DETECTING LOW-LEVEL SYNTHESIS IMPURITIES IN MODIFIED PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING LIQUID CHROMATOGRAPHY - HIGH RESOLUTION MASS SPECTROMETRY.

An LC-MS method based on the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS) for profiling oligonucleotides synthesis impurities is described.Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides.

Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals.

A series of antisense oligonucleotides (ASOs) containing either 2'-O-methoxyethylribose (MOE) or locked nucleic acid (LNA) modifications were designed to investigate whether LNA antisense oligonucleotides (ASOs) have the potential to improve upon MOE based ASO therapeutics. Some, but not all, LNA containing oligonucleotides increased potency for reducing target mRNA in mouse liver up to 5-fold relative to the corresponding MOE containing ASOs. However, they also showed profound hepatotoxicity as measured by serum transaminases, organ weights and body weights.

Human RNase H1 discriminates between subtle variations in the structure of the heteroduplex substrate.

In a previous study, we demonstrated that the sugar conformation and helical geometry of the heteroduplex substrate at the catalytic site of human RNase H1 directs the selective recognition of the substrate by the enzyme (J Biol Chem 279: 36317-36326, 2004). In this study, we systematically introduced 2'-methoxyethoxy (MOE) nucleotides into the antisense oligodeoxyribonucleotide (ASO) of the heteroduplex to alter the helical geometry of the substrate. The MOE substitutions at the 3' and 5' poles of the ASO resulted in fewer cleavage sites and slower cleavage rates compared with the unmodified substrates.

The positional influence of the helical geometry of the heteroduplex substrate on human RNase H1 catalysis.

In a companion study published in this issue (p. 83), we showed that chimeric substrates containing 2'-methoxyethyl (MOE) nucleotides inhibited human RNase H1 activity. In this study, we prepared chimeric substrates containing a central DNA region with flanking northern-biased MOE nucleotides hybridized to complementary RNA.

Synthesis of DNA using a new two-step cycle.

For the first time a new, two-step method is described for synthesizing deoxyribonucleic acid. The approach uses 5'-carbonate protected 2'-deoxynucleoside-3'-phosphoramidites as synthons and a peroxy anion buffer that removes the carbonate protecting group and oxidizes the internucleotide linkage. Following synthesis via this two-step cycle, oligomers are isolated by standard procedures.

Solid-phase oligodeoxynucleotide synthesis: a two-step cycle using peroxy anion deprotection.

A novel solid-phase phosphoramidite based oligodeoxynucleotide two-step synthesis method has been developed. Keys to this method are replacement of the 5'-dimethoxytrityl blocking group with an aryloxycarbonyl and the use of N-dimethoxytrityl protection for the exocyclic amines of adenine and cytosine. With these modifications, coupling of each 2'-deoxynucleoside 3'-phosphoramidite to the growing oligodeoxynucleotide on the solid support can be followed by treatment with an aqueous mixture of peroxy anions buffered at pH 9.