Use of Nucleases in RNA Sequence and Structural Analyses

Abstract

As might be expected, nucleases have played crucial roles in the sequence and structural analyses of RNAs. Along with other evidence, studies on the effect of pancreatic RNase and spleen phosphodiesterase on chemically synthesized nucleoside 2′- and 3′-phosphodiesters helped to establish that the phosphodiester linkages in RNA were 3′–5′ rather than 2′–5′ (Brown and Todd 1953). The availability of base-specific endoribonucleases such as pancreatic RNase and T1-RNase made possible the specific cleavage of RNAs to short oligonucleotides, a key step in sequence analysis of RNAs. Exonucleases such as snake venom phosphodiesterase and spleen phosphodiesterase have found extensive use in the sequence analysis of these oligonucleotides (Laskowski, this volume). Nuclease P1, a random endonuclease, has proved useful not only for sequencing short oligonucleotides, but also for the terminal analysis of large RNAs that are labeled at either their 5′ or 3′ ends (Silberklang et al. 1977). Base-specific nucleases are also being used for direct sequence analysis by partial digestion of end-labeled RNAs, followed by gel-electrophoretic analysis of the labeled fragments. This has provided a rapid method for sequencing up to 400 nucleotides from the labeled end of an RNA (Donis-Keller et al. 1977; Simoncsits et al. 1977; Lockard et al. 1978; 1982).

Many of the nucleases are sensitive to the secondary and tertiary structure of the RNAs. S1 nuclease from Aspergillus oryzae cleaves only single-stranded regions of RNA or DNA (Ando 1966), whereas a nuclease from cobra venom cleaves only within double-stranded regions of an RNA. These and other...