10 Artificial Nucleases

Abstract

I. INTRODUCTION
Nucleases, particularly the sequence-specific restriction endonucleases, have become indispensable tools in modern biochemistry and molecular biology. They have made possible gene isolation, DNA sequencing, and recombinant DNA technology. However, their usefulness is, in many instances, limited by their small recognition site sizes (4–8 bp) and limited availability. For example, the restriction enzymes cleave too frequently to allow analysis of large chromosomal DNAs. Most restriction enzymes have recognition sites of 4–6 bp, corresponding to cleavage frequencies of once in every 136 and 2080 bp, respectively. Because a single mammalian chromosome can contain more than 100 million base pairs, these enzymes generate far too many fragments to be analyzed by current gel electrophoresis methods. Second, although 2400 restriction endonucleases have been reported, and even more will likely be discovered over time, the number of different sequences they recognize has not exceeded 200 (see Chapter 2; Appendix A). More specificities will surely be added to the existing pool in the future, but there will remain many sequences for which no restriction endonuclease is available. A final limitation is that restriction enzymes do not in general cleave single-stranded DNA or RNA.

Currently, both chemists and biologists are focusing considerable effort on generating “artificial nucleases” capable of cleaving DNA and RNA with high selectivity at any desired site. Work has focused on both the design and synthesis of efficient DNA-cleaving agents, as well as the development of strategies for selectively recognizing nucleic acid sequences of any defined sequence and length. Oxidative...