Nuclease phosphorothioates

Figure 5.12. Chemical structure of antisense oligodeoxynucleotides (AS-ODN). Phosphorothioate and methylphosphonate AS-ODN have a sulfur atom and a methyl group respectively, substituted for a nonbridging oxygen atom to increase stability to nucleases.

Until recently, the phosphorothioate linkage had been the only modified linkage compatible with SELEX enzymes. But certain modifications at the 2 position, making RNA nuclease resistant, are accepted by polymerases. In particular, 2 -amino- and 2 -fluoro-ribopyrimidines (Figure 6.4) are substrates for the... 

Strategies to eliminate the problem of degradation by nucleases are based on chemical modification such as phosphorothioates (Kurreck et al. 2002) or meth-oxyethyl phosphorothioates (Agrawal et al. 1995). Furthermore, the co-administration of permeation enhancers such as medium chain fatty acid turned out to be a promising strategy to increase the gastrointestinal uptake (Raoofet al. 2002). 

Fig. 1 Modified nucleic acids with enhanced nuclease stability, a DNA, b phosphorothioate, c Z -OjT-C-methylene-bridged (locked) nucleic acid (LNA), d 2f-0,A -C-ethylene-bridged nucleic acid (ENA), e Z -O-methyl nucleic acid, f methylphosphonate-linked nucleic acid, g morpholino-linked nucleic acid, h peptide nucleic acid (PNA)...

The in vitro selection/amplification strategy has also been applied to modified ONs, especially where the 2 -ribose position has been changed and where phosphorothioates or other phosphate replacements have been used (Fig. 10.25, top). Several strucmres of modified ON chains that have been synthetically produced to obtain constrained sequences or sequences with higher stability to nucleases have also been reported (Fig. 10.25, bottom). Examples of biosynthetic modified ON libraries are covered in the next section. 

Monomers [180]-15a-d were successfully used for the synthesis of several stereodefined oligo(nucleoside [180]phosphorothioate)s. Their stereochemistry and isotopic enrichment were confirmed by a combined enzymatic-mass spectrometry method employing snake venom phosphodiesterase or calf spleen nuclease, and MALDI TOF mass spectrometry. 

Contrary to information that LNA oligonucleotides are resistant to digestion with nucleases [67], we have observed that diastereomer 45a of LNA dinucleoside phosphorothioate (presumably RP), obtained from/asf-44, was readily digested by snake venom phosphodiesterase. 

---