Site-specific DNA cleavage of synthetic NarL sites by an engineered Escherichia coli NarL protein-1,10-phenanthroline cleaving agent.

The NarL response regulatory protein of Escherichia coli has been engineered by covalent modification with 1,10-phenanthroline (OP) to create a set of site-specific DNA-cleaving agents. This was accomplished by introducing single cysteine amino acid replacements at selected locations within the carboxy-terminal DNA-binding domain in or nearby the helix 8 to helix 9 region of the NarL protein using site-directed mutagenesis. Of 18 modified NarL-OP proteins made, 13 retained the ability to bind DNA as evidenced by gel mobility assays, whereas 10 of the 1,10-phenanthroline-modified proteins also exhibited specific cleavage activity for a synthetic NarL recognition sequence. These DNA-cleaving agents were divided into two groups based on the location of the cleavage sites. The first class set cleaved the DNA nearby the center of a synthetic 7-2-7 sequence composed of two NarL heptamer sites separated by a 2-bp spacer element. The second class cut the DNA at the periphery of the 7-2-7 sequence. The cleavage data are consistent with the ability of two NarL monomers to recognize and bind to the DNA in a head-to-head orientation. A second set of DNA-cleaving agents was constructed using the carboxy-terminal domain of NarL called NarL(C). Similar cleavage patterns were observed whether full-length NarL or NarL(C) was used. The availability of 1,10-phenanthroline-modified NarL and NarL(C) proteins opens up the possibility to explore the position, orientation, and number of NarL recognition sites at E. coli promoters predicted to contain multiple and complex arrangements of NarL-binding sites.

Transcription inhibition using modified pentanucleotides.

Inhibition of gene expression was recently achieved by targeting the transcriptionally competent open complex using relatively short, pentameric modified oligonucleotides at approximately 60 microM. Corroborative affinity cleavage experiments using the copper complex of a phenanthroline conjugate provided the impetus to synthesize additional analogues containing substituents at the 2'-position of uridine in a derivative of 5'-GUGGA (-4 to +1), with the purpose of inhibiting transcription at lower concentrations. Conjugates of 5'-GUGGA modified at the 2'-position of uridine were convergently synthesized using a recently reported method. Seven analogues based upon the 5'-GUGGA scaffold were tested for their ability to inhibit transcription of the lac UV-5 operon. The conjugate containing a tethered pyrene showed 70% inhibition at 20 microM, and modest inhibition at as low as 5 microM. This is a significant improvement over previously tested pentanucleotides and provides direction for the preparation of a next generation of inhibitors.