Phosphoramidite

Nucleoside phosphoramidites are used to synthesise short nucleic acid chains. The chemical process allows several modifications, such as linker arms or using alternative nucleotides, such as LNA or morpholino or 2' group modified (OMe, NH₂, F) or abasic, non-canon bases (xanthine, hypoxanthine, tricyclic bases, etc) or bases with a fluorescent group, linker arm to attach a fluorescent group (aminoallyl) or biotin attached and so forth. There are a variety of alternative chemical and methods to do so, in fact Pubmed list nearly a thousand articles that modify this method [1]. The prices depend on the company and the quantity required and are around 0.17$-0.30$ a base for DNA, while higher prices for RNA 3.50-4.50$/base (technology is not that efficient) and other varaints (IDT [2], invitrogen [3]). The name nucleoside phosphoramidite comes from the phosphite group that has an NH₂ instead of an OH group. The "phosphate" group in normal nucleic acids is pentavalent, while here it is trivalent. The structure of amidophosphoric acid is present in pubchem [4]. The efficiency of the chemical synthesis is about 98% per base (see capping step). This is ideal for short oligos, but when 100 or more bases are made the method is not that good and enzymatic ligation is used.

Additional recommended knowledge

How to quickly check pipettes?

Essential Laboratory Skills Guide

What is the Correct Way to Check Repeatability in Balances?

Solid phase chemical synthesis

It is done by protection chemistry where the most reactive groups are protected to avoid unwanted products. Whereas in biological processes it is nearly a dogma that enzymes work on DNA 5' to 3', this artificial chemical process is done backwards. The 3' primer is immobilized via a linker onto a solid support (polystyrene beads or similar), allowing the chemicals to be added and washed off, while the 5' OH group is protected by DMT (dimethoxytrityl) group. The free phosphoamidite bases have on their phosphate group a diisopropylamino (iPr₂N) group and a 2-cyanoethyl (OCH2CH2CN) group. The bases also have protecting groups on the exocyclic amine group (benzoyl or isobutyryl).

• Detritylation: The DMT is removed with an acid, such as TCA, resulting in a free OH
• Coupling: a phosphoramidite nucleotide is added (or a mix) and tetrazole which removes the iPr₂N group on the incoming base that is attacked by the deprotected 5'OH of the growing oligo (these reactions are not done in water but in tetrahydrofuran or in DMSO). In RNA the 2' is protected with a TBS (butyldimethylsilyl) group or with a Me group.
• Capping: The few (1%) free 5'OH must be stopped; they are capped with acetic anhydride and 1-methylimidazole.
• Oxidation: The phosphate group is made pentavalent by adding iodine and water. This step can be substituted with a sulphorylation step for thiophosphate nucleotides.

These 4 steps are repeated n number of times. The products are cleaved and deprotected (base and phosphate) thanks to base hydrolysis (ammonium hydroxide). They are then desalted and lyophilized or purified by HPLC. Reporter groups and so on are often added post-synthesis (aminoallyl groups are a common method).

Enzymatic synthesis

T4 RNA ligase and T4 DNA ligase are used for making long oligos by ligating 2 short oligos (5'Phosphate donor and 3'OH acceptor). Template driven enzymatic synthesis is also efficient when using T7 Polymerase or the Klenow fragment and modified bases.

Microarrays

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).[5]

Uses

• siRNA
• primers for PCR and RT PCR
• 70mers for printer microarrays
• 18mers in situ synthesis of probes for a genechip

Modified Bases

Verma S, Eckstein F.1998 Modified oligonucleotides: synthesis and strategy for users. Annu Rev Biochem.67:99-134.

Reaction process

Brown T, Brown DJS. 1991. In Oligonucleotides and Analogues. A Practical Approach, ed. F Eckstein, pp. 1– 24. Oxford: IRL