21 June 2022

Possibilities with RNase inhibitors

RNase inhibitors are proteins that inhibit RNases. What else is there to know? Turns out the possibilities are quite endless. Here we have put together some things that might be worth knowing when it comes to ribonuclease inhibitors.


But first, what are RNases?

RNases are enzymes that degrade RNA into smaller components. They are also part of RNA processing, turnover, and gene regulation [ref. 1]. All organisms contain RNases, which shows that they belong to a very ancient process that is vital for life [ref. 1]. In fact, RNases are so abundant in organisms, that they often create a risk of contamination for any kind of RNA related science work, like cDNA synthesis, RT-PCR, RT-qPCR etc. There are also many different types of RNases, which are divided into two big groups: endoribonucleases and exoribonucleases [ref. 1].

When working in a lab, there are usually two RNases to think about – RNase A and B. They are both endoribonucleases – enzymes that cleave RNA molecules internally [ref. 1]. RNase A is the most used RNase in research. It specifically degrades single-stranded RNA [ref. 2] and is mostly found outside the cell, being secreted by the pancreas [ref. 3]. RNase B is also very often used in research or as a target for RNase inhibitors. RNases A and B are quite similar, only differing by one residual sugar molecule, making RNase B glycosylated unlike RNase A, which is nonglycosylated [ref. 4]. Because of that RNase B is known to be more stable of the two [ref. 5].

RNases are known to ruin experiments by contaminating and destroying the RNA that’s being researched. Yet, they are still essential for life in general. But why?

RNases have a lot of different functions and roles. For example, they play key roles in the maturation of RNA molecules [ref. 1]. They are important for cell metabolism, gene expression, cell growth and differentiation [ref. 1]. They are also the first defense against RNA viruses and part of more advanced cellular immune strategies (e.g RNA interference) [ref. 1, 6]. There is more, but we are here to focus on RNase inhibitors.

Crystal structure of wild-type bovine pancreatic ribonuclease A. From the Protein Data Bank. [ref. 7]

What are RNase inhibitors?

As mentioned at the beginning, RNase inhibitors inhibit RNase’s activity. More precisely they are proteins that bind with very high affinity to corresponding RNases [ref. 8]. Since RNase inhibitors usually occur in complex with the RNases they are inhibiting, they are important in all the same processes that were mentioned above [ref. 8]. One thing, that is specifically RNase inhibitor function, and is crucial for research and diagnostics, is protecting RNA research samples from degradation by RNases [ref. 9].

What is also good to know, is that RNases and their inhibitors tend to be species or at least class specific [ref. 9, 10]. For example, mammalian RNase inhibitors can’t bind to certain types of RNases from other non-mammalian species [ref. 10]. This knowledge has been used in cancer research, where amphibian RNases were used to kill cancer cells [ref. 8, 10]. Another application for this is RNase inhibitor therapy for allergies [ref. 8]. There are many other methods as well how RNase inhibitors could be used to cure cancer and other diseases, which continues to prove how important these little molecules are [ref. 10].

Porcine ribonuclease inhibitor. From the Protein Data Bank. [ref. 11]

Which RNase inhibitor should I get?

If you are planning on doing some experimenting using RT-qPCR, RT-LAMP or perhaps COVID-19 testing, then we recommend RiboGrip™ RNase inhibitor.

RiboGrip™ is a unique recombinant chimeric RNase inhibitor protein of mammalian origin, expressed in E. coli and purified according to state-of-theart protein purification methods. It is used for protecting RNA samples from RNase degrading activity in various RNA assays, such as cDNA synthesis, RTqPCR etc. RiboGrip™ includes also a genetic modification called Stability TAG – Solis BioDyne’s proprietary and patented polypeptide stabilization technology increasing its stability at elevated temperatures [ref. 12, 13]. Therefore, the reaction set-up and shipment can be done without ice, reducing your carbon footprint and costs.

RiboGrip™ can also be used with RT-LAMP assays in which cDNA, synthesized using reverse transcriptases (i.e. FIREScript® or SOLIScript®), is further amplified at constant temperature without the need for PCR cyclers. Also, if you are looking for RNase inhibitors that can be lyophilized, then there is a non-glycerol option available as well on request. Both the RT-LAMP assays and lyophilization of detection reagents may be desired for those developing point of care (POC) devices.

References

  1. Arraiano CM, Andrade JM, Domingues S, et al. The critical role of RNA processing and degradation in the control of gene expression. FEMS Microbiol Rev. 2010;34(5):883-923.
  2. Murchison, E.P. (2013). RNAases. In S. Maloy, K. Huges (Ed.). Brenner’s Encyclopedia of Genetics (Second Edition, pp. 270). Academic Press. https://doi.org/10.1016/B978-0-12-3749840.01355-3.
  3. Gotte G, Menegazzi M. Biological activities of secretory rnases: focus on their oligomerization to design antitumor drugs. Front Immunol. 2019;10:2626.
  4. Rudd PM, Joao HC, Coghill E, et al. Glycoforms modify the dynamic stability and functional activity of an enzyme. Biochemistry. 1994;33(1):17-22.
  5. Arnold U, Schierhorn A, Ulbrich-hofmann R. Modification of the unfolding region in bovine pancreatic ribonuclease and its influence on the thermal stability and proteolytic fragmentation. Eur J Biochem. 1999;259(1-2):470-475.
  6. Il’inskaia ON, Shakh Makhmud R. Ribonucleases as antiviral agents. Mol Biol (Mosk). 2014;48(5):707-717.
  7. Chatani E, Hayashi R, Moriyama H, Ueki T. Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease A as revealed by crystallographic study of three Phe120 mutants at 1.4 A resolution. Protein Sci. 2002;11(1):72-81.
  8. Iakovlev GI, Mit’kevich VA, Makarov AA. Inhibitors of ribonucleases. Mol Biol (Mosk). 2006;40(6):962-970.
  9. Dickson KA, Haigis MC, Raines RT. Ribonuclease inhibitor: structure and function. Prog Nucleic Acid Res Mol Biol. 2005;80:349-374.
  10. Ardelt W, Shogen K, Darzynkiewicz Z. Onconase and amphinase, the antitumor ribonucleases from Rana pipiens oocytes. Curr Pharm Biotechnol. 2008;9(3):215-225.
  11. Kobe B, Deisenhofer J. Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A. J Mol Biol. 1996;264(5):1028-1043.
  12. Kahre, O. et al. and Solis BioDyne (2015), Compositions for increasing polypeptide stability and activity, and related methods, EP2501716B1
  13. Kahre, O. et al. and Solis BioDyne (2016), Compositions for increasing polypeptide stability and activity, and related methods, US9321999B2

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