Solid-phase DNA syntheses

This section examines the synthesis of nucleosides that contain seven-membered sugar analogues in place of the deoxyribose component. Nucleosides from the last group have been further incorporated into ONs via solid-phase DNA synthesis. A physical and biochemical investigation of the oligomers thus prepared continues in the next section. The study under review culminated in the assessment of the ability of the oligomers to complex with single-stranded RNA and for the heteroduplexes so formed to serve as substrates of RNAseH. 

The phosphoramidite of 8-Ph-dG has been prepared and used to site-specifically incorporate it into oligonucleotides using solid-phase DNA synthesis. In the 12-mer 5 -d(GCGCCXGCGGTG), where X = 8-Ph-dG, the adduct behaved very similar to 8-Me-dG with of the duplex being 5.6°C lower than the normal dG dC base pair, while the of 8-Ph-dG dG was 2.1°C higher than that of the mismatched dG dC base pair. This data suggested that, similar to 8-Me-dG, 8-Ph-dG exists primarily in the anti conformation when paired with dC, while the syn conformation may pair with dGMP. ... 

Fluorous reverse phase silica gel (FRPSG) has been used in the purification of synthetic DNA fragments.In solid phase DNA synthesis, truncated sequences are often separated from the desired product after deprotection using HPLC or electrophoresis. In order to perform, parallel syntheses and separations of nucleotides the trityl-on purification procedure was developed, in which a lipophilic support material is used to separate the desired and undesired product, followed by deprotection. If the protecting group is labelled with a fluorous group, fiuorous-fiuorous interactions between the FRPSG and the protected nucleotide can be used to aid separation of the aqueous mixture. 

All these modified purine nucleosides have the advantage that they are readily converted to fluorescent building blocks for solid-phase DNA synthesis or to 5 -triphosphates for enzymatic DNA synthesis. Such compounds are useful for single DNA molecule detection in solution by laser-induced fluorescence as well as for single DNA molecule sequencing. 

Lyttle MH, Hudson D, Cook RM. A new universal linker for solid-phase DNA synthesis. Nucleic Acids Res 24 2793-2798, 1996. 

Figure 2.5 Solid Phase DNA Synthesis. 5 -dimethoxytrityl (DMT)-deprotection of resin bound 3 -terminal deoxynucLeoside residue is effected with trichloroacetic acid (TCA) (mechanism shown) Thereafter the first coupling reaction is enabled by phosphoamidite activation with tetrazole (mechanism shown) followed by oxidation of the newly formed diester linkage to a phosphodiester link. The process of 5 -DMTr deprotection, phosphoramidite coupling and then diester oxidation, continues for as many times as required (n-times), prior to global deprotection and resin removal under basic conditions.

Figure 2.5 Solid Phase DNA Synthesis Cycle (Contd.). Most frequently used base protecting groups are shown Bz Af-6 benzoyl (adenine), W-4 benzoyl (cytosine) N-2 isobutyroyl (guanine). All are base sensitive. DNA chain is built up from 3 to 5 on controlled-pore glass (CPG) bead solid support. Post global deprotection and resin removal, the desired product oligo-/polydeoxynucleotide is then separated initially by precipitation by means of an agent such as ethanol and purified finally by reversed phase liquid chromatography, or ion exchange chromatography as appropriate (see later in Chapter 2).

Figure 2.6 DNA Phosphoramidites. The four main phosphoramidite "building blocks" used in solid phase DNA synthesis are illustrated with base protecting groups where appropriate.

Figure 2.11 Fluorescent purines. Two very useful non-invasive DNA (base fluorophores that can be included by solid phase DNA synthesis are 2-aminopurine (left) and 3-methyl-isoxanthopterin (right). The fluorescence quantum yield depends on the base stacking, and both bases are useful probes of conformational change/state in DNA.

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