Depurination As a Yield Decreasing Mechanism in Oligodeoxynucleotide Synthesis

Abstract

The synthesis in our laboratory of very long (80–106 bases) oligodeoxynucleotides using the phosphite method of Matteucci and Caruthers in an automated DNA synthesizer indicates the true efficiency of this multiple step process. The coupling efficiency as measured by trityl cation release i s 99.5–99.8%. However the yield obtained is still below the theoretical yield calculated from the above efficiency. We have attempted t o elucidate yield decreasing mechanisms in long ol igodeoxynucleotide synthesis. Crude reaction mixtures of phosphate-deblocked oligonucleotides which had no 5′-trityl group and which were examined immediately after cleavage from the support show one major species by high resolution PAGE. Harsher hydrolysis generates a new pattern of products superimposed upon the initial ladder representing cleavage at purine residues internal to the full length oligonucleotide. In many cases the cleavage ladder is significantly more intense than the coupling ladder. Hydrolysis a t apurinic sites in ammonium hydroxide at 55° C is apparently quantitative. The implications of this study are that internal depurination does not interfere with chain propagation but directly reduces the isolated yield. The efficiency of the phosphite coupling, oxidation, demethylation, and base deprotection are not the yield limiting steps. Synthesis of even longer fragments may be achievable with a reduction in the depurination o f the propagating chain. More significantly, compounds isolated after proper hydrolysis will not contain any apurinic sites making these compounds suitable for biological use.