Enzymatic probing

Ribonucleases are macromolecules and therefore sensitive to steric hindrance especially from the proteins in the RNA-protein complexes and from the intramolecular structures. The analysis performed with the ribonucleases described in this section provides valuable information on the gross folding of an RNA, and in many cases the mild conditions for RNase digestions are optimal for RNA-protein complexes. 

Various RNases employed in different studies will not be described in this section because the optimum conditions for many of these are incompatible with proper folding of the RNA or protein- 

RNA interaction and therefore cannot be recommended for probing experiments (e.g. RNase SI and RNase U2 have a pH optimum of 4.5 which prevents or destabilises protein-RNA complex formation). RNase A has a high affinity for a pyrimidine-adenosine stretch (particularly UA), so it can therefore be difficult to obtain single-hit kinetics (except for the UA sequence). Furthermore, RNase A exhibits an endogenous helix unfolding property which makes structure assignment difficult. RNases such as RNase CL3 and a-sarcin are inhibited by Mg2+ which is required for the stability of many complexes and also for proper folding of RNA. 

Place 10 pmol renatured RNA or complex in 20 pi modification buffer on ice. 

01 unit RNase 77, or 0.05 unit RNase T2, or 0.15 unit RNase VI. 

---

Epe, B., Mutzel, P. and Adam, W. (1988). DNA damage by oxygen radicals and excited state species, a comparative study using enzymatic probes in vitro. Chem. Biol. Interact. 67, 149-165. 

Table 1 lists representative examples of the application of in situ hybridization, including FISH, to studies that use chromosome preparations and interphase nuclei (4,5). FISH is superior to in situ hybridization with an enzymatic probe because it provides finer resolution and higher signal intensity. This is especially important when the localization of a gene within a specific chromosome band is to be established. 

Capelo, J.L., P. Ximenez-Embun, Y. Madrid-Albarran, and C. Camara. 2004. Enzymatic probe sonica-tion Enhancement of protease-catalyzed hydrolysis of selenium bound to proteins in yeast. Anal. Chem. 76 233-237. 

M. Dong et al., Development of enzymatic probes of oxidative and nitrosative DNA damage caused by reactive nitrogen species. Mutat. Res. 594, 120-134 (2006)... 

By chemical and enzymatic probing, the solution structure of this tRNA was analyzed. tRNA is identical to Other tRNAs concerning the T-arm and anticodon arm conformations, and the joining of the D- and T-loops by... 

Labeling of nucleic acids via JV-hydroxysuccinimide (NHS) esters of fluorescein (Research Organics) or Eu -chelates is straightforward. Nucleic acid should then possess reactive amino groups. These can be introduced by transamination (Section 7.8.1) or by using amino-hexyl-dNTP or AH-NTP as precursors during enzymatic probe synthesis (Section 7.6.2.2 Folsom et al., 1989). After AH-NTP incorporation in RNA (or AH-dNTP in DNA and heat-denaturation), the nucleic acid is diluted to 1 p,g in 25 p,l of HjO and added to 25 xl of freshly prepared 0.4 M sodium bicarbonate (pH about 8.2) and then to 10 pel of ester in DMF (1.7 mg/ml of ester final concentration). After incubation for 2 h, the reaction is stopped by adding 50 g.1 of 1... 

Certain criteria must be fulfilled for a chemical or an enzymatic probe to be useful in a structural analysis of an RNA molecule. 

The unusual conformation of the isolated EcoRl-PstI SV40 fragment. The findings reported above could reveal new information about the mechanism of binding of poly(ADP-ribose) polymerase to unusual secondary structures on DNA. DNA stmcture is known to be polymorphic (5, 6). Chemical and enzymatic probes (e.g. SI nuclease) are available to... 

Experimental examinations can be used instead of structural simulations to determine the essential sequence. In fact, experimental approaches are more commonly used. These procedures require the mass synthesis and analysis of shortened aptamer sequences. Chemical syntheses, boundary analyses, and enzymatic probing and footprinting can be used to generate the shortened sequences. The disadvantage of utilizing an experimental approach is that the iterative process of truncating aptamers and measuring aptamer activity requires weeks to months to survey a small population and may become very costly. 

---