Synthesis Training July, 2011 Azco Biotech, Inc. Synthesis Training July, 2011

Synthesis Training Conclusion Applications Introduction to DNA/RNA Synthesis Review of Synthesis Chemistry Instrument Review Instruments Reagents FAQs Conclusion

Applications: Oligo Synthesis

Uses for Oligonucleotides Great! Microarray Diagnostics DNA Forensics Predisposition to Disease Detection of Infectious Agents Antisense Research Therapeutics Viral Infections Immune Disorders Cancer Gene Therapy Cystic Fibrosis Diabetes Cancer Main Applications Primers & Probes for PCR/RT-PCR Synthetic Genes - 2nd Gen Sequencing WWW.AZCOBIOTECH.COM

Oligonucleotide Synthesis Chemistry

Nucleic Acid Synthesis Developed by Marvin Carruthers, U. Colorado in 1982 WWW.AZCOBIOTECH.COM

Oligonucleotide Synthesis Basic Synthetic Cycle Steps (NOTE: Very little has changed since original Carruthers paper in 1982) The Detritylation Step The Coupling (Activation) Step The Capping Step The Oxidation Step The Deprotection and Cleavage Step WWW.AZCOBIOTECH.COM

The Detritylation Step 5’ protecting group is removed with TCA or DCA in DCM (3% w/v). There is now a hydroxyl at the 5’ carbon, which will react with the next base to be added. WWW.AZCOBIOTECH.COM

The Coupling (Activation) Step To react with the free hydroxyl of the first base, phosphoramidites are reacted with a weak acid,generally 1-H-tetrazole, ETT or DCI. The acidic conditions protonate the dialkylamino group and then tetrazole attacks as a nucleophile generating the reactive tetrazolophosphane intermediate. The result is a phosphite triester bond formed between the first and second bases. WWW.AZCOBIOTECH.COM

The Coupling (Activation) Step Water (H-O-H) Kills Coupling! B a s e D M T O 2 O O P N N C C H C H O 2 2 B a s e D M T O 2 O A C T I V A T I O N . 2 5 M E T O P B a s e N C C H C H O B a s e H O 1 2 2 O 1 O O O C P G O C P G WWW.AZCOBIOTECH.COM

The Capping Step The coupling step is not 100% efficient. Need to stop the elongation of sequences that are missing bases, e.g. failure sequences. This is done by capping the free hydroxyls through acetylation, making these sequences terminally unreactive. Capping is in two steps: acetic anhydride and lutidine to form the cap and N-methylimidazole to form the reactive intermediate. WWW.AZCOBIOTECH.COM

The Capping Step WWW.AZCOBIOTECH.COM

The Oxidation Step After the capping step is done, the unstable phosphite triester is oxidized to the stable phosphate triester. Iodine acts as the oxidant with water providing a hydroxyl. This step must occur after capping because acetic anhydride will react with trace amounts of water to form acetic acid. WWW.AZCOBIOTECH.COM

The Oxidation Step WWW.AZCOBIOTECH.COM

Post Synthesis

Deprotection and Cleavage Column Oligo The N-benzoyl and isobutyryl protecting groups are removed completely by treatment with 9N ammonia in 8 hours at 55° C or 90 minutes at 70° C. Fast Deprotect can occur in 15 minutes at 50° C temperature… Simultaneously, the first base is cleaved from the solid support. WWW.AZCOBIOTECH.COM

Deprotection & Cleavage Chip Synthesis Deprotection and Cleave Deprotection– same as normal, aminolysis with either ammonium hydroxide or AMA, regulated by amidites for Oligo Pools. EDA/EtOH for Microarray For Oligo Pools Cleave from Chip – If the oligos are for pooled applications they are cleaved during aminolysis. The chip contains a cleavable linker (same as with column based synthesis). Here are the key features. Note with this system you can 1) make long oligos 2) make a lot of oligos 3) using standard phosphoramidtie chemistry. The only difference is it uses electrochemistry for deblocking the elongating oligo. It comes standard with 6 amidies and 7 liquid reagent ports, but as with most “high end” synthesizers, this can be customized to meet your needs. You can also synthesize either DNA or RNA…your choice! WWW.AZCOBIOTECH.COM

Instruments

Instruments The instrument just automates the above steps The instrument range is based on throughput and quantity Select the instrument that meets your throughput requirements WWW.AZCOBIOTECH.COM

Azco Synthesizers New Synthesizers Refurbished Synthesizers Oligo 800 OM LS2 OligoArray Refurbished Synthesizers ABI 391-4 ABI Expedites BLP 96/192 Dr. Oligo 96/192 Polyplex

Azco Synthesizer: Oligo-800 Automated 8 column synthesizer 8 amidite/10 reagent ports Highly accurate reagent delivery via syringe pump Wide range of synthesis scales Ideal for long oligos or modified oligos including FRET probes Very Economically Priced!

Oligo-800 Plumbing Diagram

Azco Synthesizer: Oligo 96/192 96, 192 column/well format Up to 36 Amidite Ports Low volume “specials” upgradeable 10 Reagent Ports Fully controlled via Oligo v 4.0 Workstation Full process monitoring with audit tracking and error reporting Trityl monitoring capability Ideal for high throughput applications

OligoArray In-situ Synthesizer This is the system. It is an 8 chip system that uses syringes to push reagents across a semiconductor chip for in-situ oligo synthesis uf 8 x 12,000 or 8 x 90,000 oligos…. WWW.AZCOBIOTECH.COM

OligoArray Plumbing Diagram

Reagents

The key to quality synthesis is the reagents you select Synthesis Reagents Phosphoramidites Liquid Reagents Solid Supports and Columns or Chips The key to quality synthesis is the reagents you select WWW.AZCOBIOTECH.COM

DNA Phosphoramidites Standard Phosphoramidites dA, dG, dC and T Fast Deprotect Phosphoramidites Acetyl - dC dmf- dG High Quality WWW.AZCOBIOTECH.COM

T-CE Phosphoramidite Adds a “T” to an oligo Catalog Numbers: 20-8100-xx WWW.AZCOBIOTECH.COM

dG-CE Phosphoramidite Adds a “G” to an oligo Catalog Numbers: 20-8110-xx WWW.AZCOBIOTECH.COM

dA-CE Phosphoramidite Adds an “A” to an oligo Catalog Numbers: 20-8120-xx WWW.AZCOBIOTECH.COM

dC-CE Phosphoramidite Adds a “C” to Catalog Numbers: 20-8130-xx WWW.AZCOBIOTECH.COM

Acetyl-dC-CE Phosphoramidite Fast Deprotecting “C” Catalog Numbers: 20-8230-xx WWW.AZCOBIOTECH.COM

dmf-dG-CE Phosphoramidite Fast Deprotecting “G” Catalog Numbers: 20-8210-xx WWW.AZCOBIOTECH.COM

RNA Phosphoramidites Fast Deprotect RNA Phosphoramidites 2’ TBDMS (n Acetyl) – rA, rG, rC, rU Requires 2nd deprotection to remove TBDMS RNAse resistant Phosphoramidites 2’ Fluoro (nAcetyl) rC, rU, rA and rG 2’ OMe (nAcetyl) rC, rU, rA, rG ONLY USE MILD DEPROTED RNA AMIDITES!

Silyl RNA Amidites Popular for small RNA pH stability Ease of use Catalog Numbers: 28-8800-xx rU WWW.AZCOBIOTECH.COM

rU-CE Phosphoramidite Adds a “U” to an oligo Catalog Numbers: 20-8800-xx WWW.AZCOBIOTECH.COM

(Acetyl) rG-CE Phosphoramidite Fast deprotect rG Adds a “rG” to an oligo Catalog Numbers: 20-8810-xx Ac O-TBDMS

rA-CE Phosphoramidite Adds an “rA” to an oligo Catalog Numbers: 20-8820-xx O-TBDMS WWW.AZCOBIOTECH.COM

(Acetyl) rC-CE Phosphoramidite Fast Deprotecting “C” Catalog Numbers: 20-8810-xx O-TBDMS WWW.AZCOBIOTECH.COM

2’ OMe rU-CE Phosphoramidite RNAse Resistant Product Numbers: 20-5200-xx WWW.AZCOBIOTECH.COM

2’ OMe (N-Ac) rG-CEPA 2’ OMe rG Fast Deprotect RNAse Resistant Product Numbers: 20-5310-xx WWW.AZCOBIOTECH.COM

2’ OMe (N-Bz) rA-CEPA 2’ OMe rA RNAse Resistant Product Numbers: 20-5220-xx WWW.AZCOBIOTECH.COM

2’ OMe (N-Ac) rC-CE PA 2’ OMe rC Fast Deprotect RNAse Resistant Product Numbers: 20-5330-xx WWW.AZCOBIOTECH.COM

2’ F rU-CE Phosphoramidite RNAse Resistant Product Numbers: 20-9000-xx WWW.AZCOBIOTECH.COM

2’ F (N-Ac) rC-CE PA 2’ F (N-Ac) rC RNAse Resistant Product Numbers: 20-9030-xx WWW.AZCOBIOTECH.COM

Specialty Phosphoramidites Fluorescent Phosphoramidites TF and TQ dyes and quenchers Biotin Linkers and Spacers Modified and non-natural Bases WWW.AZCOBIOTECH.COM

Specialty Phosphoramidites Phosphorylation Reagent Amino Modifiers Thiolation Reagent Cholesterol Custom Phosphoramidites

Dyes and Quenchers We offer a full line of dyes and quenchers FAM, TET, HEX, Cy3, Cy5, TF Phosphoramidites (TF1, 2, 3, 4, 5, 6) TQ Quenchers (wavelength matched to TF) JOE, ROX, All other dyes as free dye and NHS esters Used in Dual Labeled Probes TeHP Probes TaqMan (FQ) Probes Molecular Beacons Other

Deprotection Depends on Amidite Amidtite Deprotect Soln Time DNA All Standard Ammonium Hydroxide 10 hours at 60C 14 hours at 50 C DNA All Fast AMA (1:1 MeNH4/NH4OH) 15 minutes at 55C DNA w/ dmf-dG 4 hours at 55C RNA (only use fast deprotect) AMA 15 minutes at 50C Std for Microarray EDA/EtOH (1:1) 1 hour at 55C Std for Oligo Pool 7 hours at 65C 14 hours at 55C Fast for Oligo Pool Same as above… WWW.AZCOBIOTECH.COM

Liquid Reagents

Acetonitrile/ Diluent Used to re-suspend amidites and wash. Supply one grade of Acetonitrile: <10ppm. Need to use only 10ppm or less. Low water content ensures quality synthesis, should also use MolSieves. Water will cause precipitation of dG. WWW.AZCOBIOTECH.COM

ETT Activator Turn key solution. Optimal Concentration 0.25M Produced in US & UK Preferred by Chip Co.’s Great for RNA (0.3M) Catalog Numbers: 17-1124-xx 17-1224-xx WWW.AZCOBIOTECH.COM

Other Activators DCI, 5-BMT Slight acidic conditions excellent for DNA and RNA synthesis. WWW.AZCOBIOTECH.COM

Cap A Solution One of Two reagents used to stop the elongation of failure sequences. Components (17-1130-xx): Tetrahydrofuran Acetic Anhydride Lutidine Other formulations: (17-1131-xx) WWW.AZCOBIOTECH.COM

Cap B Solution The second solution used to form the chemical cap. Components (17-1137-xx): Tetrahydrofuran N-Methylimidazole Pyridine Other Formulations: (17-1138-xx) WWW.AZCOBIOTECH.COM

Oxidizer Solution Used oxidize the unstable phosphite triester to the stable phosphate triester. Three formulations: 0.1M & 0.02M & 0.05M Iodine acts as the oxidant with water. Components Tetrahydrofuran Iodine Pyridine Water WWW.AZCOBIOTECH.COM

Deblock Solutions Removal of DMT group. Two formulations TCA in dichloromethane DCA in dichloromethane Many switching to Tolune WWW.AZCOBIOTECH.COM

eChem Deblock Solutions Removal of DMT group. Made prior to use, contains: Benzoquinone Hydroquinone Tetraethylammonium p-ts Lutidine In Methanol/Acetonitrile WWW.AZCOBIOTECH.COM

Miscellanous Reagents 2% Trifluoroacetate (17-1169-xx) 3% Trifluoroacetate (17-1172-xx) 20% Acetic Acid (17-1173-xx) 20% Acetonitrile (17-1174-xx) WWW.AZCOBIOTECH.COM

Solid Supports & Columns

Solid Supports Polystyrene CPG More non-polar, better for DNA Derivitized for almost any base or Specialty Various pore sizes Uses LCAA-group to CPG Long chain amino alkyl WWW.AZCOBIOTECH.COM

Columns We provide various columns for DNA synthesis Various machines Various scales Various “bases” All filled with CPG or PS Should always consider Universal Support WWW.AZCOBIOTECH.COM

CPG Basics Controlled Pore Glass is essentially glass shavings that have a nucleoside based or universal group attached through linker and amine chemistry Typically characterized by two methods Pore Size Nucleoside Loading Occasionally characterized by mesh size. WWW.AZCOBIOTECH.COM

CPG Pore Size Refers to the space between the individual shavings of the CPG particles. Common Pore Sizes 500 Angstroms (Standard Pore Size) 1000 Angstroms (Long Pore Size) 2000 Angstroms (Extra Long Pore Size) WWW.AZCOBIOTECH.COM

CPG Loading Loading refers to the amount of nucleoside base bound to the CPG particles. Commonly expressed as mmols/mg. Using this unit of measure, one can calculate the correct amount of CPG to load within a column or plate. WWW.AZCOBIOTECH.COM

CPG Loading Categories Standard: 500Å, 25-35 mmol/mg High Load: 500Å, 45-90 mmol/mg Extra High Load: 500Å, 100-200 mmol/mg Long : 1000Å, 25-40 mmol/mg Extra Long: 2000Å, 18-30 mmol/mg WWW.AZCOBIOTECH.COM

12K and 90K chips available 12K and 94K chip formats available for making 12,000 or 94,000 oligos simultaneously Can use a “mask” to divide 12K chip into smaller 2K segments Chips are distinguished base on cleave or leave applications. Fig. 4. 12K and 90K CustomArray microarrays after in situ synthesis and hybridization to fluorescently labeled DNA Chips are available in 12K and 90K formats. You can use a “mask” to further subdivide the chip into 1/4ths so 3K for the 12K chip and 23K for the 90K chip. The chips are single use, but with the mask can be reused until all spots have had an oligo made on them. The list price for these chips is $300 for the 12K chip and $550 for the 90K chip. With the 12K chip, the total cost of synthesizing 12,000 oligos is ~$360 for 100mers, or 0.03 cents per coupling!!! WWW.AZCOBIOTECH.COM

F.A.Q.’s The other new product from Azco…the OligoArray synthesizer.

Common Issues (Misconceptions) Coupling is always regulated by reagents and water kills coupling Average coupling efficiency is ~99%, order of efficiency T > A > C > G, so a long run of Gs will result in low quality synthesis, this dictates yield Specialty molecules have much lower coupling efficiencies (~80 to 90%), Purification is application dependent Synthesis is ALWAYS 3’ to 5’, can simply purification by remembering this! WWW.AZCOBIOTECH.COM

Are the SNA reagents for use on all Oligo Synthesizers? Yes, providing we have the follow input from the customer: What synthesizer do you own? What activator solutions do you use? What packaging do you prefer?

What is the shelf life of the SNA Products? Assuming that the products were stored properly: For most phosphoramidites, five years dry. For reconstituted phosphoramidites: DNA and RNA amidites between 8-10 days. For specialty amidites 3-6 days. For the liquid reagents, one year. For the solid supports, five years. It is highly unusual for us or the customer to keep these materials for long periods of time.

What are these crystals in my dG-CE amidite? dG amidite can crystallize under three conditions: High water content of the ACN diluent. High water content of the amidite itself. A long period of reconstitution.

Can I Swap Out the Different Activator Solutions? ETT, DCI, 5-BMT and 1-H-tet solutions generally are interchangeable. ETT and DCI are slightly more acidic than 1-H-tet Better for RNA synthesis. Better for longer oligo synthesis.

My trityl monitor is showing low coupling efficiency. The phosphoramidite lines may be clogged. Crystals in the amidite. Water in the diluent. Long term placement of the synthesizer. The column is clogged. The instrument may have issues.

The bottles you sent don’t fit my machine Different instruments utilize bottles with different neck sizes. This problem comes about when either the customer is unfamiliar with our catalog numbers or we don’t know what instrument they own. Occasionally occurs with new customers.

I am having issues with base deamination in my oligo. They oxidizer solution is too strong. Switch to 0.02M oxidizer. Reduce the cleavage and deprotection times. Reduce the cleavage and deprotection temperatures.

My long oligo synthesis yielded very little material This may actually be appropriate for the sequence. Questions to ask: Are you using a 1.0mmol or greater synthesis scale? Are you using long CPG? Did you try to purify the material?

Azco OligoArray Synthesizer The other new product from Azco…the OligoArray synthesizer.

Market and Competitors

Instruments - Market Size $15 to $20M Annual Market Very established and consistent Not much growth Mostly a replacement market WWW.AZCOBIOTECH.COM

Instruments Low Throughput High Throughput BioAutomation – MM4, MM12 A couple in Germany High Throughput Biolytic – “Dr. Oligo” BioAutomation – MM96, 192, 384 A Couple in Euorpe WWW.AZCOBIOTECH.COM

Instruments Production Scale GE Healthcare – OP10, OP100, OP400 We make custom Instruments WWW.AZCOBIOTECH.COM

Reagents - Market Size $95 to $140M Annual Market 2 companies control about 70% of this market (Thermo Fisher and Sigma) Not much growth – same with instruments Big part of this market dependent on pharmaceutical market – no successes means no growth… WWW.AZCOBIOTECH.COM

Reagents Large Scale DNA/RNA Amidites Laboratory Scale Thermo Fisher SIAL Chemgenes Us Laboratory Scale Glenn Research WWW.AZCOBIOTECH.COM

Reagents Specialty Amidites Other (Dyes, Supports, etc.) Glenn Research Chemgenes Us Other (Dyes, Supports, etc.) WWW.AZCOBIOTECH.COM

NOTE Azco is the worlds ONLY TURN-KEY SUPPLIER OF INSTRUMENTS REAGENTS, CUSTOM REAGENTS, SUPPORT, EVERYTHING NEEDED FOR DNA AND RNA SYNTHESIS!!! WWW.AZCOBIOTECH.COM

Conclusion

Conclusion WWW.AZCOBIOTECH.COM

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