The Use Of RNA As A Therapeutic Tool Biology Essay


Ribonucleic acid is a type of nucleic acid that is different from DNA as it contains ribose instead of deoxyribose in its structure. RNA comes in different forms, such as tRNA, rRNA, snRNA which are used directly in biological reactions unlike RNA which have to be translated to make proteins. (Clark and Pazdernik, 2009) RNA is capable to be used as a tool for various therapeutic applications from virus infection to cancer treatment. Ribozyme, a large-sub unit rRNA, can catalyse enzymatic reactions and are the most often used RNA-based therapeutic tool to knockdown gene expression and repress different types of infections. (Mulhbacher et al., 2010) There are different types of ribozymes, which can be classified by either size or catalytic properties. Large ribozymes like the Group I and Group II intron ribozyme have ability to self-splice, by cleavage and ligation of phosphodiester bond. RNase P is a large ribozyme that is able to cleave a phosphodiester bond of a variety of cellular tRNA precursors. Small ribozymes like the Hammerhead and Hairpin ribozyme are able to undergo self-cleavage of a specific phosphodiester bond.

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In this review, we will discuss about how to use ribozymes as a therapeutic tool in terms of medical and biotechnological purposes.

Hammerhead and hairpin ribozymes are small ribozymes of about 200 to 500 nucleotides long and are found in RNA satellite of pathogenic plant viruses. Hammerhead is the smallest ribozyme capable of catalyzing a self-cleavage reactions of specific phosphodiester bond (Watson, 2008). It is a small catalytic RNA that is involved in the cloning of some viroids and satellite RNAs. In the process of replicating viroids, the host cell RNA polymerase replicates the positive strand of the RNA, which produces a long continuous, same sequenced RNA molecule of negative-strand genome. The hammerhead motif cuts the RNA strand into individual pieces by cleaving the ribose phosphate backbone of RNA first, followed by ligation of the individual pieces into circular genomes (Watson, 2008). Hairpin ribozyme is found in the tobacco ring spot virus and functions identical to the hammerhead ribozyme in vivo. The addition of the hammerhead or hairpin ribozyme can aid in the stability of the structure of the antisense oligonucleotide as they are not broken down.

Analysis of ribozyme activity

Ribozyme activity can be found by detecting the cleavage product in the incubation of the ribozyme and its substrate in vitro during the gel electrophoresis analysis (Phylactou et al., 1998). It is more difficult to learn about ribozyme activity in cells as it requires the ribozyme to be inserted into the cell firstly. This can be done through three methods; by transfection with plasmid constructs designed to express the ribozyme RNA, delivery of pre-formed ribozymes by microinjection or by cationic liposomes, by infecting a cell with a retroviral vector bearing the ribozyme. Secondly, ribozyme creates cleavage products that cannot be recognized within the cells. This could be due to the intracellular ribonucleases activitiy that prevents the presence of cleaved substrate RNA and will denature them.

Ribozymes used in real life

Trans-cleaving ribozymes are the pioneer batch of ribozymes being used in phase I and II clinical trials on people who are diagnosed with cancer or infectious diseases (Tedeschi et al., 2009). These ribozymes are inserted into the patients by two methods; direct injection of a synthetic ribozyme and gene therapy methods.

Gene therapy methods to insert ribozymes

Human immunodeficiency virus (HIV) is one the targeted virus that is undergoing trials by using gene therapy to develop a ribozyme-based treatment. It can be done through the use of retroviral vectors to bring in the expression cassettes for anti-HIV ribozymes into CD4+ lymphocytes or CD34+ haematopoietic precursors ex vivo that have been extracted from HIV victims (Jose, 2002). The modified cells are then inserted into the affected person and the life cycle and fitting of the cells in the new environment is monitored closely. The results of the studies showed that both cells and the human host respond wells together. The findings from the results also suggest that the anti-HIV ribozyme-containing cells may have a upper hand in surviving in the HIV-affected victims compared to cells that are transduced using a control vector (Jose, 2002).

In using ribozyme-based antiviral methods, HIV-1 recognizes CD4 as a primary receptor and uses CXCR4 or CCR5 as co-receptor to penetrate into the cell (Berger et al., 1998). CCR5 presence is not as crucial as CD4 and CXCR4 in the cell and studies have shown that people who do not express CCR5 are resistant to HIV-1(Sheppard et al., 2002). Nazari et al. tried to downregulate CCR5 using a multimeric hammerhead ribozyme RZ1â€"7, which targets seven specialised sites within the human CCR5 mRNA (Nazari et al., 2008). His efforts proved to be useful as the ribozyme turned out to be more effective than a monomeric version of ribozyme (Nazari et al., 2008). The multimeric hammerhead ribozyme RZ1â€"7 almost fully prevented the entry of HIV-1 in previously transducted cells after being infected with R5-tropic HIV-1 (Nazari et al., 2008). It is thus a good therapeutic tool for use in HIV-1 gene therapy.

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Direct injection of synthetic ribozymes

Three types of nuclease-resistant synthetic ribozymes are being looked at in trials which use hammerhead ribozyme derivative that contains certain changes that strongly increases the ribozyme's stability in biofluids (Jose, 2002). These three synthetic ribozymes have display favourable results in cell and animal experiments where the ribozymes target RNA which has expression that is associated with the induction or progression of cancer (Pavco et al., 2000). The ribozyme that encodes the high affinity receptor for the angiogenic protein vascular endothelial growth factor (VEGF) was found to be well received and accepted by the human body with the plasma levels maintaining at appropriate levels for long hours after the subcutaneous injection (Usman and Blatt, 2000). The other two ribozyme targets the mRNA of human epidermal growth factor receptor type 2, a substance which is overly expressed in many types of cancer (Jose, 2002) and survivin, a well-known anti-apoptotic factor also commonly over expressed in all types of cancers (Mulhbacher et al., 2010). Due to the over expression, they are used as biomarkers for cancer prognosis and diagnosis. Survivin has even been choosen as a target for cancer internvention by using four hammerhead ribozyme adenoviruses to target single-strand regions of survivin mRNA (Fei et al., 2008). With three hammerhead ribozyme, the expression combined was enough to significantly reduce the surviving expression and suppress it to give the best anticancer effect. This suppression of the survivin expression will finally cause cell death via the caspase-3-dependent apoptotic pathway (Mulhbacher et al., 2010).


In conclusion, the use of ribozymes as a therapeutic tool is evident in many fields, whether through gene therapy or direct injection of the various ribozymes. It can help to suppress gene expression of human diseases and provide new solutions to deal with the diseases. The more in depth knowledge of the ribozymes can help researchers have a greater control of the use of ribozyme gene therapy in each specific disease.