Ribozyme assay

Figure 8.23 Gel assay. Ribozyme assay system. One strand of the ribozyme acts as the biocatalyst E and the other strand acts as the substrate S. S is specifically cleaved at the indicated site generating two product oligonucleotides PI and P2. The conversion of S into larger molecular weight product PI can be followed by gel electrophoresis with time.

Hampel and Burke observed that protection of hammerhead backbone sites in Mg + solutions required assembly of the full ribozyme-substrate complex. In other words, testing of ribozyme or substrate separately in the hydroxyl footprinting assay showed essentially complete hydrolysis of all nucleotides (Figure 2B of reference 56). In contrast, the fully assembled ribozyme-substrate complex showed protection of nucleotides structurally near the densely packed three-helix junction of hammerhead constructs HH16, HHal, and RNA 6. Two of the ribozyme group of protected nucleotides (Gs, Ae) are part of the conserved uridine U-turn seen in all known hammerhead constructs. (See Figures 6.10,6.11, and 6.12.) The footprinting results are collected in Table 6.5. 

For example, misfolded forms of the Tetrahymena ribozyme refold very slowly at 4 °C, and are easily separated from the native form (Pan and Woodson, 1998). However, if the ribozyme is first incubated in another ion such as Na+ that allows the RNA to come close to the native structure, these native-like intermediates are captured as the native form when the RNA encounters Mg2+ in the gel running buffer (Figure 9.3A) (Heilman-Miller et al, 2001). Similarly, the Azoarcus ribozyme rapidly forms nativelike, compact intermediates in Mg2+ concentrations below that required for catalytic activity (Rangan et al, 2003). These intermediates also appear in the folded state when assayed by native PAGE. 

Worldwide on-going work in the field of nucleic acid-based gene therapy targets the question of the delivery, transport, and in-vivo activity of nucleic acid drugs. The results obtained from these studies will be taken into consideration for the in-vivo design of twin ribozymes after the in-vitro assay has proven successful. 

Although ribozymes have been shown to work in vitro in cellular assays, there are few reports demonstrating their efficacy in vivo. One such success utilized the rabbit model of IL-l-induced arthritis. In this model, IL-1 is believed to generate the formation of a proteinase mediator called stromelysin, which participates in the inflammatory condition. The intra-articular administration of ribozymes directed against stromelysin mRNA has been reported to produce a reduction in the message for this mediator in synovial fluid. 

In vitro kinetic assays are an important tool to study the catalytic mechanisms of ribozymes. Intron mutant and deletion constructs have been used in kinetic assays to study the role of particular nucleotides and to distinguish whether a mutation causes a defect in chemistry or binding of exonic substrate or intronic components. 

As with DNA aptamers, there is a trend towards applying RNA aptamers to a particular application. Allosteric ribozyme sensors have been developed which are specific for caffeine and aspartame. " Using a fluorescence-based assay, caffeine or aspartame may be detected in solution over a 0.5 5mM concentration range. Aptamers designed to malachite green (151) or other triphenylmethane dyes have been developed that enhance the fluorescence of the dye up to 2300-fold. " A further fluorescence-based assay has been... 

Brown Augsburger, R, Yue, X.M., Lockridge, J.A., McSwiggen, J. A., Kamboj,D.,and Hillgren, K.M. (2004) Development and validation of a sensitive, specific, and rapid hybridization ELISA assay for determination of concentrations of a ribozyme in biological matrices. Journal of Pharmaceutical and Biomedical Analysis, 34, 129 139. 

The concept used in the endonuclease assay was the dimination of RET by enzymatic breakage of a covalently linked D-A pair. This concept has been applied in avariety of other circumstances, including assay of an HIV-1 endonuclease, which is involved in integrating the HIV into the host DNA. RET has also been used to measure the activity of an HIV protease, proteolytic cleavage of linked derivatives of GEP," and the activity of ribozymes in cleaving RNA and as the basis of a general assay for DNA cleavage. ... 

In vitro selection methods permit the generation of ribozymes and deoxyribozymes (catalytic DNA) with allosteric properties, wherein the binding of an effector molecule controls the catalytic function. This feature provides challenging opportunities for the development of new analytical and very specific tools using traditional assay formats or for the development of entirely new biosensing principles. 

Enzymes, as you probably know, are proteins that can make chemical reactions happen in a more selective and faster way. At the end of each reaction cycle the enzymes remain unchanged so they act as catalysts. Since they occur in the living world we call them biocatalysts. Aside from proteins, ribonucleic acids and their fragments can act as catalysts and are called ribozymes, by analogy to enzymes. Enzymes are extracted from living tissues, for example, milk, saliva, liver, muscle have to be stored under carefully maintained conditions and, once outside living tissue, lose their activity fast. Isolation and purification of enzymes and assaying their activity have been major operations in biochemical and biomedical laboratories. Today,... 

Cell Death (Apoptosis) Chromatin Structure AND Modification Mammalian Cell Culture Microanalytical Assays Ribozymes... 

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