= Phosphorothioate substitution
A \b[phosphorothioate substitution] replaces an <oxygen> of a phosphate group with <sulfur>. In a <nucleic acid> backbone, the usual experimental substitution replaces a non-bridging oxygen, while retaining the oxygen bridges connecting neighboring <nucleotides>. It changes backbone chemistry without deleting a nucleotide or changing its <nucleobase> sequence. The altered group is a phosphate analogue rather than a <disulfide bond>.
The two non-bridging positions matter. For a phosphate joining different nucleotide environments, replacing one oxygen or the other can give different configurations at <phosphorus>. Such <phosphorothioate diastereomers> can interact differently with an RNA-folding environment or a metal ion. The 2013 <U6 metal rescue experiment> tested both $R_P$ and $S_P$ substitutions at selected sites, rather than treating every sulfur replacement as equivalent.<RNA catalyses nuclear pre-mRNA splicing by Sebastian M. Fica and others>[pp.229–230, Figure2a and site-specific substitution comparisons.]{ref}
An original counting example shows the extra information required. At three independently stereogenic, substituted phosphates, two possible configurations at each site give $2^3=8$ configuration combinations. All eight can have the same nucleotide sequence and elemental formula. Measuring <molecular mass> alone therefore cannot tell which configuration combination was supplied. If every oxygen-to-sulfur replacement is made once at a selected site, each adds approximately $32-16=16$ <unified atomic mass units>; three add about $48$, regardless of those configuration choices. These rounded atomic masses are illustrative, not exact isotope masses.
Replacing oxygen with sulfur also changes metal–ligand interactions. A loss of RNA activity in <magnesium> followed by preferential rescue with a different metal can test whether the original oxygen helped position a catalytic ion. It can also reflect altered folding or assembly, so the comparison needs controls. The substitution is a targeted chemical perturbation whose interpretation depends on position, configuration and the reaction being measured.
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