Jack Pollard

08/31/98

Avery’s realizationthat DNA carries the genetic information lead chemists on a forty yearsearch which has culminated in efficient, automated oligonucleotide synthesison solid phase supports. Modern nucleic acid synthesizers utilize phosphitetriester chemistries which employ stable phosphoramidite monomers to buildthe growing polymer. These robust reactions allow both chemists and molecularbiologists to easily generate specific ribo and deoxy-ribo oligonucleotideswith a variety of labels, modified linkages, and non-standard bases attachedthroughout the chain. The synthesis of short (less than 40 nucleotides)probes and primers requires no more special expertise than the abilityto read the synthesizer’s operators manual, and longer oligonucleotides(up to 150 nucleotides) can be synthesized with a little more care.

This section providesstrategies on the maximization of synthetic yield, the generation of sequencescontaining site specific modifications, and the isolation of syntheticoligonucleotides. The protocols outline methods for deprotecting synthesizedoligonucleotides and for monitoring the progress of synthesis via the tritylassay. This unit augments the detailed instructions provided by the manufacturersof oligonucleotide synthesizers. A functional understanding of the synthesischemistries coupled to insights on the mechanical operation of the synthesizerwill allow the user to minimize input time and maximize oligonucleotideoutput.

INTRODUCTION TOCHEMICAL NUCLEIC ACID SYNTHESIS

Many hydroxyl and aminemoieties make nucleic acids very animated molecules with rich chemistriesof their own that can interfere with the phosphite triester reactions usedto couple the nucleotide monomers; therefore, protection strategies arenecessary in chemical synthesis to mask the functional groups on the monomersso that the only significant reaction is the desired 3’ to 5’ sequentialcondensation of monomers to the growing oligonucleotide. (Note that enzymaticpolymerizations occur with the opposite directionality and require no maskingof monomer functionality.) These synthetic protecting groups must be chosenso that they can be removed easily to reveal the natural nucleotides.

The fully protectedmonomers for nucleic acid synthesis are generally called phosphoramidites(Figure 1). In traditional protection schemes thenucleophilic amino functions on the bases are protected with either isobutyryl(N-2 of guanine) or benzoyl (N-6 of adenine and N-4 of cytidine) groups,both of which can be removed at the completion of synthesis by ammoniolysis.However, recent advances have lead to the wide spread use of phenoxyacetyl(PAC) protection of adenosine, dimethylformadine (DmF) protection of gaunosine,and acyl protection of cytosine to yield oligonucleotides which can bedeprotected rapidly under very mild conditions (Reddy, M. et al., 1994).The 5' primary hydroxyl of the ribose sugar is protected with a dimethoxytrityl(DMT) ether moiety which is removed by mild protic acids at the beginningof each coupling cycle. The efficiency of synthesis at each coupling cyclecan be monitored by detecting the release of the chromophore trityl cation.For the synthesis of nucleic acids with the natural phosphodiester backbone,the 3' secondary hydroxyl function of the ribose sugar is derivatized witha highly reactive phosphitylating agent. The phosphate oxygen on this moietyis usually masked by ß-cyanoethoxy and diisopropylamine protectinggroups. By insulating the phosphate oxygen with alternative groups, modifiedphosphate backbones may be accessed. Finally, for ribonucleic acids thesecondary 2’ hydroxyl of the ribose is shielded throughout the chemicalsynthesis by the tert-butyldimethyl silyl group.

figure 1

The genius of theseprotecting groups for automated nucleic acid synthesis is that they yieldnearly lesion free natural nucleic acids with high efficiency through simplehydrolysis, nucleophilic displacement, and redox chemistries. In a standardsynthesis cycle, the nucleotide chain grows from an initial protected nucleosidederiviatized via its terminal 3' hydroxyl to a solid support. Reagentsand solvents are pumped through the support to induce the consecutive removaland addition of sugar protecting groups in order to isolate the reactivityof a specific chemical moiety on the monomer and affect its stepwise additionto the growing oligonucleotide chain. This design eliminates the need topurify synthetic intermediates or unreacted reagents because they are simplyrinsed off the column at the end of each chemical step. Assembly of theprotected oligonucleotide chain is carried out in four chemical steps:deblocking, activation-coupling, oxidation, and capping (Figure2).

(1. DEBLOCKING) Thesynthesis cycle begins with the removal of the acid labile DMT ether fromthe 5’ hydroxyl of the 3’ terminal nucleoside. This is usually accomplishedby using dichloroacetic acid (DCA) in dichloromethane. The resulting tritylcation chromophore can be quantitiated to determine coupling efficiency(see Monitoring DNA Synthesis Using the Trityl Assay). After deblocking,the 5’ hydroxyl is the only reactive nucleophile capable of participatingin the subsequent coupling step. Since the nitrogenous bases of the growingDNA chain are susceptible to acid catalyzed depurination, the deblockingstep is short, and an acetonitrile rinse thoroughly removes the deblockingagent from the support. Also, coupling efficiency and accuracy are increasedby this wash since premature detritylation of the incoming phosphoramiditemonomer is prevented.

(2. ACTIVATION-COUPLING)Following deblocking of the 5’ hydroxyl group, the next protected phosphoramiditeis delivered to the reaction column along with the weakly acidic activatortetrazole (pKa = 4.8). Nucleophilic attack of the previously freed 5’ hydroxylupon the incoming monomer’s diisopropylamine-protected phosphorus, whichwas activated via protonation by tetrazole, elongates the nucleic acidchain. Because this protonated phosphoramidite is so reactive, the couplingreaction is usually complete within 30 seconds. A molar excess of tetrazoleover the phosphoramidite ensures complete activation, and a molar excessof phosphoramidite to free 5' hydroxyls of the growing chain promotes efficientcoupling. To optimize the coupling efficiency, the amounts of reagentsinjected and the coupling time can be varied (see Synthesizing Long Oligonucleotides).

(3. CAPPING) In spiteof these efficiency measures, a small percentage of the support-bound nucleoside’s5’ hydroxyls do not couple to the incoming activated monomer. They mustbe rendered inactive to minimize deletion products and simplify the purificationprocess. Usually, acetic anhydride and N-methyl-imidazole dissolved inpyridine and tetrahydrofuran (THF) act to create an acylating agent that"caps" the unextended 5’ hydroxyls. The 5' acetyl ester cap is unreactivein all subsequent cycles and is removed during the final ammonia deprotectionstep. Additional acetonitrile washing subsequent to capping can increasesynthetic yield. After coupling and capping the internucleotide linkageis a trivalent phosphite triester that is extremely unstable and must beoxidized to a phosphotriester which will ultimately yield natural DNA.

(4. OXIDATION) In thelast step of the cycle, the unstable phosphite triester linkages are oxidizedto a more stable phosphotriester by 0.02 M iodine dissolved in water:pyridine:THF.An iodine-pyridine adduct forms to the phosphite triester which is subsequentlydisplaced by water to yield phosphorus oxidized to the pentavalent state.Pyridine also neutralizes the hydrogen iodide byproduct. Because the oxidizercontains water, the support is rinsed several times with acetonitrile followingthis reaction. One cycle of monomer addition is then complete, and anothercycle begins with the removal of the 5' DMT from the previously added monomer.

At the end of the synthesis,the final trityl can either be removed with a final acid wash ("trityl-off"),or can be left on for purification purposes ("trityl-on"). The oligonucleotideitself is removed from the support with concentrated ammonium hydroxide.Additionally, this treatment deprotects the phosphorus by ß-eliminationof the cyanoethyl group and removes the protecting groups on the heterocyclicbases to yield a single stranded nucleic acid.

figure 2

RNA chemical synthesisis identical to that used for DNA except for the presence of an additionalprotecting group at the 2' hydroxyl of ribose. This position is usuallyprotected with tertbutyldimethyl silyl groups which are stable throughoutthe synthesis (Figure 1). They are removed at thefinal deprotection step by the basic fluoride ion. The remaining positionson both the sugar and the bases are protected in the same fashion as forDNA. By adjusting several parameters in the DNA synthesis protocol includingthe coupling times, monomer delivery rate, frequency of washing steps,and types of capping reagents, stepwise coupling efficiencies of up to99% can be obtained (Glick, G., personal communication) (Wincott, F. etal., 1995). However, for the casual user this yield represents the exceptionrather than the norm, and only shorter oligoribonucleotides (<20 bases)should be initially attempted.

STRATEGIES FOR NUCLEICACID SYNTHESIS

A Checklist forNucleic Acid Synthesis

Consistency and planningare the keys to reliable nucleic acid synthesis. Organizing these repetitivetasks into a standard operating protocol will streamline efficiency andproduce better quality oligonucleotides.

1. Plan syntheses tooptimize machine use. Oligonucleotides of similar size should be combinedin parallel runs since synthesizing many short oligos followed by a longerone is faster than mixing the sets on dual column synthesizers.

2. Determine the totalnumber of bases to be incorporated. Be sure there are enough of the requiredreagents and phosphoramidites available for the entire synthesis. Consultthe synthesizer manual for the amount of reagent needed for each coupling(see also Commentary). Remember that different synthetic scales (0.25-mmolvs. 1-mmol) require different amounts of reagents. Syntheses should beplanned so that a phosphoramidite is almost completely exhausted. Also,phosphoramidites that have been dissolved for more than 2 weeks shouldbe replaced.

3. Consider specialprogramming requirements. Many synthesis options regarding the scale ofthe synthesis, backbone composition, and presence of protecting groupscan be modified. Create a log for users to fill out detailing exact synthesisrequirements, and check the log prior to synthesis. A computerized logbook is especially useful and allows for an organized oligonucleotide nomenclaturesuch as R20.17 which refers to Rebecca's 20-mer, the 17th 20-mer made onthe system.

4. Setup a fractioncollector to monitor the chromophoric trityl cation release if the synthesizerlacks a trityl monitor (see Monitoring DNA Synthesis Using the Trityl Assay).

5. Initialize the system.If previous oligonucleotides were cleaved from their supports automatically,rinse the columns for 30 sec with acetonitrile to remove any remainingtraces of ammonium hydroxide. Immediately before starting the synthesis(and following the addition of fresh reagents), remove any stale reagentsor moisture from the lines by priming them if the machine has been at restfor more than 6 hours. This will maximize the first coupling step's efficiency.Check the reagents/phosphoramidite flow rates to ensure that reagents arebeing properly delivered.

6. Start the synthesis.Confirm that the flow through the lines and columns is not obstructed andwatch the first few trityl releases because an abortive synthesis of a60-mer sequence wastes much more material than a failed 3-mer run.

Synthesizing LongOligonucleotides (>100 Bases)

Modern synthesizerscan routinely produce sequences of 150 or more nucleotides in usable amounts(310 mg). Several strategies can be employed to enhance the yield fromsyntheses longer than 100 bases.

1. Exclude water fromthe system (the importance of this cannot be overestimated; see Commentarysection). Replace reagents on the machine with fresh ones before all longsyntheses. This is particularly important for the phosphoramidites andespecially for guanosine phosphoramidite since it decomposes more quicklythan the other two protected bases (Zon et al., 1985).

2. Monitor trityl releasesfor shorter runs prior to attempting the synthesis of long oligonucleotidesto limit wasting expensive reagents. In general, if the stepwise efficiencyof synthesis is <99%, alter parameters to increase the efficiency onshorter sequences before attempting to synthesize a long oligonucleotide(see Monitoring DNA Synthesis Using the Trityl Assay).

3. Use dichloroaceticacid (DCA) for deblocking as opposed to trichloroacetic acid (TCA) if thesynthesizer is compatible with this reagent. Depurination (cleavage ofthe glycosidic bond) under acidic conditions is a prominent side reactionthat ultimately limits DNA synthesis. DCA tends to show much better syntheticyields than trichloroacetic acid especially for longer oligonucleotides(Pon, R., personal communication). Use a 2 percent (v/v) DCA/ 1,2 dichloroethanemixture.

4. Modify the synthesisprotocol to increase the coupling time of the phosphoramidite. Also, additionalmethylene chloride wash steps included prior and subsequent to deblockingalong with increased acetonitrile washing subsequent to capping leads toincreased yields (Glick, G., personal communication).

5. Increase the phosphoramiditeconcentration to enhance the coupling efficiency--e.g., use a concentrationof 50 mg/ml (double the normal concentration; 20-fold molar excess overthe synthetic polynucleotide chain) for longer sequences.

6. By using a supportmatrix such as control pore glass (CPG) with a loading capacity of lessthan 40 mmol/g, yields of long oligonucleotide may be greatly increased;furthermore, the pore size of the support should be 1000 angstrom for >100-mersand 2000 angstrom for 200-mers (Gait, 1986) to alleviate molecular crowdingand steric effects. For a typical 1-mmol scale synthesis of a 150-mer,20 mg of a support with a loading capacity of 5 mmol/g is used.

Finally, if it is toodifficult to synthesize the desired sequence in a reasonable yield, orif oligomers >150 bases are desired, then the nucleic acids may be madein segments and ligated (Bartel and Szostak, 1993) following PCR with aproof reading polymerase such as Pfu or Tth. Note thoughthat since PCR is an inherently mutagenic procedure, and any products generatedshould be checked by sequencing. Also, very long synthetic oligonucleotides(300-600 bases) have also been synthesized directly, and inspite of incredelylow yields, rare full length products have been successfully amplifiedby PCR (Ciccarelli, R. B., 1991). Finally, Mutually primed synthesis (UNIT8.2) can also be a suitable option for oligomers >150 bases.

Synthesizing RNA

Since many structuraland mechanistic studies are underway concerning catalytic RNAs, the catalogueof commercially available modified RNA monomers has recently bloomed (Table1) RNA chemical synthesis has become as routine as that of DNA andtypically uses identical 5’-dimethoxytrityl-b-cyanoethyl-protected phosphoramiditessave for an additional protecting group on the 2' hydroxyl. Tert-butyldimethylsilylprotection of the 2’-hydroxyl group is the basis of most commercially availableRNA phosphoramidites since the silyl group is stable to both acid and baseand can be removed with fluoride ion. Recently however, RNA monomers with2’-acetyl groups (FPMP, CEE) have appeared and have the advantage of beingconveniently removed at pH 2 just prior to use of that particular sampleof RNA. Strategies for ribo-phosphoramidite protection are an active areaof research, and recent work with 5’ silyl ethers in conjunction with 2’orthoester protection has proven particularly interesting (Scaaringe, S.,personal communication). For the casual user of RNA, it is often easierto just purchase small quantities of the required sequence from a ribo-oligonucleotidesynthesis company such as Baron Consulting, Dharmacon Research, Genosys,or Peninsula Labs.

Isomeric purity ofthe phosphoramidites is often variable because of the difficulties inherentin distinguishing between the vicinal hydroxyls of ribose. If a homogeneouspopulation of 5' to 3' linked oligoribonucleotides is required (as in mostcases it will be), then a thin layer chromatography (TLC) or ³¹Pnuclear magnetic resonance (NMR) analysis of the starting phosphoramiditesshould be performed to establish their isomeric composition. While phosphorusNMR facilities are not generally available to molecular biologists, TLCis both inexpensive and straightforward. Recommended solvent systems toseparate the 2' and 3' ribonucleotide phosphoramidites are 1:1 ether:chloroform,or 40:58:2 or 50:46:4 dichloromethane:hexane:triethylamine (Usman et al.,1987).

With a few slight modifications,the procedures and precautions described for DNA synthesis chemistry applyto RNA as well. Since stepwise coupling efficiency is lower than that ofDNA, even greater care should be taken to exclude water completely fromthe closed system. Because the 2' hydroxyl is often protected with stericallyhindering protecting groups, reaction times for RNA reagents tend to belonger, and adjustments should be made to phosphoramidite concentrationsand coupling times, as detailed below. As is true for all RNA work, equipmentand reagents that will contact unprotected oligonucleotides should be RNasefree (UNITS 3.12 & 4.1) to avoid degradation ofthe synthesized material.

Depending on the synthesizerand coupling program used, RNA phosphoramidites are suspended in dry acetonitrileat a concentration of 0.1 to 1.0 M with a 6 to 10 fold excess of reagentsdelivered per 300 sec. coupling. S-ethyltetrazole has also been found tobe a more effective activator since it is more highly acidic than traditionaltetrazole (Sproat, B. et al, 1995). Also, additional methylene chloridewash steps included prior to and subsequent to deblocking along with increasedacetonitrile washing subsequent to capping leads to increased yields (Glick,G., personal communication).

Incorporation ofModified Nucleosides

Chemical nucleic acidsynthesis allows for the incorporation of unnatural or modified bases aswell as a variety of labelling moieties into an oligonucleotide. This canbe extremely useful for testing models of structural interactions betweenenzymes and nucleic acids, selecting labeled molecules from a populationof unlabeled ones, or gaining insights into the parameters which governnucleic acid structure and chemistry. Modified backbone chemistries suchas phosphorothioates, phosphoroamidates, and phosphotriesters are alsoreadily available. In general, the bases themselves can be obtained commerciallyand are handled like any other phosphoramidite; however, consult the companywhich supplies the analog about necessary modifications to programs orreagents (see Table 1). Typically, the only adjustmentneeded is to dissolve the modified base at a somewhat higher concentrationthan normal to overcome problems associated with reactivity. Most of themethods used to increase the yield of long and ribo-oligonucleotide maybe applied to the synthesis of modified nucleic acids.

When synthesizing modifiedoligonucleotides, compatibilities of the chemistries, placement of modificationsrelative to other chemical groups, and 5’-3’ directionality are all factorsto consider. Generally when an oligonucleotide is end-labeled/modified,a long flexible tether is added to allow greater accessibility. Stretchesof four deoxy-thymines are often used to add this flexible tether. Also,adding deoxy-thymines 5’ to the label (5’-TTTT-label-3’) can aid in separatingby size labelled molecules from unlabelled ones. Also, note that some taggingphosphoramidites allow for the enzymatic extension or kinasing of the modifiedoligonucleotide while others do not. Finally, oligonucleotides may alsobe synthesized directly on solid glass supports (Cohen, G. et al 1997)

Terminal transferasecan be used as an alternative means of incorporating modified bases atthe 3’ end of an oligonucleotide (Ratliff, 1982; UNIT 3.6).This enzyme is tolerant of a variety of substrates, and has been used toadd deoxy-nucleotide triphosphates derivatized at virtually every position(C-8 on adenine, any of the amino groups, C-5 on cytosine, O-6 on guanosine)to DNA. It also functions, though less well, with RNA bases. It can useany DNA oligonucleotide which is at least 2 bases long [d(pXpX)] and containsa free 3' hydroxyl as a primer. A potential problem in preparing homogeneouspolynucleotides using terminal transferase is that a statistically randomnumber of bases is added to the 3' end of the template (with the exceptionof molecules such as cordycepin, which act as chain terminators due tothe absence of of 3’ hydroxyl). If a single species is desired, it canbe gel purified (see UNIT 2.12). Polynucleotide phosphorylasemay also be used to incorporate modified bases at the 3’ end (Gillam S.,1980).

A more controlled meansof introducing modified nucleotides relies on T4 RNA ligase and substratesof the form A(5')ppX (where X can be virtually any molecule, including,for example, ribose or amino acids, in a pyrophosphate linkage with adenosine)(Uhlenbeck and Gumport, 1982). The minimal template for reactions of thisform is a trinucleoside containing a free 3' hydroxyl. RNA reacts muchbetter than DNA, and single-strand molecules act as better templates thandouble stranded ones. Since 3' hydroxyl groups are required, substratesof the form A(5')ppXp will undergo only a single round of addition, unlikethe similar reaction with terminal transferase. In some cases, the compoundA(5')ppXp can be generated directly by RNA ligase from pXp and ATP, althoughthe substrate requirements for X are much more strict than for the ligationreaction. Thus, while virtually any dinucleotide of the form A(5')ppX canbe added to an oligonucleotide, only a few compounds (primarily sterically"small" derivatives of natural bases) can be used by the enzyme to formA(5')ppXp from pXp.

T4 RNA ligase can catalyzethe ligation of single-strand oligonucleotides in the presence of ATP andvarious analogous (Kinoshita, Y. at al., 1997). Templates prepared by terminaltransferase or by T4 RNA ligase which contain modified nucleotides (orother adducts) at their 3' termini may be able to act as substrates inthis reaction. This would allow modified nucleotides to be introduced intothe middle of a longer chain. However, the substrate specificity of theenzyme for the 3' hydroxyl donor is highly substrate-dependent and willhave to be determined empirically.

Table 1

Phosphoramidite	Supplier
Fast/Mild Deprotecting DNA Monomers
Ac-C-3'-CED	Cruachem	Glen
DmF-G-3'-CED	Glen	SPS
Ibu-C-3'-CED	Glen	PE
N-PAC-A-3'-CED	Biogenex	Chemgenes	Cruachem
N-PAC-C-3'-CED	Chemgenes	Perceptive
N-PAC-G-3'-CED	Biogenex	Chemgenes	Cruachem
Fast/Mild Deprotecting RNA Monomers
Ac-rC-2'-tBDSilyl-3'-CED	Glen
dmf-rG-2'-tBDSilyl-3'-CED	Glen	PE	SPS
Ibu-rC-2'-tBDSilyl-3'-CED	PE
N-PAC-rA-2'-tBDSilyl-3'-CED	Biogenex	Chemgenes	Cruachem
N-PAC-rC-2'-tBDSilyl-3'-CED	Chemgenes	Sigma
N-PAC-rG-2'-tBDSilyl-3'-CED	Biogenex	Chemgenes	Cruachem
2'-FPMP-rA-3'-CED	Sigma
2'-FPMP-rC-3'-CED	Sigma
2'-FPMP-rG-3'-CED	Sigma
2'-FPMP-rU-3'-CED	Sigma
2'-FPMP-rI-3'-CED	Sigma
Modified DNA Monomers
a-anomersof deoxyribose	Appligene
06-methyl-G-3'-CED	Glen
2'-deoxy-nebularine-3'-CED (degeneratebase)	Glen
2-amino-A-3'-CED	Glen
2-aminopurine-3'-CED	Glen
2-thio-T-3'-CED	Glen
3'-deoxy-A-3'-CED	Glen
3-nitropyrrole-2'deoxy-3'-CED (M) (universalbase)	Glen
4-methyl-indole-3'-CED	Glen
4-thio-T-3'-CED	Glen
5-halogenated-C-3'-CED	Chemgenes	Cruachem	Glen
5-halogenated-dU-3'-CED	Chemgenes	Cruachem	Glen
5-Methyl-C-3'-CED	Chemgenes	Glen	Sigma
5-Methyl-iso-C-3'-CED (alternative basepair)	Glen
5-nitroindole-3'-CED (universal base)	Glen
7-deaza-A-3'-CED	Chemgenes	Glen
7-deaza-G-3'-CED	Chemgenes	Glen
8-halogenated-A-3'-CED	Glen
8-halogenated-G-3'-CED	Chemgenes	Glen
8-oxo-A-3'-CED	Glen
8-oxo-G-3'-CED	Glen
8-oxo-A-3'-CED	Glen
8-oxo-G-3'-CED	Glen
ARA-C-3'-CED	Chemgenes	Glen
C-5 propyne C-3'-CED (helix stability)	Glen
C-5 propyne U-3'-CED (helix stability)	Glen
dU-3'-CED	Chemgenes	Glen	Sigma
Etheno-A-3'-CED	Chemgenes	Glen
Etheno-C-3'-CED	Chemgenes
Inosine-3'-CED (universal base)	Chemgenes	PE	Sigma
iso-G-3'-CED (alternative base pair)	Glen
K-3'-CED (degenerate base)	Glen
N3-methyl-T-3'-CED	Chemgenes
N6-methyl-A-3'-CED	Glen
O4-methyl-T-3'-CED	Glen
O4-methyl-T-3'-CED	Glen
O6-methyl-G-3'-CED	Cruachem	Glen
P-3'-CED (degenerate base)	Glen
Purine-deoxy riboside Phosphoramidite	Chemgenes
Convertible Deoxy Nucleosides
O4-Triaz-dU-3'-CED	Glen
O4-Triaz-T-3'-CED	Glen
O6-Phenyl-I-3'-CED	Glen
S6-DNP-G-3'-CED	Glen
TMP-F-dU-3'-CED	Glen
Radiolabelled dexoyphosphoramidites	Cambridge Isotopes
Modified RNA Monomers
2',3'-Diacetyl-rG-3'-CED	Chemgenes
2',3'-Diacetyl-rU-3'-CED	Chemgenes
2'-CED-rC-3'-tBDSilyl	Biogenex
2'-CED-rU-3'-tBDSilyl	Biogenex
2'-O-Allyl-(2-amino)-rA-3'-CED	BOE
2'-O-Allyl-rA-3'-CED	BOE
2'-O-Allyl-rC-3'-CED	BOE
2'-O-Allyl-rG-3'-CED	BOE
2'-O-Allyl-rI-3'-CED	BOE
2'-O-Allyl-rU-3'-CED	BOE
2'-OMe-2-Amino-rA-3'-CED	Glen
2'-OMe-2-Aminopurine-3'-CED	Glen
2'-OMe-3-deaza-5-aza-rC-3'-CED	Glen
2'-OMe-Ac-rC-3'-CED	Glen
2'-OMe-DMF-rG-CE -3'-CED	Glen
2'-OMe-propynl-rC-3'-CED	Glen
2'-OMe-propynl-rC-3'-CED	Glen
2'-OMe-propynl-rU-3'-CED	Glen
2'-OMe-rA-3'-CED	Glen
2'-OMe-rC-3'-CED	Glen
2'-OMe-rG-3'-CED	Glen
2'-OMe-rI-3'-CED	Glen
2'-OMe-rU-3'-CED	Glen
2'-OMe-rU-CE -3'-CED	Glen
2'-OMe-TMP-5-F-rU-CE -3'-CED	Glen
2'-tBDSilyl-rI-3'-CED	Dalton	PE	Sigma
2'-tBDSilyl-rT-3'-CED	Chemgenes
5-Fluoro-3'-tBDSilyl-rU-3'-CED	Chemgenes
5-halogenated-2'-tBDSilyl-rC-3'-CED	Chemgenes
5-halogenated-2'-tBDSilyl-rU-3'-CED	Chemgenes	Glen
5-Methyl-2'-tBDSilyl-rC-3'-CED	Chemgenes
7-Deaza-2'-tBDSilyl-rA-3'-CED	Chemgenes
7-Deaza-2'-tBDSilyl-rG-3'-CED	Chemgenes
8-Bromo-2'-tBDSilyl-rA-3'-CED	Chemgenes
8-Bromo-3'-tBDSilyl-rA-3'-CED	Chemgenes
Etheno-2'-tBDSilyl-rA-3'-CED	Chemgenes
Etheno-2'-tBDSilyl-rC-3'-CED	Chemgenes
N-PAC-2'-CED-rA-3'-tBDSilyl	Biogenex
N-PAC-2'-CED-rG-3'-tBDSilyl	Biogenex
N-PAC-2'-OMe-rA-3'-CED	Biogenex	Cruachem
N-PAC-2'-OMe-rG-3'-CED	Biogenex	Cruachem
N-PAC-8-Bromo-2'-tBDSilyl-rA-3'-CED	Chemgenes
N3-(thiobenzoylethyl)-2'-tBDSilyl-rU-3'-CED	Chemgenes
N3-Methyl-2'-tBDSilyl-rU-3'-CED	Chemgenes
Labeling monomers
5'-acridine-3'-CED	Clonetech	Cruachem	Glen
5'-amino-modifers-3'-CED	Clonetech	Glen
5'-amino-modifers-3'-CED (terminal)	Glen	SPS	PE
5'-biotin-modifers-3'-CED	Biogenex (highly rigid cyclohexane spacer)	Clonetech	Glen
5'-biotin-modifers-3'-CED (terminal)	Cruachem	Glen	PE
5'-BODIPY-3'-CED (terminal)	ABI
5'-carboxy-modifer-3'-CED	Glen
5'-cholesteryl-3'-CED	Clonetech
5'-cholesteryl-3'-CED (terminal)
5'-dansyl-3'-CED (terminal)	Clonetech
5'-Digoxigenin-3'-CED	PE	Perceptive
5'-DNP-3'-CED	Clonetech
5'-DNP-3'-CED (terminal)	Clonetech	Cruachem	Glen
5'-FAM-3'-CED (terminal)	ABI	BOE	Cruachem
5'-fluorescein-3'-CED	Biogenex (highly rigid cyclohexane spacer)	Chemgenes	Clonetech
5'-HEX-3'-CED (terminal)	BOE	Cruachem	Glen
5'-JOE-3'-CED (terminal)	ABI
5'-phosphate-3'-CED	Chemgenes	Clontech	Glen
5'-psoralen-3'-CED (terminal)	Chemgenes	Clonetech	Glen
5'-pyrene-3'-CED (teminal)	Clonetech
5'-ROX-3'-CED (terminal)	ABI
5'-TAMARA-3'-CED (terminal)	ABI
5'-TET-3'-CED (terminal)	BOE	Cruachem	Glen
5'-Texas Red-3'-CED (terminal)	Genosys
5'-thio-modifers-3'-CED (terminal)	Chemgenes	Cruachem	Glen
3'-acridine-CPG	Clonetech	Cruachem	Glen
3'-amine-CPG	Chemgenes	Clonetech	Glen (photolabile)
3'-biotin-CPG	Chemgenes	Clonetech	Glen
3'-carboxyl-CPG (photolabile)	Glen
3'-cholesteryl-CPG	Clonetech	Glen
3'-dabcyl-CPG	Glen
3'-Digoxigenin-CPG	Clonetech	Cruachem
3'-DNP-CPG	Clonetech
3'-fluorescein-CPG	SPS
3'-glycerol-CPG	Glen
3'-phosphate-CPG	Chemgenes	Glen	PE
3'-TAMRA-CPG	SPS
3'-thio-CPG	Glen	SPS
Uniquely structured oligos
Branched oligonucleotide synthesis	Clonetech
cyclic oligos (up to 30)	Glen
deoxy-ribose spacer-3'-CED	Glen
C9 spacer-3'-CED	Glen
C18 spacer-3'-CED	Glen
Non-enzymatically extendable 3' ends
2',3'-dideoxy-A-CPG	Glen
2',3'-dideoxy-C-CPG	Glen
3'-C3 spacer-CPG	Glen
3'-deoxy-A-CPG	Glen
3'-deoxy-C-CPG	Glen
3'-deoxy-G-CPG	Glen
3'-deoxy-T-CPG	Glen
5'-amino-T-3'-CED	Glen
5'-OMe-T-3'-CED	Glen
Alternative backbones
H-phosphate chemistries lead to phosphorthioates,phosphoroamidates, or phosphotriesters	Glen
methyl phosphonate linkages for DNAand RNA	Chemgenes	Glen
sulfurizing reagents to convert to phosphothioates	Glen
Various 5' supports (3'-3' or 5'-5'linkages or opposite sense synthesis)	Glen
all phosphoramidites are 5' DMT and3' Cyanoethyl protected (CED)unless otherwise noted.

 
 

Synthesizing DegenerateOligonucleotides

Current combinatorialand "irrational" nucleic acid design methodologies focus on the abilityto create large pools of random sequences from which useful sequences maybe culled (Szostak, J., 1992). Also, random mutagenesis using degenerateoligonucleotides allows for the exploration of "sequence space" surroundinga given protein or RNA structure. Sequences can be produced which givea completely random distribution of nucleotides at a given position, oralternatively, the sequence can be biased or "doped" toward a particularbase with only a low level of randomization.

Most synthesizers canbe programmed for in-line degenerate mixing of bases which is useful ifonly a few positions are to be randomized. A potential problem with thismethod is that if mixing is incomplete, the sequence will be skewed towardwhichever phosphoramidite enters the column first since the reaction ofthe activated phosphoramidite with the free 5' hydroxyl is extremely fast.Therefore, while in-line mixing will generate all base substitutions ata given position, the distribution of these substitutions may not be uniform.If a statistically random distribution of nucleotides is required or iflong stretches of random sequence are to be made, it is better to manuallymix the phosphoramidites together and use this mixture for the degenerateposition. A true random distribution may be obtained by mixing in a 3:3:2:2molar ratio A:C:G:T phosphoramidites to compensate for the faster couplingtimes of G and T phosphoramidites (Bartel, D., personal communication).On synthesizers where phosphoramidites are loaded without detaching thebottle, the mixing generated by sequential loading of each phosphoramiditeinto the extra bottle is sufficient to generate randomized sequences.

Oliphant and Struhl(1986, 1989) have constructed degenerate oligonucleotides using mixed phosphoramidites,but have modified the synthesis protocol by deleting the capping step duringthe random additions. This increases the overall yield of long oligonucleotides,since sequences which fail to elongate are not terminated, and the sizeof the final product is more heterogeneous. This method is particularlyuseful if deletions, as well as randomized bases, are required in a givensequence.

Hermes et al. (unpublishedresults) have developed a detailed protocol for producing statisticallymutated oligonucleotides. This method employs in-line mixing between purephosphoramidite contained in separate bottles and equimolar mixtures ofthe four bases contained in an additional bottle. The obvious advantageof such a method is that doped and clean sequences can be synthesized onthe same oligonucleotide. Whether or not this method is employed, it isan example of how to dissect the chemistry of mixed-site oligonucleotidesynthesis. Hermes et al. (1989) have shown that mutations introduced bythis method are indeed statistically random.

Regardless of the strategyemployed, the level of misincorporation of an oligonucleotide should bedecided in advance by the mutagenesis frequency desired. Quantitatively,this level is given by the probability distribution:

P (n,m,x)= [m!/(m - n)!n!][x]ⁿ[1 - x]^m-n

where P is theprobability of finding n errors in an oligonucleotide m inlength with x level of misincorporation (fraction "wrong" nucleotidesdelivered). This equation describes a Poisson distribution. If primarilysingle mutations are desired then x should be maximized for n= 1; if multiple mutations (e.g., doubles, or triples in a single oligonucleotide)are necessary then x should be correspondingly higher. If the mixis optimized for n mutations then "n-1" and "n+1"mutations will occur in roughly equal amounts and "n" mutationswill be the most frequent.

Cloning randomizedoligonucleotides can be difficult, since a complementary wild-type sequencewill generate mismatches which may result in biased correction in vivo.To avoid this problem, second-strand synthesis can be primed from a nonrandomportion of the sequence, or mutually primed synthesis (UNIT 8.2)can be utilized. Alternatively, Derbyshire et al. (1986) describe the directcloning of doped single-strand material with "sticky ends" into a double-strandedcloning vector. Finally, Reidhaar-Olson and Sauer (1988) describe the synthesisof complementary oligonucleotides containing inosine (which can pair withany of the four natural bases) directly across from randomized codons.This method resulted in the successful introduction of a wide variety ofmutations into the gene for lambda repressor, although there was a slightcompositional bias in cloned sequences.

STRATEGIES FOR OLIGONUCLEOTIDEPURIFICATION

Introduction

Deprotected nucleicacids may be purified and isolated by a variety of methods. The methodof choice will depend on the availability of resources, the purity required(some methods cannot separate (n-1)-mers from n-mers), andtime considerations. Any of the methods described below can be used toclean up crude material.

Isolation Methods

Precipitation.Direct precipitation of the nucleic acids constitutes a fast andefficient purification from contaminating small molecules such as ureaand phenol, but does not allow for purification of abortive synthesis productsfrom the full length one. If oligonucleotide size separation is required,this method should be used in conjunction with some of the other methodsdescribed. Resuspend the whitish pellet obtained post-deprotection in water.Add MgCl2 to a final concentration of 10 mM and mix along with 5 vol ethanol.Precipitation should be immediate. Freeze the sample briefly at -20°or -70°C. Centrifuge the precipitated material, wash with 95% ethanol,dry, and resuspend in water.

Precipitated deoxyoligonucleotidescan be used for sequencing or cloning. They can also be used in PCR reactions,although the efficiency of amplification may be reduced as compared togel purified oligonucleotides. If the DNA is to be phosphorylated, a morethorough purification procedure is necessary since T4 polynucleotide kinaseis inhibited by lingering ammonium ions.

Sizing columns.Size exclusion chromatography is extremely useful as a final purificationstep especially when small molecule contamination occurs with otherwisepure oligonucleotides, but here again, it does not allow for purificationof abortive synthesis products from from the full length one. If oligonucleotidesize separation is required, this method should be used in conjunctionwith some of the other methods described. Oligonucleotides purified byPAGE (UNIT 2.12) might contain small amounts of low molecularweight contaminants such as as urea or phenol that might inhibit enzymaticreactions; sizing columns are a simple way decontaminate these samples.Spincolumns (UNIT 3.4) are simple to prepare and use, but gravityflow columns give better, more reproducible separation. The type of resinused should be adjusted based on the size of the oligonucleotide beingpurified (G-25 for 25-mers or less, G-50 for longer sequences).

Reversed-phasecartridges. A hydrophobic matrix may be used to separate full-lengthfrom abortive synthesis products if the final trityl group is left on followingthe final monomer coupling reaction. The resulting hydrophobically taggedfull-length "trityl-on" oligonucleotide can be separated easily from failuresequences which lack trityl groups and do not efficiently bind the hydrophobicmatrix. Several companies supply columns designed specifically for thepurification of "trityl-on" oligonucleotides (e.g., Applied Biosystemsoligonucleotide purification cartridges). The procedure is simple and canbe performed on a number of samples in parallel within only a few minutes.The yield from such columns is excellent, often >80% of the applied sample.However, although a majority of failure sequences are removed using thismethod, many shorter sequences are co-purified with the desired full-lengthmaterial. Some of these fragments are due to cleavage of depurinated DNA.These apurinic molecules can be eliminated prior to cleavage from the columnby treatment with lysine (Horn and Urdea, 1988). In addition, if care isnot taken to wash and elute samples from these columns slowly, some inhibitors(particularly of ligation reactions) may co-elute.

Denaturing PAGE.Denaturing polyacrylamide gel electrophoresis resolves oligonucleotideswith single residue resolution and is the method of choice for purificationof full-length oligonucleotides (see UNIT 2.12). However,the compatibility of chemistries of modified nucleotides incorporated intothe nucleic acids and acrylamide matrix should be checked (thiolated oligonucleotidesseem to undergo Micheal Addition to the acrylamide thereby rendering themirreversibly capped).

HPLC. Liquidchromatography can also separate oligonucleotides with single residue resolution,but its chief advantage is speed. Total run time can be as short as 30min. The use of alkali perchlorate salts has made ion-exchange the HPLCmethod of choice given that long oligonucleotide (>40 residues) may beeasily purified in large scales (3 25 mmol) (Sproat, B. et al, 1995) (Warrenand Metofs in Molecular biology, Protocols for oligonucleotide Conjugates,humana press). However, secondary structural migration anomalies are generallymore severe than those found with PAGE. The amount of oligonucleotide thatcan be purified in a single chromatographic run depending on the systememployed can be comparable to PAGE, and sample recovery is typically greaterthan 70 percent. For labs with an HPLC system and a need to routinely purifyshort oligonucleotides with no secondary structure, this method is ideal.

Oligonucleotides canbe purified with HPLC by charge differences through ion exchange or hydrophobicityif the final trityl group is left on following the ultimate monomer couplingreaction. Only the "trityl-on" systems use buffers that can be lyophilized.A more complete treatment of the complexities of oligonucleotide purificationby HPLC can be found in the Applied Biosystems User Bulletin #13 on oligonucleotidepurification.

Confirming the OligonucleotideSequence

Most oligonucleotidesthat are used for cloning need not be checked immediately after synthesis,since the clones themselves will be checked after biological or enzymaticamplification. However, in cases where a sequence will be used in a structuralapplication such as mobility shift assays (UNIT 12.1), filter bindingassays (UNIT 12.8), or crystallography, it is desirable to confirmthe sequence. For almost all oligonucleotides, this usually requires chemicalsequencing (UNIT 7.5). Although sequencing will confirm that thecorrect product was made, it cannot determine the homogeneity. How unnaturalor protected bases will react during chemical sequencing, or how they willaffect mobility on a sequencing gel, is not predictable. In order to determinewhat fraction of molecules contain only the natural bases (A,T,C,G), itis necessary to digest the DNA enzymatically to completion and to examineits composition by a comparison with standard bases separated by HPLC.At least one HPLC buffer system has been developed specifically to examinemodified nucleosides in chemically synthesized oligonucleotides (Eadieet al., 1987). Finally, matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS) has recently emerged as a new biotechnology tooland may be used to determine the sequence of deoxy and ribo-oligonucleotidesof up to 60 bases (Zhu, Y. et al., 1997).

PROTOCOL: MONITORINGDNA SYNTHESIS USING THE TRITYL ASSAY

Introduction

A trityl cation isreleased from the 5' end of the growing oligonucleotide during each synthesiscycle, and the yield of each step of the synthesis can be determined bymeasuring spectrophotometrically the amount of trityl cation liberated.This procedure is the simplest and most rapid means available for the identificationof problems with synthesized oligonucleotides prior to deprotection andpurification. Also, in-line quantitative trityl monitors can be interfacedto most synthesizers (Ana-Gen Technologies).

Trityl Assay

Materials

0.1 M para-toluenesulfonic acid (TSA; monohydrate) in acetonitrile

15-ml glass tubes (graduated,if possible)

1. Collect the tritylcation solution in 15-ml glass tubes after each step.

It is helpful touse graduated tubes so that a uniform final volume of acetonitrile canbe attained before assay.

2. Dilute the firstthree and last three fractions to 10 ml with 0.1 M TSA in acetonitrile.Mix thoroughly.

Although 2.5 mlof deblocking solution is initially released, acetonitrile is a volatileliquid and the volume may change during the course of a synthesis. Fractionsmay sit for several days before being assayed without affecting the results.Samples that have evaporated to dryness must be thoroughly redissolved.The acid ensures protonation of the trityl group making them more stronglycolored. It is misleading merely to visualize the yellow/orange color ofthe fractions since variable volumes of differing acidity are often released.

CAUTION: Handlethe solution consisting of dichloroacetic acid (or trichloroacetic acid)in dichloromethane and acetonitrile with gloves because it is corrosiveas well as toxic. Avoid prolonged contact with toxic acetonitrile fumes.DO NOT PIPET BY MOUTH.

3. Dilute each sample20- to 50-fold in the same solution. Measure the absorbence at 498 nm versusacetonitrile/TSA.

These dilutionsare necessary because most spectrophotometers cannot accurately measureabsorbancies >2.

4. Calculate the stepwisecoupling efficiency and absolute yield for the synthesis as a whole. Thestepwise efficiency is given by:

stepwise efficiency =
 

(average absorbance of last three fractions)

_____________________________________________

(average absorbance of first three fractions)

where n is thenumber of trityl nucleotides in the oligonucleotide (equals the lengthof the oligonucleotide for trityl-off syntheses).

The absolute yieldof product is given by:

mmol DMT =
 

(absorbance of last fraction)x(dilution factor)x(10ml)

_______________________________________________________

70 ml /mmol (which is the extinction coefficient ofDMT)

The average stepwiseefficiency is useful in determining the relative efficiency of each cycleof the synthesis. The absolute yield is useful for determining how manymilligrams of product are present for subsequent purifications althoughnot all of the product will be of the desired length.

The average absorbanceof the first and last few steps is used to avoid discrepancies in individualtrityl assays. Clearly, if the synthesis was performed on a 1-mmolcolumn, the absolute yield in the first few fractions should be close to1-mmol. A low absolute yield (absorbance) in the first trityl release,followed by higher absolute yields (absorbances) in the next few fractionsis sometimes observed. This is because some spontaneous detritylation occursduring storage of the columns and the trityls are subsequently rinsed offduring the prime lines program. In this case, the average stepwise efficiencyshould be calculated with fractions 2 through 4. It should be noted thatvisual assessment of the trityl fractions cannot begin to detect subtledifferences (<5%) which may be critical in terms of overall yield.

Using the TritylAssay for Troubleshooting

If the average stepwiseefficiency for the oligonucleotide synthesis is low, each fraction shouldbe assayed in order to diagnose the problem (what counts as a "low" yielddepends on the length and quantity of oligonucleotide desired). In general,synthetic efficiency should be >99% per step, although lower stepwise efficienciescan be tolerated for short oligonucleotides (<40 bases) or where yieldis not critical.

Three classes of failurescan be detected by trityl assays. A low absolute yield at the first stepfollowed by similarly low absorbances that results in a low overall yieldis commonly due to inefficient purging of activator or phosphoramiditelines prior to the synthesis. Such a problem frequently occurs when a synthesizerhas not been used for several days. Purge the lines with dry reagents priorto starting the run to avoid inefficient synthesis. Most machines havea priming program precisely for this purpose.

If the stepwise efficiencyof each step is low, there is a systematic problem with one of the commonreagents, such as the acetonitrile. Often, this is due to moisture in oneor more of the reagents and is more likely if reagents have not been recentlyreplaced. The phosphoramidies should be used until they are almost completelyexhausted during a series of syntheses, so that fresh chemicals will bediluted as little as possible by older, potentially wet material (see Commentary).

Finally, individualtrityl assays are most useful in determining when phosphoramidites havebecome defective. In this case, drops in stepwise efficiency will onlybe seen at the coupling steps involving the phosphoramidites in question.

Many problems, suchas inefficient oxidation or product depurination, cannot be detected bythe trityl cation assay. Therefore, the trityl assay procedure should beused in conjunction with HPLC or gel electrophoresis for product analysis,especially if a homogenous population of oligonucleotides is essential.

PROTOCOL: DEPROTECTIONOF DNA OLIGONUCLEOTIDES

After synthesis iscomplete, the DNA may be cleaved from the column and protecting groupsremoved by treatment with ammonia. Although very extended treatment inbase can harm DNA, hours of ammoniolysis are still preferred to ensurecomplete deprotection since a homogeneous population of "natural" oligonucleotidesat slightly lower yield is better than a mixed population of partiallydeprotected, "unnatural" molecules.

To cleave the DNA fromthe support matrix and remove protecting groups completely, the supportbound product must be treated with concentrated ammonia at 55° to 60°Covernight (312 hr). Even with such extended treatment, deoxyguanosine maynot be completely deprotected (Schulhof et al., 1987). Raising the temperatureat which oligonucleotides are deprotected has been reported to speed upthe process (35 hr at 70°C).

Also, phosphoramiditeswith more labile protecting groups such as phenoxyacetyl or dimethylformadinemasking adenosine and guanosine have recently become commercially available(Table 2.11.1). These allow essentially complete deprotection within 30to 60 min at 70°C (Reddy, M. et al., 1994). Also, by replacing thetraditional benzyl protection of cytosine with acetyl and using a 1:1 mixtureof aqueous ammonium hydroxide and aqueous methylamine, oligonucleotidessynthesized with traditional purine protections may be completely deprotectedin 5 minutes at 65°C (Reddy, M. et al., 1995). Finally, anhydrous ammoniagas phase deprotection of oligoncletotides has recently been describedand provides a convenient method for parallel deprotection of as many columnswill fit in a reactor. Since no water is present, the fully-deprotectedoligonucleotides remain adsorbed to the column matrix thereby guaranteeingno cross-contamination. The oligonucleotides can then be eluted with waterand desalted or further purified. Using PAC-protected monomers, the cleavageand deprotection processes can be completed in ~30 minutes (J.H. Boal etal., 1996).

Materials

Concentrated ammoniumhydroxide (14.8 normal)

Triethylamine

n-butanol

1. In a screw-cap plasticvial, suspend the synthesis support matrix or the already support cleavedoligo in ~1.0 ml vol concentrated ammonium hydroxide for a 1-mmol synthesis.

Depending on thesynthesizer or nucelic acid provider, the oligo may come attached to asupport matrix or free in an ammonium hydroxide solution. The volume ofammonium hydroxide in which product is eluted from the synthesizer is variable.The ammonium hydroxide used should not have been diluted by excessive vaporloss.

2. Place the samplein heat block or oven for 312 hr at 55° to 60°C.

Seal vial tightlywith parafilm (if not fitted with a rubber o-ring).

3. After cleavage fromthe support and deprotection are complete, spin the sample briefly in atabletop centrifuge to pool the ammonia and support that may have collectedin the cap. Let the vial cool to room temperature before opening it toavoid sample boil over.

4. Filter off the supportby passing the liquid through an 0.2 micron filter and wash the filterwith 0.3 ml of 3:1 (v/v) ammonium hydroxide/ethanol.

5. Precipitate theoligonucleotide from the resulting supernatant by adding 10 volumes ofn-butanol and vortexing for 15 sec. Centrifuge for 10 min at 16000xG and4°C.

6. Remove and discardthe single water containing n-butanol phase to reveal the white oligonucleotidepellet.

Oligonucleotides20 residues or shorter with good trityl responses are typically suitablefor use directly in DNA sequencing, PCR amplification, and gel shift analysis.However, if more assuredly homogenous material is required, methods suchas denaturing PAGE and HPLC may be employed to further purify the fulllength product.

7. If a yellowish liquidor crusty pellet remains, rather than a white powder, resuspend the pelletin 0.1 ml distilled water and precipitate with n-butanol again as describedabove. Generally, it is not necessary to add additional salt for preciptation.

Further extractionwill aid in removing any residual ammonia, volatile organics, etc. If theyellow color does not disappear, it will ultimately be removed by

by almost any ofthe standard purification methods.

8. Lyophilize the sampleto dryness in a speedvac evaporator.

This deprotectionis primarily intended for oligonucleotides with the trityl group off. Whenthe trityl group is left on, care must be taken that it is not prematurelyhydrolyzed from the DNA by acid conditions. During lyophilization, a dropof triethylamine must be added regularly to the sample in order to maintainits basicity. (Applied Biosystems User Bulletin #13). Avoid heating thesamples.

PROTOCOL: DEPROTECTIONOF RNA OLIGONUCLEOTIDES

Materials

Ethanol

3:1 (v/v) ammoniumhydroxide (14.8 normal)/ethanol

3 M ammoniumacetate, pH 5.2

triethylamine trihydrofluoride

Sephadex G-25 column

1. In a screwcap plasticvial, suspend the synthesis support matrix or the already support cleavedoligo in ~1.2 ml of 3:1 (v/v) ammonium hydroxide/ethanol for up to a 1-mmolscale.

Depending on thesynthesizer or nucelic acid provider, the oligo may come attached to asupport matrix or free in an ammonium hydroxide solution. The volume ofammonium hydroxide in which product is eluted from the synthesizer is variable.The ammonium hydroxide used should not have been diluted by excessive vaporloss.

2. Place the samplein a heat block or oven for 12-16 hr at 55° to 60°C.

Seal vial tightlywith parafilm (if not fitted with a rubber o-ring).

if fast cleavingphosphoramidites such as PAC-protected purines and acyl -protected cytosineare used, the deprotection time of the bases can be a little as 10 minin methyl amine at 65°C (Wincott, F. et al., 1995).

3. After cleavage fromthe support and base deprotection are complete, spin the sample brieflyin a tabletop centrifuge to pool the ammonia and support that may havecollected in the cap. Let the vial cool to room temperature before openingit to avoid sample boil over.

4. Filter off the supportby passing the liquid through an 0.2 micron filter and wash the filterwith 0.3 ml of 3:1 (v/v) ammonium hydroxide/ethanol.

5. Evaporate the combinedsolutions to dryness in the speedvac without heating. Resuspend the pelletin 0.2 ml of absolute ethanol and speedvac to dryness without heating.

6. Treat the driedresidue with triethylamine trihydrofluoride (0.3 ml for 0.2-mmol or 0.5ml for a 1-mmol synthesis). Allow to rotate in a foil covered screw-capvial in the dark for at least 24 but no more than 48 h.

Alternative methodsexist to remove the 2’ silyl protecting groups either under dilute acidicconditions (Kawahara, S. et al., 1996) or with anhydrous triethylamine/hydrogenflouride in N-methylpyrrolidinone (Wincott, F. et al., 1995).

7. To desalt via ethanolprecipitation, add an equal volume of water to the triethylamine trihydrofluoridesolution and immediately dilute with 1/10 volume 3.0 M sodium acetatepH 5.2. Add 3 volumes of absolute ethanol to precipitate by chilling to-80 °C for ~ 20 minutes. Centrifuge for 10 minutes at 16000xG and 4°C.

8. Remove and discardthe single ethanol layer to reveal the white oligonucleotide pellet.

9. If a yellowish liquidor crusty pellet remains, rather than a white powder, resuspend the pelletin 0.1 ml distilled water and add 1/10 volume 3.0 M sodium acetatepH 5.2. Add 3 volumes of absolute ethanol to precipitate by chilling to-80 °C for ~ 20 minutes. Centrifuge for 10 minutes at 16000xG and 4°C.

The RNA may be desaltedon a desalting matrix such as Sephadex (UNIT 3.4).

REAGENTS AND SOLUTIONS

Most reagents may bepurchased from the synthesizer manufacturer or from companies that specializein reagents for nucleic acid synthesis; however, some labs choose to maketheir own reagents to reduce costs. Notes on the preparation and storageof certain reagents are provided below. Other reagents should probablybe purchased from commercial sources, as the preparation of anhydrous materialsis more difficult and expensive than most molecular biology labs can support.An appropriate text should be consulted on the medical dangers of all solutionsand reagents used in DNA synthesis (see Key References of this unit).

Acetonitrile.Since large volumes are used for rinsing lines and dissolving other reagents,acetonitrile is one of the most costly reagents in nucleic acid synthesis.If the DNA synthesizer is used infrequently, it may be useful to producesmall amounts of dry acetonitrile immediately prior to synthesis sincethe acetonitrile will accumulate water after the bottle has been opened.Moisture greatly diminishes synthetic yield. In this case acetonitrilecan be prepared in any laboratory equipped for routine distillations. However,the time and effort involved in setting up and maintaining a still shouldbe balanced against the cost of obtaining dry acetonitrile. The use ofmolecular sieving pouches that do not release metal ions is recommend;they are often available from the synthesizer manufacturer and should keepthe acetonitrile to less than 20 PPM water. Some commercial suppliers nowmarket bulk solvents specifically for nucleic acid synthesis (e.g., Baker"low water" acetonitrile, 0.002% or 20 ppm water; Burdick and Jackson acetonitrile,0.001% or 10 ppm water). The cost of these special dry reagents is aboutthe same as that of HPLC-grade acetonitrile (which typically contains 0.01%or 100 ppm water), which is the starting material for a laboratory distillation.

CAUTION: Acetonitrilevapor is poisonous.

Ammonium hydroxide.Ammonia is volatile and its concentration (14.8 normal) will decrease onrepeated opening, until it is no longer completely functional as a deprotectingreagent. When this occurs, use a fresh supply of the reagent. Purchaseammonium hydroxide in 0.5- to 1.0-liter bottles, and store it at 4°Cto reduce the ammonia gas found in the vapor phase above the liquid.

CAUTION: Concentratedammonium hydroxide is extremely caustic. Breathing the vapors is harmful.Always use this compound in the fume hood, as it is possible to be quicklyovercome by ammonia fumes and blinded.

Oxidizer.Oxidizer may be prepared from 0.02 M iodine in 7:2:1 (v:v:v) THF:pyridine:water.Most commercial sources of THF contain BHT, a free-radical scavenger thatprevents the buildup of explosive peroxides. This compound has no effecton synthesis chemistry. Any commercial grade of pyridine is acceptable.Use resublimed iodine.

CAUTION: Pyridineis toxic in both liquid and vapor forms.

CAUTION: Iodineis harmful if inhaled. Beware of spills containing both iodine and ammoniasince explosive compounds can be formed.

Phosphoramidites.Some companies recommend dissolving phosphoramidites to give equal molaritiesof the four bases, while others recommend a standard weight/volume ratiothat will give slightly different final molar concentrations. In general,follow the procedure recommended for your synthesizer. The actual phosphoramiditeconcentration may matter for some applications, e.g., when making mixed-siteoligonucleotides. Extremely dry, commercially sealed acetonitrile is requiredto dissolve the phosphoramidites. Also, phosphoramidites that have beendissolved from more than 2 weeks should be replaced.

COMMENTARY

Critical Parameters

By optimizing the reagentsand protocols used in oligonucleotide synthesis, it is reliably possibleto make products of greater length and yield, while minimizing the costsassociated with unproductive runs.

Excluding waterfrom solvents.

The most critical factorin any synthesis is how the reagents are handled to excluded water fromthe system. From the moment a bottle is opened, it is in contact with waterin the air, and all the solvents used are hygroscopic and will absorb watervapor, which reduces yields. This problem is so severe that it is advisableto avoid large-scale, lengthy, or important runs on rainy or high humiditydays.

Special anhydrous reagentscan be purchased from most manufacturers of nucleic acid synthesizers.Additionally, some chemical suppliers are now beginning to market solventsspecifically for DNA synthesis (see Commentary). Adding molecular sievingpouches to the activator and acetonitrile used before oxidation are extremelyuseful in preserving the anhydrous environment of the phosphoramidite couplingreaction.

Phosphoramidites aremost sensitive to water contamination because they are easily hydrolyzedthereby rendering them unreactive. Precautions must be taken to avoid exposingthem to even small amounts of water. Very dry acetonitrile (<0.003%water), kept as a separate stock and sealed under argon, should be usedto dissolve the phosphoramidites. The acetonitrile should be introducedthrough the septum on the amidite bottles via a syringe. Glass syringescan be dried in a 300°C drying oven, then cooled in a desiccater priorto use. Plastic syringes can be dried in a 45°C vacuum oven. Disposableplastic syringes from air-tight sterile casings are dry enough to use withacetonitrile to be dispensed into phosphoramidites with no additional precautions.The syringe should be filled with argon or helium prior to drawing up theacetonitrile, so that wet air is not introduced into the system. On somesynthesizers, this can be done directly via the phosphoramidite ports.Otherwise, an argon line should be used.

An argon line is generallya helpful tool for DNA synthesis chemistry, and it consists of an argontank with a regulator connected to a piece of plastic (Tygon) tubing. Thetubing is then fitted with either a Pasteur pipet or a syringe and needle.Gas flow can be roughly determined via an in-line "bubbler" (e.g., Aldrich#Z101214) can be installed. Argon from a tank is dry enough so that anin-line desiccator is unnecessary. It is necessary to flush the line severalseconds prior to use. Empty bottles can be dried under an argon stream,which will help to exclude condensation from the air. Partially used reagentsare sealed under an argon layer to prevent equilibration with water vaporsince argon is heavier than air.

Choosing synthesiscolumns. Automated DNA synthesis generally takes place on a solidsupport made of controlled pore glass (CPG). This is a porous, nonswellingparticle, 125 to 177 mm in diameter that is derivatized with a terminalnucleotide attached to a long spacer arm. The accessibility of the growingend of the oligonucleotide chain is influenced by the pore size of theparticle. It has been recommended that oligonucleotides up to 50 basesin length should be synthesized on CPG with ~500 angstrom pores, whilelonger oligonucleotides should be synthesized on CPG with ~1000 or largerangstrom pores. Be sure that the column geometry is compatible with thetrityl moniter. Also, when packing columns be sure to purge the columnfirst with dry acetonitrile and then with dry argon to ensure that allCPG particles are caught between the filters since loose particles candamage the synthesizer.

CPG columns are availablethat contain ~0.2 to 10.0-mmol of linked nucleotides. Larger loadings areused In general for larger oligonucleotides since as the size of the oligonucleotideincreases, the overall yield decreases. Also, subsequent purification stepsinvariably involve losses, and the amount of product of correct lengthmust be kept high to ensure that there is enough material. Oligonucleotides>35 bases should be synthesized on 1.0-mmol CPG columns. See section onsynthesis strategies for choosing a column when synthesizing 3100-baseoligonucleotides.

Alternately, columnsthat are more heavily derivatized with the 3’ terminal nucleotide are availableas solid supports (e.g., Fractosil), and a higher yield of product percolumn (and per amount of reagent delivered) can be obtained. However,Fractosil is not recommended for synthesizing oligonucleotides >20 baseslong.

Synthesis chemistry.Regardless of reagent and final product purity, there are inherentlimitations in the chemistry used for the synthesis of oligonucleotides.Therefore, it is necessary to understand the differences between chemicallysynthesized and biologically derived DNA.

Failure to produce"natural" DNA can be due to the synthesis chemistry used. Methylation ofsome bases can occur during deprotection with thiophenol in methyl phosphoramidite-basedsyntheses. Under standard synthesis conditions, these methylated basescan account for up to 7% of the nucleotides present, with N-3-methyl-dTbeing the primary modified base (Farrance et al., 1989). However, thisproblem does not always occur, as such syntheses have been reported whichcontain 99.9% dA, dG, dT, and dC. In general, this problem can be avoidedby increasing the time of the thiophenol deprotection step to between 60and 90 min.

With both methyl andb-cyanoethyl chemistries, the glycosidic bond of N-protected purine phosphoramiditesis subject to hydrolysis during DMT removal with acid. Such depurinationeventually leads to strand cleavage during the ammonia deprotection step.N-protected adenosine is more sensitive to depurination than guanosine,and it is most sensitive when located at the 5' end of an oligonucleotidechain (Tanaka and Letsinger, 1982).

In order to maintainlow levels of depurination, the deblocking step should not be longer than~1 min. Additionally, the mildest effective acid (DCA rather than TCA)should be used. Occasionally, however, protected nucleotides are suppliedas monomethoxytrityl (MMT) esters, which are ten-fold more resistant toacidic detritylation. Oligonucleotides synthesized with MMT as a protectinggroup are more susceptible to depurination, since the deblocking step mustbe correspondingly lengthened to achieve complete detritylation and highyields. In cases where only one base is being added as the MMT compound,it is recommended that this step be performed manually, so that the nucleotidesbe subjected to only one lengthy acid treatment.

Advances in oligonucleotidesynthesis chemistry may mitigate the problems associated with depurination.Recently, nucleotides derivatized with protecting groups that render synthesizedmaterial less sensitive to depurination have become commercially available(Pharmacia, 1989; Schulhof et al., 1987).

Troubleshooting

Most synthesizer problemsrequire a visit from the service engineer. However, syntheses during whichreagent or solvent bottles run empty can sometimes be rescued. Certaininstruments respond to depleted reagents during a synthesis by continuingthe synthesis or stopping. For instruments that stop, oligonucleotidesmay be recovered depending on which reservoir was depleted. It is importantthat the lines be rinsed after an empty reservoir has been detected (somemachines perform this step automatically); otherwise, the oligonucleotideshould be resynthesized. Many machines have the capacity to restart inthe middle of a cycle, and if not, the cycle can be finished manually.The exact procedure will vary depending on which reagent has been exhausted.

Acetonitrile.This is quite serious. The lines cannot be rinsed of whatever reagent theylast contained; thus, determine whether to continue with the synthesisbased on the reactivity of the last reagent in the lines.

Deblocking reagent.Refill the reservoir. The machine can continue at the beginning of thecycle that was interrupted but full length yield will greatly suffer.

Phosphoramidites.Refill the reservoir and perform manual coupling. In this way, the chainsthat did elongate will not form n+1 products, and those that didnot will have a chance to elongate. Programmed synthesis can be resumedwith the next cycle.

Capping reagent.Perform a manual capping and complete the cycle manually, at which pointprogrammed synthesis can resume. This ensures that capping is efficientfor this cycle and avoids accumulation of n-1 sequences.

Oxidizer.Running out of oxidizer is particularly dangerous, because the unstablephosphite linkages remain on the column for long periods. It is best todiscard the material; otherwise, perform a manual oxidation and continuewith the synthesis.

Ammonium hydroxide.Some material may be left on the column if ammonium hydroxide runs outduring the course of synthesis. Refill the bottle and pump more ammoniumhydroxide through the column, or remove the column and treat with ammoniumhydroxide.

Thiophenol.Refill the thiophenol reservoir. Restart from the deprotection step. Atworst, a small amount of product may have cleaved because of inefficientmethyl deprotection.

Routine maintenance.To avoid other purely mechanical problems with a DNA synthesizer,create a regular maintenance schedule to perform the following minor maintenancetasks: Change the various frits and filters that remove debris before theyenter the system; furthermore, remove the deposits of salt and debris asthese may affect flow rates and decrease volumes delivered. Change thefilters on the acetonitrile bottle more often because the flow of thisreagent is much greater than any other. Also, change any rubber o-ringsevery few months. Rinse all the instrument's lines with base and organicsolvents thoroughly every 500 hr of machine use or approximately every3 months. Finally, rinse the lines extensively with dry acetonitrile. Finishthe general maintenance by checking the flow rates of the instrument aftercleaning and then synthesize a small oligonucleotide. Monitor the tritylscarefully in order to confirm that the cleaning did not affect any aspectof the synthesis cycle. If this regimen is followed, many minor delaysencountered in the synthesis schedule can be avoided.

Time Considerations

On a typical day whenmost of the bottles on the synthesizer need refilling, it may take 30 to60 min to fill the bottles, empty waste, install columns, and rinse andprime the lines prior to starting a synthesis. The most time-consumingsyntheses are those involving doped oligonucleotides which require a highlevel of precision in distributing the various phosphoramidites to theappropriate bottles. A computer interfaced with the instrument can increasesynthesis accuracy and throughput. Instruments take varying amounts oftime to synthesize oligonucleotides depending on the number of columnsin use, the length of the oligonucleotide, and the synthesis program. Deprotectionand isolation of a standard DNA oligonucleotide will probably take oneworking day, while RNA oligonucleotides take longer because of the extradeprotection and desalting steps.

Literature Cited

Derbyshire, K.M., Salvo,J.J., and Grindley, N. 1986. A simple and efficient procedure for saturationmutagenesis using mixed oligodeoxynucleotides. Gene 46:145-152.

Eadie, J.S., McBride,L.J., Efcavitch, J.W., Hoff, L.B., and Cathcart, R. 1987. High-performanceliquid chromatographic analysis of oligodeoxyribonucleotide base composition.Anal. Biochem. 165:442-447.

Farrance, I.K., Eadie,J.S., and Ivarie, R. 1989. Improved chemistry for oligodeoxyribonucleotidesynthesis substantially improves restriction enzyme cleavage of a synthetic35mer. Nucl. Acids Res. 17:1231-1245.

Hermes, J.D., Parekh,S.M., Blacklow, S.C., Kuster, H., and Knowles, J.R. 1989. A reliable methodfor random mutagenesis: The generation of mutant libraries using spikeddeoxyribonucleotide primers. Gene In press.

Horn, T. and Urdea,M.S. 1988. Solid supported hydrolysis of apurinic sites in synthetic oligonucleotidesfor rapid and efficient purification on reverse-phase cartridges. Nucl.Acids Res. 16:11559-11571.

Hubner, P., Iida, S.,and Arber, W. 1988. Random mutagenesis using degenerate oligodeoxyribonucleotides.Gene 73:319-325.

Oliphant, R., 1989.Functional Sequences from Random DNA. Harvard University Thesis, Boston,Mass.

Oliphant, R., Nussbaum,A.L., and Struhl, K. 1986. Cloning of random-sequence oligodeoxynucleotides.Gene 44:177-183.

Pharmacia, 1989. Analects.Vol. 17, No. 2 (see APPENDIX 4).

Ratliff, R.L. 1982.Terminal deoxynucleotidyltransferase. In The Enzymes, Vol. XV (P.D.Boyer, ed.) pp. 105-118. Academic Press, San Diego.

Reidhaar-Olson, J.F.and Sauer, R.T. 1988. Combinatorial cassette mutagenesis as a probe ofthe informational content of protein sequences. Science 241:53-57.

Schulhof, J.C., Molko,D., and Teoule, R. 1987. The final deprotection step in oligonucleotidesynthesis is reduced to a mild and rapid ammonia treatment by using labilebase-protecting groups. Nucl. Acids Res. 15:397.

Tanaka, T. and Letsinger,R.L. 1982. Syringe method for stepwise chemical synthesis of oligonucleotides.Nucl. Acids Res. 10:3249.

Uhlenbeck, O.C. andGumport, R.I., 1982. T4 RNA ligase. In The Enzymes, Vol. XV (P.D.Boyer, ed.) pp. 31-58. Academic Press, San Diego.

Usman, N., Ogilvie,K.K., Jiang, M.Y., and Cederagren, R.J. 1987. Automated chemical synthesisof long oligoribonucleotides using 2'-O-silylated ribonucleotide 3'-O-phosphoramiditeson a controlled-pore glass support: Synthesis of a 43-nucleotide sequencesimilar to the 3' half molecule of an E. coli formylmethionine tRNA.J. Am. Chem. Soc. 109:7845-7854.

Zon, G., Gallo, K.A.,Samson, C.J., Shao, K., Summers, M.F., and Byrd, R.A. 1985. Analyticalstudies of "mixed sequence" oligodeoxyribonucleotides synthesized by competitivecoupling of either methyl or b-cyanoethyl-N, N-diisopropylaminophosphoramidite reagents, including 2'-deoxyinosine. Nucl. Acids Res.13:8181-8196.

Reddy, M.P., Hanna,N.B., and Farooqui, F. 1994. Fast cleavage and deprotection of oligonucleotides.Tetrahedron Lett. 35:4311-4314.

Wincott F., DiRenzoA., Shaffer C., Grimm S., Tracz D., Workman C., Sweedler D., Gonzalez C.,Scaringe S., Usman N. 1995. Synthesis, deprotection, analysis and purificationof RNA and ribozymes. Nucleic Acids Res . 23:2677-2684.

Bartel, D. Szostak,J.W. 1993. Isolation of new ribozymes from a pool of random sequences.Science .261:1411-1418.

Ciccarelli, R. B.,Gunyuzlu, P., Huang, J., Scott, C., and Oakes, F. T. 1991. Constructionof synthetic genes using PCR after automated DNA synthesis of their entiretop and bottom strands. Nucleic Acids Res. 19:6007-6013.

Sproat, B., Colonna,F., Mullah, B., Tsou, D., Andrus, A., Hampel, A., Vinayak, R. 1995. Anefficient method for the isolation and purification of oligoribonucleotides.Nucleosides Nucleotides . 14:255-73.

Cohen, G., Deutsch,J., Fineberg, J., Levine, A. 1997. Covalent attachment of DNA oligonucleotidesto glass. Nucleic Acids Res. 25:911-912.

Gillam S., Smith M.,1980. Use of E. coli polynucleotide phosphorylase for the synthesis ofoligodeoxyribonucleotides of defined sequence. Methods Enzymol.65:687-701.

Szostak J. 1992. InVitro Genetics. Trends in Biochem. Sci. 17: 89-93.

Zhu, Y., He, L., Srinivasan,R., Lubman, D. 1997. Improved resolution in the detection of oligonucleotidesup to 60-mers in matrix-assisted laser desorption/ionization time-of-flightmass spectrometry using pulsed -delayed extraction with a simple high voltagetransistor switch. Rapid Commun. Mass Spectrom. 11:987-992.

Reddy, M.P., Farooqui,F., Hanna, N. B.1995. Methylamine deprotection provides increased yieldof oligoribonucleotides. Tetrahedron Lett. 36:8929-8932.

Boal, J.H., Wilk, A.,Harindranath, N., Max, E.E.,Kempe, T.,Beaucage, S.L. 1996. Cleavage ofoligodeoxyribonucleotides from controlled-pore glass supports and theirrapid deprotection by gaseous amines. Nucleic Acids Res. 24:3115-3117.

Kawahara, S., Wada.T., Sekine, M. 1996. Unprecentended mild acid-catalyzed desilyation ofthe 2-O-tert-butyldimethylsilyl group from chemically synthesized oligoribonucleotidesintermediates via neighboring group participation of the internucleotidephosphate residue. J. Amer. Chem. Soc. 118:9461-9468.

Kinoshita Y., NishigakiK., Husimi Y. 1997. Fluorescence-, isotope- or biotin-labeling of the 5'-end of single-stranded DNA/RNA using T4 RNA ligase. Nucleic AcidsRes . 25:3747-3748.

Key References

Applied BiosystemsUser Bulletin #13. 1988 (see APPENDIX 4).

A well organizedoverview of synthetic oligonucleotide synthesis, purification and quantitation.

Bretherick, L., 1986.Hazards in the Chemical Laboratory, 4th ed. Alden Press, Oxford.

A guide to hazardouschemical handling.

Gait, M.T. (ed.) 1986.Oligonucleotide Synthesis: A Practical Approach. IRL Press, Oxford.

The seminal texton synthetic oligonucleotide synthesis that provides critical insight.

Internet Resources

Introduction to Solid-phaseOligonucleotide Chemistry

http://www.interactiva.de/oligoman/intro_c1.html#b1

Web site detailingsynthesis chemistries, procedures, and reagants.

Tips For OligonucleotideSynthesis

http://www.medstv.unimelb.edu.au/ABRFNews/1994/December1994/dec94ponoligo.html

Web site providinggeneral advise on oligonucleotide synthesis

Reagants and Phosphoramidites

Most suppliers ofmaterials have a fairly extensive list of products available on-line.