|Chemistry of Oligonucleotide Synthesis |
With the advent of whole genome sequencing projects, demand for custom synthetic oligonucleotides has soared. Most techniques in modern molecular biology employ synthetic oligos, including the polymerase chain reaction (PCR), DNA sequencing, site directed mutagenesis, and single-nucleotide polymorphism (SNP) assays. Unlike other reagents used in molecular biology, oligos are not generally available as stock items but are custom made to each user's specifications (e.g., sequence, scale, purity, and modification). In recent years, improvements in oligonucleotide synthesis chemistry and processing technology have led to higher quality with lower cost. A decade ago, oligos cost as much as $5.00/base and could take a week to deliver. Today, prices have dropped by 90% and primers can be obtained with rapid 24-hour turnaround [CLICK HERE to order your oligos with 24 hour turnaround.] ]. Furthermore, broad ranges of chemistries are now available, including DNA, RNA, or chimeras, all of which can be modified in a variety of ways (such as fluorescent-labeled probes, and nuclease resistant antisense oligos) [CLICK HERE to visit our extensive catalog].
Figure 1. Commercial Nucleic Acid Synthesizer, Model ABI394
In spite of this ready availability, oligonucleotide synthesis remains a complex, multi-step process. A modern oligonucleotide synthesis facility may produce over 10,000 oligos every day, and each and every oligo is made as a unique, custom synthesis that requires precision and attention to detail. At IDT we use only the highest quality reagents and innovative synthesis techniques so that the efficiency of the chemical reactions are maintained above 99%. A small drop to 98.5% or 98% coupling efficiency will have a significant negative impact on oligo quality [CLICK HERE for a Technical Bulletin on the relationship between yield and quality].
Most oligonucleotides are made on commercial nucleic acid synthesizers (Figure 1) using phosphoramidite chemistry. Alternative chemistries (such as the H-phosphonate method) exist but are not in routine use at IDT. The Practical Approach series has reviewed phosphoramidite and alternative synthetic strategies (Brown, T., and Brown, D. J. S. 1995).
Oligonucleotide phosphoramidite synthetic chemistry was introduced nearly 20 years ago (McBride and Caruthers, 1983). The building blocks used for synthesis are commonly referred to as "monomers"e;, which are activated DNA phosphoramidite nucleosides that are modified with a trityl leaving group on the 5'-end, a ß-cyanoethyl protected 3'-phosphite group, and may also include additional modifiers that serve to protect reactive primary amines in the heterocyclic ring structure (to prevent branching or other unwanted side reactions from occurring during synthesis). The structures of the four DNA monomers are shown in Figure 2.
The phosphoramidite approach to oligonucleotide synthesis proceeds in four steps that are schematically outlined in Figure 3. Automated synthesis is done on solid supports, usually controlled pore glass (CPG) or polystyrene. CPG is loaded into a small column that serves as the reaction chamber. A loaded column is attached to reagent delivery lines on a DNA synthesizer and the chemical reactions proceed under computer control. Bases are added to the growing chain in a 3' to 5' direction (opposite to enzymatic synthesis by DNA polymerases). Although "universal" supports exist, synthesis is more often begun using CPG that is already derivatized with the first base, which is attached via an ester linkage at the 3'-hydroxyl [A].
Synthesis initiates with cleavage of the 5'-trityl group [B] by brief treatment with acid [dichloroacetic acid (DCA) or trichloroacetic acid (TCA) in dichloromethane (DCM)]. Activated monomer (a DNA phosphoramidite in tetrazole) is coupled to the available 5'-hydroxyl [C] and the resulting phosphite linkage is oxidized to phosphate by treatment with iodine (in a THF/pyridine/H2O solution) [D]. This completes one "cycle" of oligonucleotide synthesis.
Ideally, the nucleoside condensation reaction proceeds with around 99% efficiency. If left untreated, the remaining 1% of molecules could participate in subsequent reactions and will result in unwanted truncation deletion mutants. To help prevent this unreacted 5'-OH groups are blocked by acetylation ("capping") with acetic anhydride before the oxidation step. Truncated oligos that are capped remain as short species that are easily removed by a variety of purification methods. Some molecules fail to cap and continue to participate in additional cycles of synthesis, resulting in near full-length molecules that contain internal deletions, the so-called (n-1)-mer species. These molecules will usually work in PCR or sequencing but are unwanted and will lead to deletion mutants if used in cloning or other demanding applications; this species can be removed by rigorous purification using PAGE or HPLC.
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Figure 3. Solid Phase Oligonucleotide Synthesis Cycle, Phosphoramidite Chemistry
A more detailed discussion of phosphoramidite synthetic chemistry follows, which may help you in designing compounds that exploit the full potential afforded by this technology as well as better understand its limitations.
Chemical synthesis of oligonucleotides is a complex process in which four building blocks (base phosphoramidites) are connected as a linear polymer in precise order. In addition to the component bases, a number of reagents are required to assist in the formation of internucleotide bonds, oxidize, cap, detritylate, and deprotection. Automated synthesis is performed on a solid support matrix that serves as a scaffold for the sequential chemical reactions; a series of valves and timers to deliver the reagents to the matrix, and finally a post-synthesis processing stream that includes purification, quantification, product QC, lyophilization, and shipping.
Three of the four DNA bases (G, C, and A) contain primary amines that are reactive and must be blocked ("protected") so as to not participate in unwanted reactions during synthesis. Further, all four phosphoramidites contain a phosphorus linkage that similarly needs to be protected. Chemical groups used to protect these sensitive sites must remain intact during all phases of the DNA synthesis cycle yet must be readily removed after synthesis so that normal, unmodified DNA results. A number of different protecting strategies have been developed. At IDT, we employ phosphoramidites with b-cyanoethyl protected phosphorus. For the heterocyclic bases, protection of primary amines is often provided by a benzyol group for adenine and cytosine and either a dimethylformamidine or isobutyrl group for guanine (Fig 5). Thymine, which lacks a primary amine, does not require base protection. These protecting groups are stable under conditions used during synthesis but are rapidly and effectively removed by treatment with ammonia.
It is also necessary to block the 5'-OH of the base-phosphoramidites so that activated monomers do not react with themselves but can only react with the 5'-OH on the growing oligo chain tethered to the solid support. Current chemistry employs a dimethoxytrityl (DMT) group (Fig. 6). After condensation, the DMT group is cleaved from the newly added DNA base by treatment with acid. Released DMT cation is orange colored and progress of the DNA synthesis reaction can be monitored by spectrophotometric reading at 490 nm.
The 3' hydroxyl group of the deoxyribose sugar is derivatized with a highly reactive phosphitylating agent. The phosphate oxygen on this group is usually masked by the ß-cyanoethyl moiety (Adams et al, 1983, McBride and Caruthers, 1983) that can be removed by ß-elimination using mild ammonia treatment (Sinha et al, 1983). Although reactive, these derivatives are to a certain extent stable towards oxygen and atmospheric moisture at room temperature, easy to purify on silica gel and stable at room temperature.
|Figure 5. Heterocyclic Base Protecting Groups |
|Figure 6. Detritylation - Deblocking of the 5'-Hydroxyl |
Synthesis starts with the first base attached to the CPG solid support and elongates in a 3' » 5' direction. CPG particles are relatively large and are porous, containing channels that greatly increase the surface to volume ratio, allowing the reaction to be done in a small reaction chamber using small volumes of reagents. The CPG is positioned in a "column" between two filter frits; with a reagent entry port on one end and an exit port (waste) on the other. A DNA synthesis column is shown in Figure 7 and the linkage between CPG solid support and the 3'-base is shown in Figure 8.
The synthesis cycle begins with the removal of the DMT group from the 5' hydroxyl of the 5'-terminal base by brief exposure to dichloroacetic acid (DCA) or trichloroacetic acid (TCA) in dichloromethane (DCM). The yield of the resulting trityl cation can be measured to help monitor the efficiency of the synthetic reaction. Protection of the reactive species (primary amines and free hydroxyls) on the nucleoside building blocks insures that the exposed 5'-hydroxyl is the only reactive nucleophile capable of participating in the a coupling reaction (next step).
Off-the-shelf DNA phosphoramidites are not reactive and are converted to active form by treatment in tetrazole prior to coupling. These processes occur through the rapid protonation of the phosphoramidite followed by the reversible and relatively slow formation of a phosphorotetrazolide intermediate (Stec and Zon, 1984; Berner et al, 1989).
Coupling reactions with activated deoxyribonucleoside-phosphoramidite reagents are fast and efficient. We use an excess of tetrazole over the phosphoramidite (to ensure complete activation) and an excess of phosphoramidite over reactive oligo, under these conditions coupling efficiencies of >99% can be achieved.
Since the base-coupling reaction is not 100% efficient, a small percentage of molecules will fail to extend and result in undesired, truncated species. Unless blocked, these truncated oligos can continue to function as a substrate in later cycles, extend, and result in near full-length oligos with internal deletions - an (n-1)-mer species. These "reaction failures" can be prevented from participating in subsequent synthesis cycles by "capping" (Figure 10), which involves acetylation of the free 5'-OH with acetic anhydride and N-methylimidazole (Farrance, et al, 1989).
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Figure 10. Capping - block unreacted 5'-OH groups by acetylation
At this point, the DNA bases are connected by a potentially unstable trivalent phosphite triester. This species is converted to the stable pentavalent phosphotriester linkage by oxidation (Figure 11). Treatment of the reaction product with dilute iodine in water/pyridine/tetrahydrofuran forms an iodine-phosphorous adduct that is hydrolyzed to yield pentavalent phosphorous. The oxidation step completes one cycle of oligo synthesis; subsequent cycles begin with the removal of the 5'-DMT from the newly added 5'-base.
After synthesis is complete, the oligonucleotide is cleaved from the solid support with concentrated ammonium hydroxide at room temperature. Continued incubation in ammonia at elevated temperature will deprotect the phosphorus via ß-elimination of the cyanoethyl group and also removes the protecting groups from the heterocyclic bases. In Figure 12, treatment of a benzoyl-dA ß-cyanoethyl-condensation product with ammonia yields deprotected DNA plus benzamide and acrylonitrile.
During synthesis, both full-length oligo and truncation products remain attached to the CPG support. Following synthesis, all species are similarly cleaved and recovered so that the final reaction product is a heterogeneous mixture of wanted and unwanted species. Impurities accumulate to a greater degree as oligo length increases. Furthermore, cleaved protecting groups are also present. At this point, oligos are traditionally "desalted", a process in which small molecule impurities (protecting groups and short truncation products) are removed using gel filtration or organic phase extraction methods. As no salt is used during oligo synthesis, this step actually removes organic impurities (benzamide and acrylonitrile) and not salt. See the FAQ on "desalting" for more details.
Use of desalted oligos with no additional purification is economical and is appropriate when using short primers in simple applications, such as routine PCR or DNA sequencing. However, truncation species can interfere with reactions when longer oligos are used in more complex procedures. IDT recommends that added purification be done for all oligos >40 bases in length and for any application involving cloning, site directed mutagenesis, or quantitative gene detection (Dual-labeled Probes or Molecular Beacons).
Figure 13. PAGE Purification of Oligonucleotides
Many researchers incorrectly assume that synthetic oligonucleotides come exactly as ordered. From the above discussion on how oligos are made, it is clear that the reaction product must include unwanted species as well as the desired sequence. Oligo synthesis efficiency is 98-99% for each cycle of chemistry (see the Technical Bulletin on "Yields". for additional discussion), so for each cycle about 1-2% of the reaction products will be 1 base shorter than expected. Some truncated species fail "capping" and continue to participate in additional cycles of DNA synthesis. For a 60-mer oligo, less than 50% of the final product will be the desired full-length molecules (see our Purification Technical Bulletin for a more complete description). The final synthesis product will include a mixed population of (n-1)-mer and (n-2)-mer (etc.) molecules which represent a heterogeneous collection of sequences, effectively a pool of deletion mutants at every possible position. A recent report dramatically illustrates the problems associated with using unpurified oligos. The authors found that in a gene construction protocol every construct sequenced had small point deletions when using unpurified oligos (Chalmers and Curnow, 2001); a similar experiment done using PAGE purified oligos resulted in the desired end product (Au et al, 1998). It is therefore important to purify full-length product when using long oligos for more demanding applications.
Figure 14. HPLC Purification of Fluorescently Labeled Oligonucleotide
IDT offers high-level purification using polyacrylamide gel electrophoresis (PAGE) and high performance liquid chromatography (HPLC). PAGE purification (Figure 13) is recommended for oligos used in cloning, mutagenesis, gel-shift assays, gene construction or anytime an oligo>50 bases in length is needed. HPLC purification (Figure 14) is recommended for unmodified oligos under 50 bases in length, dye-labeled oligos, antisense oligos, and other specialty modified oligos. See the Technical Bulletin on Purification for more details and our online catalog for pricing information.
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