Book of Abstracts: Albany 2003
June 17-21 2003
Site-Specific Nicking of Duplex DNA using PNAs and Restriction Endonucleases
A variety of research procedures in molecular biology and biochemistry include as a fundamental step the selective cleavage of a designated sequence in only one strand of double-stranded DNA (DNA nicking). To achieve the goal, restriction endonuclease-like nicking enzymes can be employed. However, very few nicking enzymes (nickases) are available and they recognize short sequences of ≤ 7 bp. It is therefore highly desirable to develop new nicking systems with much higher sequence selectivity.
We design such systems using homopyrimidine peptide nucleic acids (PNAs). In our design we take advantage of the ability of homopyrimidine PNAs to sequence-specifically invade duplex DNA. When such PNAs are targeted to two short homopurine stretches on the same strand of duplex DNA, the opposite DNA strand becomes accessible for hybridization with an oligonucleotide (1). We demonstrate that the thus formed secondary DNA duplex (see Figure 1) can serve as a substrate for a restriction endonuclease, provided that it contains the recognition sequence of the enzyme. As a result, only one strand of the parent DNA is cleaved leading to nicked duplex DNA after removal of PNAs.
Figure 1: Design of our artificial DNA-nicking system. A pair of linked homopyrimidine PNAs (shown in green) selectively displaces a DNA segment that is then hybridized to an oligonucleotide. The resulting secondary DNA duplex is cut with a common restriction enzyme symbolized by scissors yielding, after removal of PNAs and cleaved oligonucleotide, a site- and strand-specific nick in duplex DNA. The light blue lines indicate a recognition sequence for the restriction enzyme.
All restriction endonucleases tested by us (AluI, BbsI, BglII, KpnI, SbfI, and SphI), have worked well in our design leading to quantitative yield of the nicked product. Together with the fact that the typical target site in our protocol spans about 20-25 bp, our results indicate that a vast class of semi-synthetic rare-cleaving DNA nickases has been generated.
This work has been supported by the PIF award from Boston University and by grants (GM59173 and CA89833) from the NIH.
Center for Advanced Biotechnology and Department of Biomedical Engineering