DL-Dithiothreitol is a potent reducing agent widely exploited in molecular biology as an enzyme stabilizing agent and can be found in the supplied reaction buffers of many commercially available DNA modifying enzymes as well as in their storage buffers. The main role of DL-Dithiothreitol in molecular biological assays is to keep proteins in a reduced state. Thiol containing compounds have, however, also been shown to be very effective at protecting DNA from irradiative damage, which is thought to be due to their ability to scavenge oxygen and nitrogen radicals.

Synthesis of DL-Dithiothreitol

Add the second solid to 200ml tetrahydrofuran, control the reaction temperature to 0°C, Add 5g of lithium aluminum hydride in batches. After the addition of lithium aluminum hydride, Continue to stir at a temperature of 0°C for 2h, and then heat to 25°C, Stir for 16h, quench the reaction with dilute hydrochloric acid solution; Then add 25ml water/50ml ethyl acetate for extraction, Removing the solvent to obtain a first organic layer and a first inorganic layer; Then use 50ml ethyl acetate to extract the first inorganic layer, Remove the solvent to obtain a second organic layer and a second inorganic layer, Combining the first organic layer and the second organic layer to obtain a total organic layer; The obtained total organic layer is subjected to vacuum distillation, 8.6 g of DL-Dithiothreitol was obtained, the purity was 99%, and the yield was about 79%.[1]

The Effects of Dithiothreitol on DNA

In recent years, game changing technical advancements within the field of biosensors have enabled researchers to measure the activity of DNA modifying enzymes with ultra-high sensitivity and to detect even a single DNA modification event. With the emergence of these tools for studying rare events, the importance of unexpected and infrequent side reactions mediated by additives such as DL-Dithiothreitol and not the main reactants, such as the DNA modifying enzyme itself, becomes increasingly important.

Ironically, in addition to its role as a DNA protective radical scavenger, DL-Dithiothreitol is also a potent inducer of DNA damage since, at certain concentrations, thiols in general have the ability to produce oxidative species, such as the hydroxyl radical, which has been shown to induce DNA breaks as well as other kinds of damage on DNA molecules. In agreement with this finding, thiols have been linked to chromosome damage and apoptosis in cells. Thiol induced generation of hydroxyl radicals is believed to be due to thiols boosting a Cu2+ catalyzed mechanism analogous to the oxygen radical generating reaction known as the Haber–Weiss reaction. By the proposed mechanism, Cu2+ catalyzes a reaction where thiols are oxidized by molecular oxygen which, as a result, is reduced to O2?. By reaction with H+, O2? is subsequently converted into the highly reactive hydroxyl radical in a reaction including an H2O2 intermediate. Cu2+/thiol induced DNA damage has been shown for both monothiols and dithiols but, in the present study, the focus is on the dithiol DL-Dithiothreitol due to its extensive use in DNA based studies.[2]

Motivated by the emergence of single molecule detection methods and the presence of DL-Dithiothreitol in virtually all traditionally used DNA modification protocols, we set off to elucidate the effect of DL-Dithiothreitol on DNA and thereby its influence on the results of highly sensitive DNA based assays. Examples of such assays include polymerase-based amplifying protocols such as PCR and the Rolling Circle Amplification (RCA) method, which has been developed into a single molecule detection scheme by combining it with fluorescence labelling. Surprisingly, we found that DL-Dithiothreitol is able to introduce single stranded nicks in covalently closed double stranded DNA circles even without any addition of catalyst. These DL-Dithiothreitol generated nicks were shown to be able to function as unintended starting points for RCA which forms the basis of many modern ultrasensitive assays. Furthermore, DL-Dithiothreitol was able to immobilize DNA to NHS-ester coated microscopy slides used for single molecule studies of DNA modifications. The consequence of these unexpected side-effects of DL-Dithiothreitol was highlighted by its ability to increase the background in a single molecule detection assay using a DNA based sensor system developed to measure retroviral integrase (IN) activity.[3]

References

[1] SUZHOU YACOO SCIENCE - CN112028799, 2020, A

[2] Stougaard M., Lohmann J.S., Mancino A., Celik S., Andersen F.F., Koch J., Knudsen B.R. Single-molecule detection of human topoisomerase I cleavage-ligation activ

[3] Flusberg B.A., Webster D.R., Lee J.H., Travers K.J., Olivares E.C., Clark T.A., Korlach J., Turner S.W. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat. Methods. 2010;7:461–465.