Hi everyone,
I would highly appreciate it if anyone could answer how phosphorothioates will affect DNA/RNA's synthesis and purification. If there are more than one phosphorothioates present in the DNA/RNA strand, will there be crosslinking? Can phosphorothioates form disulfide bonds? If no, what could be the major crosslinking format？
Thanks,
Yiqun
Phosphorothioates, where oxygen in the phosphate backbone is replaced by sulfur, alter the chemical properties of DNA/RNA. This modification can enhance stability against nucleases, affecting degradation susceptibility. During synthesis, phosphorothioates can be incorporated, influencing base-pairing kinetics. Purification methods may need adjustments due to altered physicochemical characteristics.
Your question is too general to be answered in a few sentences. PS do not influence the synthesis to a major extent except for two points:
1. The capping step is conducted after the sulfurization.
2. If an oligo contains both PS and PO linkages, the oxidation to the PO should be done under milder conditions to prevent desulfurization. Iodine should not exceed 0.05 M concentration. Alternatively, tBuOOH/MeCN can be used, with the contact time of 15 min.
In reversed-phase purification, 5'-DMT oligos with PS usually give more than one peak. IE HPLC, in contrast, gives a single peak. In IE, PS oligos are eluted slower than the respective PS. To elute an oligo with high percentage of PS, the use of a strong chaotropic salt (e.c. NaBr) plus 10 to 15% MeCN in buffer B is mandatory.
Formation of intra- or intermolecular disulfides during the synthesis, deprotection, or isolation is not observed. This can probably be done by a mild activation of one pf the phosphorothioates with, for instance, dipyridyl disulfide. I however think that the disulfide formed might be unstable to mild basic hydrolysis to form one PS plus one PO.
There is plenty of literature on the subject of the synthesis and purification of PS oligos. Getting familiar with the subject will save you a lot of time and grief in the lab.
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