Comments
Ribonuclease A (RNase A) is an endoribonuclease, that specifically cleaves single-stranded RNA 3' to pyrimidine residues (cytosine, uracil). Thereby, it generates pyrimidine-3'-phosphate or oligonucleotides with terminal pyrimidine-3'-phosphates. The pH-optimum is in the range of 7.0 - 7.5. RNase A is used for the purification of RNA-free DNA, for the removal of non-hybridized regions of RNA : DNA-hybrides or as a molecular weight marker. The enzyme is inhibited by diethyl pyrocarbonate (DEPC), guanidinium salts (4 M GuaSCN), β-mercaptoethanol, heavy metals, vanadyl-ribonucleoside-complexes, RNase-inhibitor from human placenta and competitively by DNA, respectively. Regarding the latter, the effect of denatured DNA is higher than by native nucleic acids. Nevertheless, RNase A is very active under very different conditions and difficult to inactivate. At low salt-concentrations (up to 100 mM NaCl), RNase A cleaves single- and double-stranded RNA and RNA in RNA : DNA- hybrides. Under high salt concentrations (>300 mM NaCl) single-stranded RNA is cleaved only. To remove the enzyme from samples, it has to be digested by proteinase K (frequently, SDS at a final concentration of 0.6 % is added) and several phenol extractions are required. (Applications: Enzymatic manipulation of DNA and RNA: ref. 1 Suppl. 8 p. 3.13.1; minipreps of plasmid-DNA: ref. 1 Suppl. 24 p. 1.6.6; inSitu-hybridisation of cellular RNA: ref. 1 Suppl. 7 p. 14.3.8; removal of RNA from plasmid preparations: ref. 2 p. 1.51) Stock solutions are prepared at concentrations from 1 - 10 mg/ml in 10 mM Tris · HCl, pH 7.5; 15 mM NaCl or in 10 mM Tris · HCl, pH 7.5; 1 mM EDTA, pH 8.0 (TE buffer). The recommended working concentration is 10 μg/ml (removal of RNA from plasmid preparations; 1 hr, RT) or 100 ng/ml (preparation of "blunt ends" of double-stranded cDNA).Unit-definition: One unit of activity is defined as that amount of enzyme which causes the hydrolysis of RNA to yield a velocity constant, k = 1, at 25°C and pH 5.0 (Kunitz-Unit). Inactivation of DNase activity: A protocol (ref. 2) suggests to dissolve 10 mg/ml RNase A in 0,01 M Sodium acetate (pH 5,2), to heat to 100°C for 15 minutes in a water bath and to cool down to room temperature very slowly. The pH value is equilibrated by adding 0.1-fold the volume of 1 M Tris-Cl (pH 7,4). Caution: Heating solutions of RNase A to inactivate DNase may not be satisfactory since RNase activity may be lost if precipitate formation occurs. For applications that require DNase-free RNase A we recommend our product A3832, RNase A (DNase-free). Stability: RNase A aggregates during lyophilizing and storage. It has a high affinity to glas surfaces, which has to be taken into consideration. At neutral pH (e. g. in PBS pH 7.4) and high concentrations (> 10 mg/ml) the enzyme will precipitate. At \+4°C (lyophilized) it is stable for several years (dry storage), in solution (-20°C) several years or (\+4°C) several weeks.
Literature
(1) Ausubel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. & Struhl, K. (eds.) (1995) Current Protocols in Molecular Biology. Page 3.13.1 Suppl. 8; Greene Publishing & Wiley-Interscience, New York (2) Sambrook, J. & Russel, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition Page A4.39. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. (3) Melton, D.A. et al. (1984) Nucleic Acids Res. 12, 7035-7056. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing the SP6 promoter. (4) Winter, E. et al. (1985) Proc. Natl. Acad. Sci. USA 82, 7575-7579. A method to detect and characterize point mutations in transcribed genes.
