Overviews of RNase and RNase Inhibitors

What is a RNase?

RNA ribonucleases (RNases) are nucleases that hydrolyze RNA, primarily by cleaving the phosphodiester bonds between nucleotides. RNAase molecules have stable structures, containing disulfide bonds, allowing them to maintain their activity even under conditions of high temperature or the presence of denaturing agents, without the need for divalent cations. RNases can be categorized into endogenous and exogenous types, where endogenous RNases may be released simultaneously upon cell rupture, making the elimination of endogenous RNases crucial during RNA extraction processes. Exogenous RNases are widely distributed in the environment, such as in air, human skin, hair, saliva, etc., and are one of the significant reasons for the easy degradation of RNA.

Cartoon representation of the NMR structure of RNase A. (D, M, Perrin, 2012)

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What are the Different Types of RNase?

Ribonuclease A

Ribonuclease A (RNase A) originates from bovine pancreas and is an endonuclease that specifically targets the 3' end of pyrimidine residues on RNA, cleaving the phosphodiester bond between cytidine or uridine and the adjacent nucleotide. The final products of the reaction are 3' pyrimidine nucleotides and oligonucleotides terminated with a 3' pyrimidine nucleotide.

Ribonuclease T1

Ribonuclease T1 (RNase T1) is derived from Aspergillus oryzae. It acts specifically on the 3' phosphate of guanosine, cleaving the phosphodiester bond between the 3' phosphate of guanosine and the 5' hydroxyl of the adjacent nucleotide. The final products of the reaction are 3' guanosine nucleotides and oligonucleotide fragments terminated with a 3' guanosine nucleotide.

Ribonuclease H

Ribonuclease H (RNase H) was first discovered in bovine thymus tissue, and its encoding gene has been cloned into Escherichia coli. It specifically degrades the RNA strand within DNA:RNA hybrid double strands, producing oligonucleotides and mononucleotides terminated with a 3' hydroxyl and 5' phosphate end. It cannot degrade single-stranded or double-stranded DNA or RNA.

Roles and Uses of RNase

Ribonuclease catalyzes the degradation of ribonucleic acid (RNA) and can now be synthesized artificially. Medicinal ointments are used topically to treat wounds and joint pain. Ribonuclease can alter host cell metabolism, inhibit viral synthesis, and in vitro, it can suppress influenza virus proliferation, and in chicken embryos, it can inhibit the formation of vaccinia and herpes viruses.

What is a RNase Inhibitor?

RNase inhibitors have a broad spectrum of inhibitory effects on RNases, including inhibition of neutral eukaryotic RNA. The effective concentration for RNase inhibitors to bind with RNases is approximately 10-14M. Furthermore, the kinetic binding of RNase inhibitors is very rapid, ensuring rapid binding with RNases and prompt inhibitory effects.

Guadinine thiocyanate (a protein denaturant): Currently considered the most effective RNase inhibitor, it not only disrupts tissues but also deactivates RNases. It can disrupt cellular structures, causing nucleic acids to dissociate from nucleoproteins, and has a strong denaturing effect on RNases.

Guanidine hydrochloride, urea (protein denaturants): Guanidine hydrochloride and urea, in high concentrations (4-8 mol/L) in aqueous solutions, can break hydrogen bonds, leading to varying degrees of protein denaturation, making them potent inhibitors of nucleases.

Phenol, chloroform (protein denaturants): These substances denature proteins, inhibiting the degradation activity of RNases. Phenol's denaturing effect on proteins is much greater than that of chloroform.

8-hydroxyquinoline (reducing agent): 8-hydroxyquinoline is a reducing agent and an incomplete inhibitor of nucleases and a weak chelating agent for metal ions. Its yellow color helps to distinguish between organic and aqueous phases. It serves several purposes: (1) reducing phenol oxidation, (2) providing color indication for easy phase separation during extraction, and (3) exerting some inhibition on RNase and DNase activity.

β-mercaptoethanol, dithiothreitol (reducing agents): These are used to disrupt disulfide bonds in RNase proteins, leading to their denaturation.

Trizol: Contains phenol, guanidine thiocyanate, 8-hydroxyquinoline, β-mercaptoethanol, etc. The main component of Trizol is phenol, which helps to lyse cells, releasing protein and nucleic acid material. Although phenol can effectively denature proteins, it cannot completely inhibit RNase activity. Therefore, Trizol also contains 8-hydroxyquinoline, guanidine thiocyanate, β-mercaptoethanol, etc., to inhibit both endogenous and exogenous RNases. 0.1% 8-hydroxyquinoline can inhibit RNase, and its combination with chloroform can enhance the inhibitory effect. Guanidine thiocyanate is a chaotropic agent, a potent protein denaturant that dissolves proteins and eliminates secondary protein structures, causing degradation of cellular structures and rapid dissociation of nucleoproteins from nucleic acids. The main function of β-mercaptoethanol is to disrupt disulfide bonds in RNase proteins.

Proteinase K: Proteinase K is a serine protease with broad cleavage activity that cleaves carboxyl-terminal peptide bonds of aliphatic and aromatic amino acids. Proteinase K degrades proteins into peptides or amino acids, inhibiting RNase activity.

SDS (anionic detergent): SDS is a highly efficient surfactant that can dissolve almost all proteins. It disrupts proteins' non-covalent bonds (hydrogen bonds and hydrophobic interactions) and binds to the hydrophobic regions of proteins, denaturing them and inhibiting RNase activity.

EDTA (metal ion chelator): Some RNases require divalent cations for their activity, so EDTA can inhibit RNase activity by chelating divalent cations.

Diethylpyrocarbonate (DEPC): DEPC is a potent but incomplete RNase inhibitor. It denatures proteins by binding to the imidazole ring of histidine residues in the active sites of RNases, thereby inhibiting enzyme activity. Its action is enhanced by heparin. It is unstable in Tris and quickly decomposes into carbon dioxide and ethanol. DEPC is sensitive to moisture, soluble in ethanol, ether, acetone, and hydrocarbons, sparingly soluble in water, and undergoes hydrolysis, producing ethanol and carbon dioxide. After adding DEPC to water, it does not immediately dissolve but forms small spherical droplets, which need to be thoroughly stirred until the droplets disappear to ensure complete mixing. The commonly used concentration is 0.1% DEPC as an RNase inhibitor. The concentration should not be too high; otherwise, the residual DEPC byproducts in the solution after sterilization may inhibit some enzyme reactions. For example, DEPC byproducts inhibit in vitro transcription reactions.

Oxygen vanadyl ribonucleoside complex (RVC): The complex formed by vanadium ions and nucleosides (obtained by mixing equimolar mixtures of four rNTPs with vanadium IV oxide using the Berger method) acts as a transitional substance when bound to RNases, almost completely inhibiting RNase activity. As an exogenous RNase inhibitor, the vanadyl complex is widely used in the separation, purification, and detection processes of various RNA samples to inhibit RNA degradation. It can also inhibit RNase activity during cell lysis and cytoplasmic fractionation using sucrose gradients.

Applications of RNase Inhibitor

Preserving RNA Integrity: In laboratory studies, the inclusion of RNAse inhibitors can prevent RNA breakdown in experimental settings, thereby safeguarding the integrity of RNA molecules. This is particularly crucial for RNA extraction, analysis, and various applications, especially in fields like transcriptomics, transcriptional regulation, and RNA sequencing where RNA isolation is necessary.

Enhancing RNA Stability: Adding RNAse inhibitors can increase the stability of RNA within cells or tissues, thwarting degradation processes. This becomes particularly significant when investigating RNA stability and functionality across diverse biological contexts.

Antiviral Strategies: RNAse inhibitors can effectively impede the replication and propagation of RNA viruses. Numerous RNA viruses, such as influenza and hepatitis B viruses, rely on RNAse for their replication. By inhibiting RNAse activity, the replication cycle of these viruses can be disrupted, offering potential therapeutic benefits in combating viral infections.

Biotechnology and Drug Discovery: RNAse inhibitors hold versatile applications in biotechnology and pharmaceutical research. For instance, in gene expression regulation and RNA interference methodologies, specific gene modulation can be achieved through RNAse activity inhibition. Moreover, these inhibitors can be instrumental in the development of novel therapeutics, particularly targeting diseases associated with RNAse dysregulation.

Reference

1. D, M, Perrin. 9.02 - Lifelike but Not Living: Selection of Synthetically Modified Bioinspired Nucleic Acids for Binding and Catalysis. Frontiers in Cardiovascular Medicine. Polymer Science: A Comprehensive Reference. 2012, 9: 3-33.