https://zenodo.org/record/1259717/files/article.pdf

Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach

Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models.

Publisher Summary This chapter discusses the chemical synthesis of deoxy oligonucleotides by the phosphoramidite method. It discusses the synthesis methodology; detailed protocols for preparing the silica support, the phosphoramidite, and deoxy oligonucleotides; and the purification of synthetic deoxyribo nucleic acid (DNA). The phosphite triester approach to DNA synthesis using deoxynucleoside phosphoramidite as synthons has become the method of choice for the preparation of deoxy oligonucleotides. The general synthetic strategy involves adding mononucleotides sequentially to a deoxynucleoside, attached covalently to a silica-based insoluble polymeric support. Reagents, starting materials, and side products are then removed simply by filtration. At the conclusion of the synthesis, the deoxy oligonucleotide is chemically freed of blocking groups, hydrolyzed from the support, and purified to homogeneity by either polyacrylamide gel electrophoresis (PAGE) or high-performance liquid chromatography (HPLC). These preformed synthons are especially attractive for preparing DNA on automatic or semiautomatic DNA synthesizers, or for those who plan to manually synthesize a large number of deoxy oligonucleotides.

Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method.

The intracellular distribution and metabolism of microinjected fluorescently-labeled oligonucleotides (ODNs) have been evaluated using confocal fluorescence microscopy. Fluorescent phosphodiester ODNs, microinjected into the cytoplasm of mammalian cells, rapidly accumulate within the nucleus; the fluorescence disappears with a half-life of 15-20 minutes. Microinjected fluorescent phosphorothioate ODNs remain in the nucleus for more than 24 hours. The persistence of fluorescence depends on the length of the ODN. Modification of the 3' end of phosphodiester ODNs does not significantly slow the rapid disappearance of fluorescence, although certain 3' modifications localize ODNs into the cytoplasm. Using specially designed ODNs, endonuclease activity is demonstrated to exist in the cytoplasm and nucleus. Modification of the 2' position of the ribose rings of a fluorescent phosphodiester oligodeoxynucleotide with O-methyl or O-allyl does not alter its intracellular distribution; however, the 2'-O-allyl modification stabilizes the persistence of fluorescence more than 60-fold compared to the 2'-deoxy control. Thus, the experiments indicate that somatic cells contain nucleolytic activities which degrade microinjected ODNs; however, chemical modification can dramatically circumvent this process.

Intracellular disposition and metabolism of fluorescently-labeled unmodified and modified oligonucleotides microinjected into mammalian cells.

Chemical synthesis of DNA and DNA analogs

High-performance liquid chromatographic analysis of oligodeoxyribonucleotide base composition

Novel activating and capping reagents for improved hydrogen-phosphonate DNA synthesis

Oligodeoxynucleotides 18–150 bases in length were synthesized with both 0-(2-cyanoethyl)- and 0-(methyl)-phosphoramidites. After enzymatic degradation of the purified products, base modification and composition were evaluated by HPLC. Additionally, synthesis of 5′-d[GCGCGCTT] with O-(methyl) phosphorus protection generated 3-methylthymidine when thiophenoxide was omitted from the deprotection protocol.

J. W. Efcavitch

Papers

High-performance liquid chromatographic analysis of oligodeoxyribonucleotide base composition

Novel activating and capping reagents for improved hydrogen-phosphonate DNA synthesis

Base Modification and the Phosphoramidite Approach