Michael Gerster

Sequence verification of oligodeoxynucleotides and oligophosphorothioates using electrospray ionization (tandem) mass spectrometry

The collision induced dissociations of oligodeoxynucleotides, 5′ and 3′-terminal modified oligodeoxynucleotides and oligophosphorothioates, have been studied by using pneumatically assisted electrospray ionization mass spectrometry on a triple quadrupole instrument. Fragment ions were either produced in the collision gas cell of the triple quadrupole mass spectrometer or by nozzle-skimmer fragmentation. Sequence information was obtained for oligomers up to 21 bases and for 5′- and 3′-terminal modified 15-mers. Main fragments observed for oligodeoxynucleotides resulted from a-B- and w-type cleavages, and were most prominent when the lost base was guanine or cytosine. Positions containing thymine exhibited a low signal intensity for the corresponding a-B- and w-type fragment ions. Internal fragment ions containing from one up to four bases resulted from two subsequent a-b- and w-type cleavages, and were most prominent when both bases lost were guanine and/or cytosine. For oligophosphorothioates fragments resulting from a-B-, w-, b-, c-, x- and y-type cleavages were observed. In contrast to oligodeoxynucleotides none of the fragmentation reactions seems to be favoured, resulting in more sequence specific fragment ions. Compared to oligodeoxynucleotides, internal fragment ions were less prominent for oligophosphorothioates, possibly due to the many low energy fragmentation reactions possible for the intact oligomer and each fragment ion.

Growth inhibition of pancreatic tumor cells by modified antisense oligodeoxynucleotides

Introduction: Pancreatic adenocarcinomas are largely resistant to apoptosis. More than 50% of pancreatic tumors reveal mutations in the p53 tumor suppressor gene. Methods: We investigated the growth of pancreatic tumor cells after downregulation of p53 protein expression by antisense oligodeoxynucleotides. Results: Proliferation and p53 expression of PancTu-I cells overexpressing mutant p53 protein were inhibited by antisense oligodeoxynucleotide treatment. When analyzed, two of three other pancreatic tumor cell lines with mutated p53 were also inhibited in their growth. Two of two wild-type (wt) p53 pancreatic tumor cells were not significantly influenced by p53 expression and were, only to a lesser extent, affected in their proliferation. K562 cells (lacking p53 mRNA) and normal human skin fibroblasts used as a target mismatch control showed no changes in proliferation rates with treatment. The different biological effects in the various cells were not caused by differences in the uptake of the oligodeoxynucleotides as monitored by confocal laser-scanning microscopy. Conclusions: Truncation and 5′- and 3′-lipophilic modifications of the oligodeoxynucleotides drastically enhanced the growth inhibition of PancTu-I cells, which were resistant to apoptosis-inducing agents. Furthermore, a higher sequence-specificity of the observed effects was achieved with these compounds.

OPTIMIZED LARGE SCALE SYNTHESIS OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES ON PS-PEG SUPPORTS

Sulphurization is a crucial step during synthesis of phosphorothioate oligonucleotides. Insufficient reaction leads to inhomogeneous products with phosphodiester defects and subsequently to destabilization of the oligomers in biological media. To achieve a maximum extent of sulphur incorporation, various sulphurizing agents have been investigated. Solely, the use of Beaucage reagent provided satisfactory results on PS-PEG supports. Based on our investigations in small scale synthesis (1 μmol) with continuous-flow technique, upscaling to the 0.1-0.25 mmolar range has been achieved using a peptide synthesizer. The syntheses were performed in batch mode with standard phosphoramidite chemistry. Additionally, large scale synthesis of a phosphodiester oligonucleotide has been carried out on PS-PEG with optimized protocols and compared to small scale synthesis on different supports. Products were analysed by 31P NMR, capillary gel electrophoresis and electrospray mass spectrometry. An extent of sulphurization of 99% and coupling effiencies of more than 99% were obtained and the products proved to have similar purity compared to small scale syntheses on CPG