Chemically synthesized zinc finger molecules as nano-addressable probes for double-stranded DNAs

Nanostructures based on DNA have been reported previously. Generally it is a prerequisite to generate single stranded DNA (ssDNA) molecules to open the possibility of probing with oligonucleotides. However, the production of long ssDNA may not be an easy task. ssDNAs are more sensitive to physical handling, tend to form stable secondary structures and require considerable effort to obtain sufficient quantities intact molecules. An alternative approach relies on the use of peptide-nucleic acids (PNAs), which are capable of recognizing dsDNAs. The recent reports have demonstrated that this approach is not always practical and PNA binding is not always reliable [1]. Another potential approach is to use existing cellular mechanisms involved in the control of the production of proteins on the level of nucleic acids. Transcription factors which bind to dsDNA target sequences are switching specific genes "on" or "off" by recognizing and binding to short specific DNA fragments. Zinc finger proteins are transcription factors which have concise and simple structure [2], can be made synthetically and might therefore provide alternative probes for dsDNA recognition [3].In our experiments we used one of the three zinc finger motifs of the SP1-zinc-finger as a template for our chemically synthesized zinc finger probes, which were of the C2H2 type (Figure 1). The DNA-binding region was altered in two places to enable the folded zinc finger to bind either at the "AAA"-DNA sequence site or at the "GCG"-DNA sequence site (see M & M, bold and underlined). The custom-made peptides were modified with a fluorophore (Dye633) at their amino-termini to allow quantitative detection. The double-stranded DNA targets were made of custom-made complementary oligonucleotides carrying the appropriate DNA recognition-sequence and a phosphate modification at the 5'-end of one of the complementary oligonucleotides for their immobilization.Detection was done by fluorescence on a fibre-optical-device de