Oligonucleotide synthesis is the non-biological, chemical synthesis of defined short sequences of nucleic acids. It is extremely useful in laboratory procedures covering a wide range of molecular biology applications. Automated synthesizers allow the synthesis of oligonucleotides up to 160 to 200 bases. Typically, synthesized oligonucleotides are single-stranded DNA molecules around 15-20 bases in length. They are most commonly used as primers for DNA sequencing and amplification, as probes for detecting complementary DNA or RNA via molecular hybridization, and for the targeted introduction of mutations and restriction sites, allowing for the synthesis of artificial genes.
Oligonucleotides are chemically synthesized using phosphoramidites. A phosphoramidite is a normal nucleotide with protection groups added to its reactive amine, hydroxyl and phosphate groups. These protection groups prevent unwanted side reactions and force the formation of the desired product during synthesis. The 5' hydroxyl group is protected by DMT (dimethoxytrityl), the phosphate group by a diisopropylamino (iPr2N) group and a 2-cyanoethyl (OCH2CH2CN) group. The bases also have protecting groups on the exocyclic amine group (benzoyl or isobutyryl). In RNA synthesis, the 2' group is protected with a TBS (butyldimethylsilyl) group or with a methyl group. With the completion of the synthesis process, all the protection groups are removed.
Whereas enzymes synthesize DNA in a 5' to 3' direction, chemical DNA synthesis is done backwards in a 3' to 5' reaction. Based on the desired nucleotide sequence of the product, the phosphoramidites for the bases A, C, G, and T are added sequentially to react with the growing chain in a repeating cycle until the sequence is complete. In each cycle, the product's 5' phosphate is deprotected and a new base is added for extension. However, incorrect reactions may occur and therefore the process is only suitable for short oligonucleotides as the number of errors increases with the length of the oligonucleotide sequence that is being synthesized. Products are often purified by HPLC to isolate the products with the proper sequence.
In solid-phase synthesis, the 3' end of the oligonucleotide is bound to a solid support column on which all reactions take place. The 3' group of the first base is immobilized via a linker onto a solid support (polystyrene beads or similar). This allows for easy addition and removal of reactants. In each step, the solutions with the nucleotides for the next reaction are pumped through the column from an attached reagent delivery system and washed out before the next nucleotide is added. In modern synthesizers, reagent delivery and washing steps are controlled via computer based on the desired sequence. At the end of the synthesis program, the oligonucleotide is cleaved off the solid support and eluted from the column.
Oligonucleotide synthesis is done via a cycle of four chemical reactions that are repeated until all desired bases have been added:
- Step 1 - De-blocking (detritylation): The DMT is removed with an acid, such as TCA, and washed out, resulting in a free 5' hydroxyl group on the first base.
- Step 2 - Base condensation (coupling): A phosphoramidite nucleotide (or a mix) is activated by tetrazole which removes the iPr2N group on the phosphate group. After addition, the deprotected 5' OH of the first base and the phosphate of the second base react to join the two bases together in a phosphite linkage. These reactions are not done in water but in tetrahydrofuran or in DMSO. Unbound base and by-products are washed out.
- Step 3 - Capping: About 1% of the 5' OH groups do not react with the new base and need to be blocked from further reaction to prevent the synthesis of oligonucleotides with an internal base deletion. This is done by adding a protective group in the form of acetic anhydride and 1-methylimidazole which react with the free 5' OH groups via acetylation. Excess reagents are washed out.
- Step 4 - Oxidation: The phosphite linkage between the first and second base needs to be stabilized by making the phosphate group pentavalent. This is achieved by adding iodine and water which leads to the oxidation of the phosphite into phosphate. This step can be substituted with a sulphorylation step for thiophosphate nucleotides.
After synthesis is complete, the oligonucleotides are cleaved off the column and deprotected (base and phosphate) by base hydrolysis using ammonium hydroxide at high temperature. This removes all remaining protection groups, resulting in a reaction mixture containing the wanted product. For some applications, additional reporter groups are added post-synthesis.
The oligonucleotide can be purified further from this mix by desalting through ethanol precipitation, size exclusion chromatography, or reversed-phase chromatography. To eliminate unwanted truncation products, the oligonucleotides can be purified via polyacrylamide electrophoresis or HPLC.
An interesting development of this technology has allowed genechips to be made, where the probes are synthesised on the silicon chip, and not printed, allowing a higher resolution. This can be done via a mechanical mask where thin silicon rubber capillaries are put on a glass slide and the probes synthesised. More high-tech versions employ photolayable products and Photolithographic mask or micromirrors. The 1cm2 surface of silicon is coated with a linker and a photoprotecting group such as nitroveratryloxycarbonyl is used and the mask exposes to a lamp the spots that will receive the subsequent nucleotide: this step is repeated for all four bases, but only one correct one is added to the growing probes on each spot (www.affymetrix.com). Thanks to digital light processing (DLP) technology (that give HD TVs) micromirrors were developed which have more detail and speed compared to masks, allowing the generation of microarray chips having one million and more features (www.nimblegen.com). DLP technology and improved synthesis chemistry is the basis for an extremely fast and flexible DNA microarray synthesizer, recently commercialized for cutting-edge research projects (www.febit.com).