Modified nucleosides designed to function as universal nucleic acid bases will be synthesized and their suitability as constituents of oligonucleotide probes determined in physicochemical and molecular biological studies. The candidate structures include 2-carbamoyl-4-beta-D-deoxyribofuranosylimidazole, 5-carbamoyl-3-beta-D-deoxyribofuranosyl-1,2,4-triazole, and 6-amino-4-pyrimidone linked to deoxyribose via three atom spacers between C-5 and C-l'. The compounds are designed to base-pair through two hydrogen bonds to dA, dC, dG, or T. The four-fold degeneracy is obtained by constructing molecules wherein independent rotation of two bonds is sufficient to shift both base position and hydrogen bond donor and acceptor sites. The specific studies to be pursued include the following: 1) The nucleosides will be synthesized and incorporated into oligonucleotides by way of standard phosphoramidite chemistry. 2) The melting curves and thermodynamic properties of representative oligonucleotides containing the universal bases will be determined. 3) The ability of universal base modified oligomers to function in hybridization assays for the detection of low levels of mRNA will be determined. 4) Oligomers containing the universal base will also be assessed for their ability to serve as sequencing primers, as primers in the polymerase chain reaction (PCR). 5) Finally, experiments will be done to determine whether oligonucleotides which contain the modified bases can be used as templates for site-directed mutagenesis. It is clear that a universal base would have extensive and direct application to biochemistry and molecular biology. Its impact on molecular genetics, and biomedical research could be profound.
| Liu, H; Nichols, R (1994) PCR amplification using deoxyinosine to replace an entire codon and at ambiguous positions. Biotechniques 16:24-6 |
