Oligonucleotide Synthesis: Methods and Applications by Piet Herdewijn

By Piet Herdewijn

A set of strong new innovations for oligonucleotide synthesis and for using transformed oligonucleotides in biotechnology. one of the protocol highlights are a singular two-step strategy that yields a excessive purity, more cost-effective, DNA, the synthesis of phosphorothioates utilizing new sulfur move brokers, the synthesis of LNA, peptide conjugation easy methods to enhance mobile supply and cell-specific focusing on, and triple helix formation. The purposes contain utilizing molecular beacons to watch the PCR amplification method, nuclease footprinting to review the sequence-selective binding of small molecules of DNA, nucleic acid libraries, and using small interference RNA (siRNA) as an inhibitor of gene expression.

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To be absolutely sure that you are using anhydrous acetonitrile, distillation from calcium hydride immediately before use is recommended. Another source of water is its adsorption on the synthons during storage. This can be minimized by storage at –20°C under dry argon in a desiccator equipped with Drierite. Prior to opening the desiccator, it should be allowed to attain room temperature. Following transfer of the synthons to vials for use on the synthesizer, these samples should be dried under vacuum overnight to remove final traces of water.

Oligonucleotide Deprotection A two-stage rapid deprotection strategy is employed to remove phosphate backbone protection, release the oligonucleotide from the solid support, and remove the exocyclic amine protecting groups on A, G, and C. The treatment also removes the acetyl moiety from the acetoxyethyl orthoester, resulting in the 2'-bis-hydroxyethyl protected intermediate that is now 10 times more labile to final acid deprotection. In the first deprotection step, S2Na2 is used to selectively remove the methyl protection from the internucleotide phosphate, leaving the oligoribonucleotide attached to the polystyrene support (8).

3. , which in addition converts the product into the sodium form. 4. ). Fig. 4 shows the trace of the anion-exchange HPLC purification of the 58-mer oligoribonucleotide that was initially purified trityl-on by reversed-phase HPLC (see Fig. 3). The peak was collected from 49 to 52 min. 4. Desalting by HPLC For oligonucleotides purified by anion-exchange HPLC it is necessary to remove the excess salt, buffer, and EDTA from the RNA regardless of the intended application. This is readily achieved using a desalting column such as the Hi-Prep 26/10, which is filled with Sephadex G-25.

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