Also called catalytic RNA, ribozymes are found in the ribosome where they join amino acids together to form protein chains. Ribozymes also play a role in other vital reactions such as RNA splicing, transfer RNA biosynthesis, and viral replication.
The first ribozyme was discovered in the early 1980s and led to researchers demonstrating that RNA functions both as a genetic material and as a biological catalyst. This contributed to the worldwide hypothesis that RNA may have played a crucial role in the evolution of self-replicating systems. This is referred to as the RNA World Hypothesis and today, many scientists believe that ribozymes are remnants of an ancient world that existed before the evolution of proteins. It is thought that RNAs used to catalyse functions such as cleavage, replication and RNA molecule ligation before proteins evolved and took over these catalytic functions, which they could perform in a more efficient and versatile way.
Ribozymes have been extensively investigated by researchers to try and determine their exact structure and function. Scientists have developed synthetic ribozymes in the laboratory that are able to catalyze their own synthesis under specific conditions. One example is the RNA polymerase ribozyme. Using mutagenesis and selection, scientists have managed to develop and improve variants of the Round-18 polymerase ribozyme from 2001. The best variant so far is called B6.61, which can add up to 20 nucleotides to a primer template over a period of 24 hours. After 24 hours, the hydrolysis of its phosphodiester bonds causes the ribozyme to decompose.
Such detailed studies of RNAs have led to rules being established regarding how they achieve target recognition and based on those rules, scientists have managed to adjust ribozymes so that they target and cleave new RNA molecule targets that would not usually undergo cleavage by ribozymes. This raises the exciting possibility that artificial ribozymes could be used as a therapeutic agents to target RNA molecules that cause diseases such as HIV. In models of such diseases, ribozymes have been successful at achieving this and a ribozyme that has been shown to target and break up the RNA that makes up the HIV virus has already been approved for testing in patients with HIV. In the future, ribozymes may also be used as therapeutic agents in the correction of genetic disorders. They could be used to eliminate abnormal proteins before they even exist by attacking and breaking up the molecules of RNA that are needed for their translation and transcription.
An association between hammerhead ribozymes and non-autonomous, long terminal repeat retrotransposons is uncovered in plants, shedding light on the biological function of genomically encoded ribozymes.
Five different self-cleavage ribozymes have been discovered since 1986: hammerhead , hairpin , hepatitis delta virus (HDV) , Varkud satellite , and glmS . More recently, four more self-cleavage ribozymes were reported in a short period of time: twister , twister sister , pistol , and hatchet . It is believed that many self-cleaving ribozymes are still working in living organisms after several billions of years .
A question of debate intrinsically connected to the different hypothesis proposed to explain the origin of catalysis of RNA-enzymes is the role assigned to the magnesium cations that have been identified in the cleavage site in some enzymes . Some authors propose that the folded structure of the RNA itself contributes more to the catalytic function and that the divalent cations would play a structural role . In contrast, other authors suggest that the catalysis in self-cleaving ribozymes is basically due to the role of the Mg2+ cations [15, 16]. It seems that hairpin, VS, and twister use a guanine as a base and an adenine as an acid, although in the later the proton donor role is played by the N3 atom and not by the N1 atom [17, 18]. In the env22 twister ribozyme, X-ray diffraction structures show that both an invariant guanosine and a Mg2+ are directly coordinated to the non-bridging phosphate oxygens at the self-cleavage site . Further theoretical studies on the twister ribozyme suggest that the general acid must be the Mg2+-bound water molecule . Other theoretical studies have been also focused on the role of the cation in hammerhead  and glmS ribozymes .
As mentioned above, a critical question of debate about the activity of the small ribozymes is the role of Mg2+ cations. The direct participation of the divalent metal ion are ambiguous . An strategy of testing the role of the Mg2+ was based on the replacement of the nonbridging oxygens of the scissile phosphate by sulfur atoms, since the S-Mg2+ interaction is much weaker than the O-Mg2+. This sulfur-containing pistol ribozyme showed a hinder activity , which was interpreted to conclude that the pistol ribozyme does not require inner-sphere coordination of divalent cation for catalysis .
In the present work, a computational study of the reactivity of the pistol ribozyme has been carried out by means of classical MD simulations with AMBER and TIP3P forcefields for modelling the ribozyme and solvent, respectively, and QM/MM hybrid calculations with the QM sub-set of atoms described at semiempirical (AM1d) and DFT (M06-2X) level of theory. Different mechanisms for the self-cleavage RNA reaction have been explored with special attention to the role of the active site Mg2+ cations. The results have been compared to those previously obtained from other ribozymes such as hammerhead ribozyme. The study was initiated with a deep insight into the possible protonation state of the titratable species in the active site by preparing different models and an analysis of the evolution of the geometries along the corresponding unconstraint MD trajectories. This is a key step for the following exploration of the mechanism because of the uncertainty derived from the previous structural studies in this ribozyme. Analysis of the MD simulations was used to test whether G40 and G32 appear in a proper position to act as base and acid, respectively, as proposed in previous studies [27, 28, 34] from analysis of X-ray structures [23, 24, 31, 32]. Our results reveal that only the model where the protonation of the nucleotide base was according to the canonical state renders reactive conformations of the active site. Moreover, it is important to note that the analysis of the evolution of the geometries along the MD trajectory shows a change of the inner sphere of the Mg2+ cation. In particular, the interaction with N7 of G33 is lost and a new interaction is established with the proRP oxygen atom of the phosphate group. This result is in contrast with recent experimental and theoretical studies [32, 35, 36] suggesting that Mg2+ is kept in the inner-sphere of N7 of G33, which means to be in the outer-sphere of the proRP oxygen atom.
5KTJ PDB structure can be downloaded from www.rcsb.org/structure/5KTJ. VMD v1.9.2 can be downloaded free of charge from www.ks.uiuc.edu/Research/vmd. NAMD v2.14 can be downloaded free of charge from www.ks.uiuc.edu/Research/namd. PROPKA3 v3.2 can be freely downloaded from github.com/jensengroup/propka. MOLDEN v5.8.1 can be downloaded under academic license from www3.cmbi.umcn.nl/molden. Antechamber is part of AmberTools20 that can be get from ambermd.org/AmberTools.php. Gaussian 09 D01 can be purchased from gaussian.com. fDYNAMO v2.2 can be freely downloaded from www.pdynamo.org/downloads
Telomerase activity is found in almost all malignant tumors . Human telomerase RNA (hTR) is associated with the activity of telomerase, immortalized cancer cells retain the highest level of hTR [4, 5]. In recent years, hammerhead ribozymes were used to inhibit the telomerase activity by targeting the template region of telomerase RNA in malignant tumors [6, 7]. Yet, there is no report about HDV ribozyme for inhibition of telomerase activity.
Ribozymes are catalytic RNA molecules which can be designed to specially cleave a target RNA sequence by incorporating the flanking sequence complementary to the target. Like other ribozymes, HDV ribozyme has this property. So it may have a potential application in gene therapy in which an engineered ribozyme is directed to inhibit gene expression by targeting a specific mRNA molecule.
As hepatocellular carcinoma is often associated with the infection of HBV and HDV, The facts that HDV ribozyme derived from HDV and that pathogen naturally infects and replicates in hepatocytes suggest that it can be used to control gene expression in human cells. The HDV ribozyme is active in vitro in the absence of any proteins, it is the only known example of a catalytic RNA associated with an animal virus. there are no known homologues of HDV ribozymes, and sequence variation of the HDV ribozymes in clinical isolates is minimal.
Targets of different Biochemical Prevention and Treatment strategies. Antibodies (Ab) or soluble receptors (Rc) can inhibit the viral entry. Antisense oligonucleotides (AS-ONs), ribozymes (Rz) or siRNA (SI) pair with their complementary target genomic DNA, RNA or mRNA. AS-ONs can block recombination, transcription, translation of the mRNA or induce its degradation by RNaseH. Rz possess catalytic activity and cleave their targets. SiRNAs (SI) induce degradation of the target mRNA via RNA-induced silencing complex (RISC).
Targeting viral mRNA is one of the most active areas of research and development. Several strategies have emerged over the years and are being tested pre-clinically and clinically. They include: antisense-oligonucleotides (AS-ONs), ribozymes, and recently, RNA interference (RNAi). All these strategies share the features of conceptual simplicity, straightforward drug design and quick route to identify drug leads. However, the challenges have been to improve potency, pharmacokinetics and, most importantly, intracellular delivery of the drug candidates. As the oldest strategy, AS-ON technology has produced to date one drug in the market place, Vitravene®. A number of clinical trials of drug candidates from these technologies are currently ongoing. 2b1af7f3a8