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Duplex Stability and Nuclease Resistance

Modifications in Detail

The driving force for the search for novel chemical modification groups compatible with Watson-Crick hybridization of oligonucleotide was based on the observation of the short stability of naturally occurring oligonucleotides with phosphodiester bonds. Oligonucleotides with natural phosphodiester bonds are highly susceptible to rapid degradation by cellular nucleases. Cellular nucleases have endonuclease activity as well such that 3 and 5 end caps are not sufficient to prevent from degradation.

Modifications of the phosphodiester bond by replacing one of the non-bridging oxygen by sulfur imparts resistance to nuclease degradation, but in general hybridize to the target sequences with lesser affinity than the phosphodiester counter part.

The sulfur-substituted oligonucleotides have a phosphorothioate linkage and are termed as phosphorothioates or simply as S-oligo. Phosphorothioate oligos are synthesized by Gene Link using the Beaucage sulfurizing reagent. The sulfurization reaction is rapid and is performed on automated DNA synthesizers yielding greater than 96% phosphorothioate linkages; the remainders are phosphodiester linkages. Custom phosphorothioate oligonucleotides synthesized by Gene Link can be specified to have all the diester bonds substituted or only some selected diester linkages depending upon the researchers experimental requirement. Substitution of all diester linkage is recommended to provide greater nuclease resistance.

Phosphodiester Linkage Phosphorothioate Linkage (PS)
Chimeric Phosphorothioate Linkages
(DNA,RNA, LNA, 2O Me and other bases)
RNA phosphorothioate Linkage
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