DTT and Spermidine for RNA Buffers and Vaccine Production

Since Iris Biotech was founded, we have been supplying our customers in the peptide world with a long-established reducing agent: DL-Dithiothreitol “DTT” - also known as Cleland's Reagent.

We have already explained the reducing effect of DTT in previous blog posts. 

Furthermore, DTT has long been a component in a completely different area: in buffers for nucleic acid amplification, and in particular in transcription buffers for RNA production. Also present in typical transcription buffers is the triamine spermidine.

Have you ever wondered why these two reagents are used? Many suspect that DTT is to prevent oxidation. However, this is not the main task here. In transcription buffers, DTT is primarily added to inactivate any ribonucleases (RNases) that may be present.

Free RNA is considered dangerous by most organisms because it is potentially infectious. As a result, most living organisms produce and secrete ribonucleases, making these RNases ubiquitous. Thus, we have them on our skin and as a consequence on many surfaces we touch. Traces of RNases can therefore also be found in reaction vessels – even in those that we use for RNA production. DTT inactivates those RNases by reducing their surface-exposed disulfide bridges. As a result, RNases lose their active conformation and are no longer able to degrade the RNA that you want to produce in a translation reaction.

The role of spermidine is far more diverse. It was first described in 1878 as a component of semen. Due to a favorable spacing of its positively chargeable amino groups, it is able to complex the negatively charged phosphate groups of DNA and RNA and thus stabilizes these nucleic acids. In addition, it can improve the transcription efficiency of several RNA polymerases and is therefore added as a component in many buffers for RNA synthesis and RNA purification.

So far, the production of mRNA has usually only been carried out in minimal quantities. This changed dramatically with the SARS-CoV-2 pandemic and the first approval of mRNA-based vaccines. Within shortest time, RNA production scales reached previously unthinkable dimensions. Consequently, this also meant a sudden need for DTT and spermidine with a suitable specification for mRNA production and corresponding documentation for use in pharmaceutical processes. Iris Biotech contributed to making this supply possible and is proud of having served numerous vaccine production sites worldwide in the meantime.

Benefit from our DTT and spermidine in "mRNA grade" - proven hundreds of times in use for the production of vaccine mRNA!

References:

Enzymatic and Chemical-Based Methods to Inactivate Endogenous Blood Ribonucleases for Nucleic Acid Diagnostics; A. T. Bender, B. P. Sullivan, L. Lillis, J. D. Posner; J Mol Diagn 2020; 22: 1030-1040. https://doi.org/10.1016/j.jmoldx.2020.04.211

The Effects of Dithiothreitol on DNA; S. Fjelstrup, M. B. Andersen, J. Thomsen, J. Wang, M. Stougaard, F. S. Pedersen, Y. P. Ho, M. S. Hede, B. R. Knudsen; Sensors (Basel) 2017; 17. https://doi.org/10.3390/s17061201

Ueber eine neue organische Basis in thierischen Organismen; P. Schreiner; Liebigs Ann. Chem. 1878; 194: 68-84. https://doi.org/10.1002/jlac.18781940107

Studies on DNA-dependent RNA polymerase from Escherichia coli. 1. The mechanism of polyamine induced stimulation of enzyme activity; K. A. Abraham; Eur J Biochem 1968; 5: 143-6. https://doi.org/10.1111/j.1432-1033.1968.tb00348.x

Purification and Some Properties of a Soluble DNA-Dependent RNA Polymerase from Nuclei of Human Placenta; R. Mertelsmann; Eur J Biochem 1969; 9: 311-318. https://doi.org/10.1111/j.1432-1033.1969.tb00610.x

The Effect of Spermidine on the Transcription of DNA by RNA Polymerase; R. B. Tanguay; Department of Biochemistry 1971; Ph D Thesis; University of New Hampshire

The effect of spermine on transcription of mammalian chromatin by mammalian deoxyribonucleic acid-dependent ribonucleic acid polymerase; G. Moruzzi, B. Barbiroli, M. S. Moruzzi, B. Tadolini; Biochem. 1975; 146: 697-703. https://doi.org/10.1042/bj1460697

Stimulation of in vitro transcription of T4 DNA by the polyamine spermidine; D. L. Nuss, E. J. Herbst; ABB 1975; 169: 513-521. https://doi.org/10.1016/0003-9861(75)90194-0

Synthetic polyamines stimulate in vitro transcription by T7 RNA polymerase; M. Frugier, C. Florentz, M. W. Hosseini, J.-M. Lehn, R. Giegé; Nucleic Acids Res. 1994; 22: 2784-2790. https://doi.org/10.1093/nar/22.14.2784