For controlling the structure (sequence) of defined nucleic acids used in biosensors, nucleic acids could be obtained by bond cleavage of longer segments, but they are now more commonly synthesized by chemically polymerizing individual nucle-otide precursors. Chemical oligonucleotide synthesis is carried out from the 3' to 5' direction, which is opposite to enzyme synthesis. Synthesis starts from a solid support which is then processed by a step-by-step addition of nucleotide residues to the 5'-terminus of the elongating oligonucleotide chain until a desired sequence has been built. Here the general DNA/RNA synthesizer is taken as an example. Solid supports are nucleoside phosphoramidate (starting nucleotide residue) connected to controlled-pore glass via a carbon linker; they end up with a N, N-dimethyltryptamine (DMT) group which is essential to chain elongation. The following addition of each nucleotide residue is referred to as a synthetic cycle (Fig. 4), which consists of four basic reactions:
Step one, deblocking: the acid labile protecting group DMT is removed with an acid, such as trichloroacetic acid or dichloroacetic acid, in an inert solvent (dichlo-romethane or toluene) and washed out, resulting in a 5' free hydroxyl group ready for coupling.
Step two, coupling: a nucleoside phosphoramidite is mixed with an acidic azole catalyst, 1H-tetrazole, 4,5-dicyanoimidazole, 2-ethylthiotetrazole, or other activators, brought in contact with the oligonucleotide precursor whose 5'-hydroxyl group is unprotected. The coupling reaction allows connecting of incoming nucleoside phosphoramidate to oligonucleotide precursors which are linked to a solid support, resulting in one nucleotide elongated phosphite triester. This coupling requires highly anhydrous conditions and often happens in anhydrous acetonitrile, which is an inert polar solvent. The final washing removes unconnected reagents and by-products.
Step three, capping: after the coupling reaction, there is still a small portion of solid support-bound 5' hydroxyl groups present. To prevent elongation of undesired sequences, those free 5' hydroxyl groups need to be blocked permanently. This reaction is done by treating the solid support with acetic anhydride/pyridine in tetrahydrofuran (THF) to form acetylation products of hydroxyl groups.
Step four, oxidation: phosphite triester generated in the coupling step is not naturally formed with confined stability under the condition following oligonucleotide elongation. Oxidation is achieved by treating the solid support-bound oligonucle-otide with iodine water and pyridine in THF solution. The result is to transform phosphite trimesters into phosphate trimesters whose oxidation state is the same as native DNA.
Eventually, after the synthetic steps and purification steps, the designed DNA sequence is readily obtained.
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