What is a phosphorothioate linkage?

Phosphorothioate Bond The phosphorothioate (PS) bond substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of an oligo. This modification renders the internucleotide linkage resistant to nuclease degradation.

What is phosphorothioate used for?

Phosphorothioate oligonucleotides are indispensable tools for probing nucleic acid structure and function and for the design of antisense therapeutics. Many applications involving phosphorothioates require site- and stereospecific substitution of individual pro-RP or pro-SP nonbridging oxygens.

What is phosphorothioate oligonucleotides?

Phosphorothioate oligonucleotides downregulate gene expression by hybridizing to a target mRNA, which in turn either inhibits mRNA maturation, enables RNase H-mediated degradation of the transcript, or blocks translation3.

How do you make phosphorothioate?

A phosphorothioate bond is produced by using Beaucage reagent to add a sulfer to the phosphate. Once either the sulfer or the oxygen has been attached to the phosphate, the bond is stabilized and will not be affected by the subsequent cycles of chemistry.

What is PS DNA?

Phosphorothioate deoxyribonucleotides (PS-DNA) are among the most widely used antisense inhibitors. PS-DNA exhibits desirable properties such as enhanced nuclease resistance, improved bioavailability, and the ability to induce RNase H mediated degradation of target RNA.

How do Morpholinos work?

Function. Morpholinos do not trigger the degradation of their target RNA molecules, unlike many antisense structural types (e.g., phosphorothioates, siRNA). Instead, Morpholinos act by “steric blocking”, binding to a target sequence within an RNA, inhibiting molecules that might otherwise interact with the RNA.

What are antisense Morpholinos?

Morpholino oligos are advanced tools for blocking sites on RNA to obstruct cellular processes. A Morpholino oligo specifically binds to its selected target site to block access of cell components to that target site.

How does the phosphorothioate modification change the phosphate linkage?

On the right, we can see the phosphorothioate linkage in which a sulfur atom replaces one of the oxygens in the phosphate linkage between bases in the oligo. The phosphorothioate bond oligo modification alters the phosphate linkage by replacing one of the non-bridging oxygens with a sulfur atom.

How is phosphorothioate used as a hydrolysis primer?

The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization The effect of phosphorothioate bonds on the hydrolytic activity of the 5′–>3′ double-strand-specific T7 gene 6 exonuclease was studied.

How is phosphorothioate primer used in single stranded PCR?

The phosphorothioated strand is protected from the action of this enzyme, whereas the opposite strand is hydrolyzed. When the phosphorothioated PCR primer is 5′ biotinylated, the single-stranded PCR product can be easily detected colorimetrically after hybridization to an oligonucleotide probe immobilized on a microtiter plate.

How does the phosphorothioate bond protect the internucleotide?

The phosphorothioate (PS) bond substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of an oligo. This modification renders the internucleotide linkage resistant to nuclease degradation.