The ability of DNA sequences to adopt unusual structures under the superhelical torsional stress has been studied. Sequences that are forced to adopt unusual conformation in topologically constrained pBR322 form V DNA (Lk = 0) were mapped using restriction enzymes as probes. Restriction enzymes such as BamHl, Pstl, Aval and HindlIl could not cleave their recognition sequences. The removal of topological constraint relieved this inhibition. The influence of neighbouring sequences on the ability of a given sequence to adopt unusual DNA structure, presumably left handed Z conformation, was studied through single hit analysis. Using multiple cut restriction enzymes such as Narl and FspI, it could be shown that under identical topological strain, the extent of structural alteration is greatly influenced by the neighbouring sequences. In the light of the variety of sequences and locations that could be mapped to adopt non-B conformation in pBR322 form V DNA, restriction enzymes appear as potential structural probes for natural DNA sequences. INTRODUCTIONOver the last decade our understanding of DNA structure as a rigid uniform double helix has significantly changed to that of a sequence-dependent dynamic conformation (1,2). Although several crystal structures of oligonucleotides have shown that the DNA conformation is indeed sequence-dependent (3), paucity of data does not enable us to arrive at a generalised rule. However, the influence of ionic environment and topological strain (resulting from supercoiling) in altering DNA secondary structure has provided valuable information with regard to structural transition from B to Z form (4-8). Enzymes such as DNase I and SI nuclease have been extensively used to probe DNA conformation in solution (2,9-11). Although restriction endonucleases are believed to be only sequence-specific and not structure sensitive, recent studies have shown that some restriction enzymes fail to recognize and cleave DNA in left handed Z conformation (12)(13)(14).