PNA Directed Genome Rare Cutting: New Developments

This chapter deals with the PNA-assisted rare cleavage (PARC) of duplex DNA. The technique is a variety of the general ‘Achilles’ heel ́ cleavage strategy and uses PNA oligomers to protect very few sites on genomic DNA against enzymatic methylation. As a result, the PARC technique makes it possible to convert common restriction enzymes into a pool of infrequent genome cutters. These artificial genome cutting systems cover the range of recognition specificities, where very few, if any, cutters are now available. Here, we present the PARCbased method for robust purification of yeast artificial chromosomes (YACs) from host chromosomes with similar lengths and demonstrate the PARC potential for rare fragmentation of human DNA. Further progress in the PARC approach also includes the use of pseudocomplementary PNAs (pcPNAs) as sequence-unrestricted duplex DNA-binding ligands. Frank-Kamenetskii and Demidov 2 PA GE P RO OF S Background DNA rare fragmentation is used in genomics for genome analysis and may find applications in DNA technology for handling with large DNA molecules. However, a limited number of rare-cutting restriction enzymes (1-4) and homing endonucleases have been identified (4-7). To extend the range of natural DNA rare cutters, their directed modification or selection (8, 9), coupling with DNA methylases (10) and employment of specially inserted sites (11, 12) could be used. Also, artificial ‘restriction enzymes ́ combining a variety of DNA binding and cleavage functions have been created (13-15). Still, the progress in this direction is limited, especially for creation of 8-12 bp cutters (16). Considering that this range of recognition specificities corresponds to the most useful sizes of large DNAs–from several dozen kilobases to few megabases, further expansion of approaches for rare cutting the genomes is highly desirable. In 1996, we proposed a variety of the ‘Achilles’ heel ́ rare cleavage strategy (17, 18), the PARC approach (19-21; see refs. 16 and 20 for schematics), which is based on targeting the double-stranded DNA (dsDNA) with cationic pyrimidine bis-PNAs (22-27). A major idea underlying this method is that sequence-specific binding of bis-PNA to relatively short dsDNA sequences should block DNA recognition Figure 1. Schematics of the PARC method involving pcPNAs. In contrast to triplexforming pyrimidine bis-PNAs, sequence-unrestricted recognition of dsDNA by pcPNAs via the double-duplex invasion makes it now possible to completely cover the desired methylation/restriction site. For the original similar PARC schematics with bis-PNAs, see refs. 16 and 20. genomic DNA bind pcPNAs methylate inactivate methylase disrupt PNA/DNA complex digest by restriction enzyme Legend: site cleavable by restriction enzyme methylated site, not cleavable