dễ dàng bởi hệ thống máy vi tinh.

• Một công trình trong vòng 30 phút đến 1 tiếng sẽ cung cấp đầy đủ kết quả chính xác để đi đến kết luận

• Vì đặc điểm trên, Amplification đã trở thành một ngành quan trọng cho : Clinical Medicine, Genetic Desease Diagnostic, Forensic Science, Evolutionary biology

Tinh chế DNA, PCR Amplification1. Thu thập tế bào: Các tế bào chứa DNA được lấy bằng sự cọ

xát nhẹ nhàng những que thử, ở trong miệng, trên gần má.

2. Tinh chế tế bào: Tế bào dính trên que thử được ngâm vào trong dung dịch buffer, dùng máy ly tâm để làm cô đọng các tế bào.

3. Tinh chế DNA: Tế bào được hâm nóng để phá màng tế bào, và DNA được ra khỏi tế bào, và hòa tan vao dung dịch 

4. PCR Amplification: Khi ở nhiệt độ cao, DNA sẽ dãn  ra, và các primers, mang tín hiệu marker, sẽ bám vào đầu của chuỗi DNA, anneal, nhờ điều kiện Thermal Cycler thay đổi, và nhờ enzyme TAQ Polymerase, các vùng quan trọng trên DNA được sinh sản, và trong 1 giờ có thể sinh ra 1 triệu copies.

Phản ứng của công trình Amplification

• PCR Amplification là công trình dùng polymerase enzyme đặc biệt để sinh sản chuỗi complementary từ chuỗi đối tượng DNA, RNA.

• Trong dung dịch thử DNA, RNA có a) 4 dNTP, b) 2 primers và c) target sequence.

• Phản ứng của công trình có 3 giai đoạn

1. Tăng nhiệt độ lên 95° C, để tách rời hai chuỗi của target double helix DNA. 20 giây.

2. Hạ nhiệt độ xuống 55° C, để primers tìm và bám vào vùng thích hợp của chuỗi đã được tách ra. 20 giây.

3. Polymerase enzyme sẽ sinh sản từ primers ra chuỗi complementary mới ở 72° C. 30 giây.

• Vòng công trình sản xuất này sẽ diễn ra trong vòng 1 tiếng sẽ sinh sản 1 triệu copies.

Real-Time Transcription-Mediated Amplification

(RT-TMA) Detection System

Vũ Mạnh HuỳnhTiến Sĩ Hóa Học

Transcription-Mediated Amplification

• TMA is an RNA transcription amplification system. • Utilizes two enzymes to drive the reaction:

1. T7 RNA polymerase

2. Reverse transcriptase • Amplifies RNA to RNA via DNA intermediates• Isothermal reaction, the entire reaction is

performed at the same temperature in a water bath or heat block (no thermal-cycler required)

• Has very rapid kinetics resulting in a billion fold amplification within 15-30 minutes.

Target Capture

• Purified rRNA by automated Target Capture on King Fisher.

• The lysate is transferred to a tube with :1. Magnetic beads-T142. Capture Oligo3. Buffer

• Heat• Cell debris, proteins,

non-specific DNA, RNA are washed

• The purified rRNA on the beads is resuspended in Amplification Reagent.

The Mechanism of Transcription-Mediated Amplification (TMA) Reaction

1. The 1st T7 anneals to the target and is extended by reverse transcriptase (RT).

2. The RNA strand is degraded by RNase H, and a 2nd nonT7 anneals to the DNA strand and is extended by RT, yielding a double-stranded DNA template with an active T7 promoter region.

3. T7 RNA polymerase binds to this promoter and transcribes the template strand, yielding 100 to 1000 RNA copies.

4. The nonT7 anneals to each of these RNA copies, is extended by RT, and the RNA strand is degraded by RNAse H.

5. The promoter-primer then binds to the DNA strand, and extension with RT produces the dsDNA template with an active T7 promoter.

6. More RNA copies are made, and this cycle continues in an autocatalytic fashion.

Transcription-Mediated Amplification (TMA)

RNAse HActivity

RNAse HActivity

RT2

Promoter-Primer

RNA Target1

3 DNARNA

4

5

6dsDNA Template

7 RNA Pol12

Promoter-Primer

RT

11

109Primer 2RT

8100-1000 copies RNA Amplicon

Primer 2RT

Assay Conditions for RT-TMA Test

• Assay Model System : Legionella pneumophila on the KingFisher96 with TMA' on Chromo4.

• Target Capture Volume : 150 µl • Sample Volume : 600 µl [300 µl Milli-Q water sample +

300 µl UTM] • Capture oligo conc. : 20 pmoles/rxn • Magnetic Bead conc. : 20 µg/rxn • TMA' Volume : 100 µl • T7 primer : 16 pmoles/rxn • Blocker oligo : 8.5 pmoles/rxn • non-T7 primer : 32 pmoles/rxn • Molecular Beacon Torch (MBT) : 10 pmoles/75 µl/rxn

Real-Time Transcription-Mediated Amplification (RT-TMA) Detection System

for Pseudomonas aeruginosa

• P. aeruginosa is common organism found in purified water system, or water for injection.

• Biofilms from P. aeruginosa can buil up in closed plumbing systems, and equipment.

• Contaminations can happen in processes, raw materials, or final product formulations.

• The endotoxins of P. aeruginosa can cause serious disease.

• It can result in significant quality issues, costly decontamination processes.

Target Capture

• Purified rRNA by automated Target Capture on King Fisher.

• The lysate is transferred to a tube with :

1. Magnetic beads-T14

2. Capture Oligo

3. Buffer• Heat• Cell debris, proteins, non-

specific DNA, RNA are washed• The purified rRNA on the

beads is resuspended in Amplification Reagent.

Amplification & Detection

• Amplification is transferred to a microplate.

• Place in PTI FluoDia T70 microplate reader.

• Target rRNA is amplified and detected in real-time by hybridization of Molecular Torch.

• Molecular Torch are non-fluorescent, unless bound to amplified target.

• Fluorescent signal is generated• Signal is measured and recorded

for the duration of the reaction.

Real-Time TMA