The synthesis and study of modified oligonucleotides

• Main Author: Lough, David M.
• Published: University of Edinburgh 1996
• Subjects: 547.7
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.654047

2-Aminoadenine or 2,6-diaminopurine (D), is an adenine analogue where three hydrogen bonds are formed in a base pair with thymine, instead of only two hydrogen bonds in the A.T. base pair. Hence the thermodynamic stability of duplexes containing D.T base pairs should be increased relative to those containing A.T. More subtle interactions affect the structural transitions of duplexes containing diaminopurine. Previous attempts at incorporating diaminopurine into DNA during routine oligonucleotide synthesis were hindered due to acid catalysed depurination (dependent on N<SUP>6</SUP>-amino protection) and very poor lability of the N<SUP>2</SUP>-amino protecting group during ammonia deprotection. An improved N<SUP>2</SUP>-amino and N<SUP>6</SUP>-amino protection strategy was developed for the synthesis of the diaminopurine monomer. This monomer was used successfully during routine machine synthesis to make diaminopurine-containing oligonucleotides. Derivatised solid support was synthesised to allow 3'-incorporation of diaminopurine. A novel synthetic route to the free diaminopurine nucleoside was also developed. The ability of nucleic acids to form very stable duplexes has many important applications in diagnostic and therapeutic molecular biology. The hybridisation of DNA probes or PCR primers to their targets may be increased, and hence the specificity; or allow shorter probes/primers to be employed. The same strategy may be applied to antisense therapy, or DNA sequencing primers. In order to predict the physical properties of such duplexes, the thermodynamic stability (UV melting) of duplexes containing diaminopurine was determined, using oligonucleotides with all combinations of nearest neighbours. This data revealed that when diaminopurine replaced adenine, stability of the duplex was increased, with the exception of the TD/AT nearest neighbour interaction. This showed that the additional hydrogen bond does not always increase stability - more complex interactions are responsible for overall duplex stability.