Engineering Of The Inihibitors Of Nicking Endonuclease Bspd6l Using Synthetic Dna Fragments

Nicking endonucleases (NE) recognize specific sites in double-stranded DNA and cleave only one strand in predetermined position. These enzymes have become widely used in molecular biology and genetic engineering. NEs are possible to consider as powerful molecular tools for DNA cleavage near defective gene that may further result in its repair by cell mechanisms. The absence of non-specific DNA hydrolysis is a key requirement of this method. The targeting regulation of NEs activity represents a critical task at present: it is necessary to prevent the undesirable cleavage of DNA since it can have harmful effects for the cell. Using NE BspD6l as a model we developed the strategy that allows to "switch" on and off the enzymatic activity at any given point in time by means of external signal (changing of temperature). The approach is based on using thermosensitive analogues of enzyme's substrate. We constructed 13 and 15 b.p. non-modified synthetic DNA duplexes that can block the enzymatic activity at 20 degrees C. Temperature increase causes dissociation of duplexes-inhibitors and results in enzymatic hydrolysis of long length DNA at similar to 40-50 degrees C. The proposed approach can be used for optimization of isothermal strand displacement amplification of DNA, as well as for hydrolytic activity modulation of chimeric nucleases used in gene therapy.