The effects of modified nucleosides on DNA duplex and triplex stability

To date, the single most effective method of improving base pairing affinity and binding selectivity of PCR primers, fluorescent probes and triplex forming oligonucleotides (TFO) whilst destabilising mismatch base pairs has been the incorporation of modified nucleoside analogues into these oligonucleotide structures. As a consequence, significant improvements have been made in the areas of human identity testing, forensic science analysis, pharmacogenetics/pharmacogenomics and anti-gene therapy. In an effort to improve the stability of these DNA duplexes and DNA triplexes further, we have synthesised and incorporated a series of cytosine, 7-deaza adenine, thymine and 3H-furo-[2, 3-d] pyrimidin-2-one base analogues. By using a combination of UV melting analysis and fluorescence melting experiments, we have demonstrated that each of the base analogues gives a significantly higher base pairing affinity and binding selectivity when compared to their corresponding natural base. In addition, we have also incorporated these base analogues into PCR primers (7-deaza adenine) and fluorescent probe sequences (cytosine, 7-deaza adenine, thymine and 3H-furo-[2, 3-d] pyrimidin-2-one). Results fro the PCR experiments show that the 7-deaza adenine base analogue does not adversely affect the functioning of Taq polymerase during amplification and therefore at the very last behaves similarly to adenine with a PCR primer sequence. In addition, all of the fluorescently labelled base analogues (cytosine, 7-deaza adenine, thymine and 3H-furo-[2,3-d] pyrimidine-2-one) show a significantly higher level of base pairing affinity and binding selectivity for complementary target sequence over a mismatched sequence.