Studies on phosphate ester cleavage and development of oligonucleotide based artificial nucleases (OBAN’s)

Abstract

Five different Oligonucleotide Based Artificial Nuclease (OBAN) systems have been synthesized. OBAN's may be regarded as a development of traditional antisense methodology where inhibition of gene expression can be achieved by hybridizing a synthetic oligonucleotide to natural mRNA and thus inhibiting further translation into protein products. The OBAN's have a hydrolytic transesterification agent covalently attached via a linker to the oligonucleotide scaffold. In contrast to antisense methodology cleavage of substrate mRNA can then be obtained without the assistance of cellular enzymes like RNase H.

Efficient synthetic methodologies for the synthesis of the OBAN's are presented. A highly convergent approach was used for the synthesis of the OBAN's were the catalytic neocuproine unit was introduced in aqueous buffer to the fully deprotected oligonucleotide. Isolated yields for the conjugation step were as high as 80% after purification on RP-HPLC. The substrates for the five Zn (II) dependent 11 or 12-mer OBANs were RNA sequences that upon association with the OBAN's formed bulged RNA structures. By changing the number of nucleosides in the bulged out region, structures having bulges varying from 0-5 nucleosides in the bulge were obtained. Degradation of these structures were studied to allow direct comparisons between different append points, directionalities and length of the linkers. The proximity factor is one of the most important design factors in construction of an efficient system. This was shown both in comparisons of the different OBANs and in the preference for certain bulge sizes for each OBAN. The top-performing OBAN, called OBAN 1 in this thesis, was shown to degrade a 4-nt bulge in a catalytic fashion. Cleavage was also demonstrated at Zn (II) concentrations that are present in human serum.

The acidity of the secondary hydroxyls of ATP, deoxy ATP and three 2' modified analogues of ATP was determined in aqueous buffers. In addition, the pKa values for secondary hydroxyls of adenosine, 2' and 3'-O-methyl adenosine in water, methanol and DMSO were determined. The results suggested that a hydrogen bond between the 2'-hydroxyl and a 3'-oxyanion, if present, is virtually energy neutral. These studies provide data useful in mechanistic studies of phosphate esters and for design of catalysts for cleavage of phosphate esters.

Phosphate ester cleavage was also studied in RNA model compounds. Several Furanosides 5(diphenylphosphate)'s, including a 2-deoxy-2-aminoderivative, were synthesized and the transesterification where the oxygen displaces a phenyl group was studied in presence of several different metal ions.