Nuclease applications

It should be pointed out that when using ethidium bromide the sensitivity of the assays varies depending on the physical state of the nucleic acids (see Table I). Ethidium does not discriminate between RNA and DNA, although dyes are available which bind DNA exclusively, so the relative amounts of each may be determined by taking two sets of measurements. Alternatively, nucleases (DNA-ase or RNA-ase) can be used to exclusively remove one or the other in a mixture. Nucleic acids from different sources (see Table II) also show a variation in sensitivity, and the fluorescence assay lacks the selectivity of the hybridization technique. Nevertheless, for rapid screening or quality-control applications the fluorescence assay is still the method of choice. 

Plitnik, L.M., et al., Cytokine profiling for chemical sensitizers Application of the ribo-nuclease protection assay and effect of dose. Toxicol. Appl. Pharmacol., 179, 145, 2002. 

O. (2004) Synthetic metallonucleases for RNA cleavage. Curr. Opin. Chem. Biol., 8, 192. (e) Molenveld, P., Engbersen, J.F.J. and Reinhoudt, D.N. (2000) Dinuclear metallophosphodiesterase models application of calix[4]arenes as molecular scaffold. Chem. Soc. Rev., 29, 75. (f) Kimura, E. (2000) Dimetallic hydrolases and their models. Curr. Opin. Chem. Biol., 4,207. (g) Williams, N.H., Takasaki, B. and Chin, J. (1999) Structure and nuclease activity of simple dinuclear metal complexes quantitative dissection of the role of metal ions. Acc. Chem. Res., 32, 485. (h) Hegg, E.L. and Burstyn, J.N. (1998) Toward the development of metal-based synthetic nucleases and peptidases a rationale and progress report in applying the principles of coordination chemistry. Coord. Chem. Rev., 173, 133. 

Several other types of backbone modification have also been proposed, which produce nuclease-resistant oligos. Of these, a-oligos have been extensively studied. In a-oligos the base is transposed from the natural P-orientation to the unnatural a-orientation to form a parallel duplex with target sequence. This parallel duplex is nuclease-resistant, but does not elicit RNase H activity (Cazenave et al., 1989). These modifications have generated limited interest and application in antisense research. 

DEAE-dextran. Like the calcium phosphate co-precipitation method, the DEAE-dextran technique was originally developed to increase the viral infectivity of animal cells, and its application was later extended to transfection processes. Although it is simple, efficient, and appropriate for transient expression, its use for stable transfections has not given satisfactory results. The transfection efficiency of this method can be increased by treating cells with glycerol or DMSO. The DNA is incorporated by endocytosis, and thus exposed to extreme pH levels and cellular nucleases, which may explain, to a certain extent, the high frequency of mutations observed when transfecting by this method (Calos et al., 1983). This transfection technique can be applied to both adherent and suspension cell lines. For detailed transfection protocols, the works by Keown et al. (1990) and Kaufman (1997, 2000) are recommended. 

FIGURE 21.5 Chemical structures of modified nucleotides used in the SELEX procedure. Aptamers containing 2 F- and 2 NH2-modified nucleotides are resistant against degradation by nucleases and therefore suitable for in vitro and in vivo applications. Aptamers containing modifications (I or Br) at the C-5 position of pyrimidine can be photo-cross-linked to their protein targets. Arrows indicate the respective chemical modifications. 

Kay, L.E., Torchia, D. A., and Bax, A. (1989) Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy application to Staphylococcal nuclease, Biochemistry 28, 8972-8979. 

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