Modified oligonucleotides as a tool for DNA sequencing, fingerprinting and mapping
The synthesis of modified oligonucleotides bearing 5-methylcytosine and 2aminoadenine instead of natural counterparts, which are characterised by enhanced strength of complementary DNA binding (strong binding oligonucleotides, SBO) has been developed. Advantages at SBO applications are described as hybridisation probes, probes for physical mapping, primers for DNA sequence and PCR amplification. The progress, achieved over the last 15 years in the field of chemistry of oligonucleotide synthesis, resulted in appearance of great variety of techniques using the synthetic oligonucleotides in different areas of molecular biology. All the techniques depend on formation of duplexes "oligonucleotide-complementary DNA', the stability of which has a considerable effect on efficiency of such methods. Stability of the complementary complexes could be increased as a result of modification of the heterocyclic bases by means of changing cytosine and adenine by 5-methylcytosine and 2-aminoadenine (2,6diaminopurine) respectively (1). It is known that stability of diaminopurine-thymidine pair becomes approximately equal that of the G-C pair due to appearance of new hydrogen bond (2). The increase of stability in case of 5-substituted pyrimidines is explained by hydrophobic interaction of methyl or others hydrophobic groups (bromine, propinyl) in the large groove of DNA helix (3). Fig. 1 represents the schemes of synthesis of modified amidite components for oligonucleotide synthesis: a) initial 5-DMTrT is converted to 5-methyl-5'-DMTr-cytidine by standard procedure via 4substituted triazolide intermediate. The further blocking of aminogroup by benzoyl chloride and phosphitilation of 3'-hydroxy group by p-cyanoethyl diisopropylamino phosphochloridite result to 5-MeC amidite. b) synthesis of 2,6-diaminopurine monomer appeared to be more complicated and time consuming. The yield on the stage of dG conversion to diaminopurine does not exceed 50%, and there are some problems with blocking both aminogroups due to high acid lability of this nucleoside. The best result was achieved with dimethylacetamidine protection group. The structure of both monomers was confirmed by NMR spectroscopy. Solid phase oligonucleotide synthesis was carried out by standard amidite technique using the ASM-102 automatic synthesizer