We have shown here that both third-strands designed for this project are able to bind to their respective target sequences in deoxyoligonucleotide duplexes cloned into plasmids of modest size. Moreover, that binding is specific to the target. Below saturating concentrations, binding is directly proportional to the concentration of added third-strand. For the third-strand targeted to the histone cluster, saturation was reached at a modest excess (30-fold) of third-strand. Saturation was not reached with the GAGA target because the similar amounts of third-strand used were not high enough.
Results between duplicate experiments were not entirely consistent in the case of the histone target, probably because of a phosphoimager problem. Further work can probably correct this problem. Nevertheless, the binding data obtained with the protocols used showed that third-strand binding to the two targets can be achieved to saturation.
The GAGA third-strand system provides an insight into the way third-strands interact with their targets and with each other. The apparent KD for the entire 14-target system is 105 nmolar. For the binding of a single third-strand, KD is roughly 7-8 nmolar. Since , these measurements seem to indicate that third-strand binding events at neighboring but not fully contiguous targets are additive, with no inhibition or synergistic effects apparent. It therefore seems probable that multiple third-strand binding events to targets in such close proximity are independent.
The methodology used in this project involves solution equilibrium third-strand binding to supercoiled plasmids. Kinetic and pulse-chase experiments are required to better characterize the rates and efficiency of binding. The next logical step is to evaluate the binding efficiency to linear DNA strands of different sizes and to check binding specificity to ensure that the third-strand is indeed binding uniquely to its target.
We have designed an in vitro assay that measures the position of the bound third-strand relative to a restriction enzyme site. Supercoiled plasmids are linearized near the third-strand target. Binding is allowed to occur and Bal-31 exonuclease is then used to digest back the ends of the DNA towards the target site. The bound third-strand should inhibit nuclease activity and yield a DNA strand of characteristic size which can be visualized on an ethidium bromide stained agarose gel.
The plasmid pMJ13 was designed for this purpose. It contains a lone AvaI restriction site fifty-five residues upstream of the target site. Consequently, exonuclease inhibition studies can be used to show that third-strand binding is specific to the targeted region of DMHISTS. Similar assays can be used on larger targets to insure target specificity.
The next phase of this project will be to examine binding to nuclear spreads of metaphase Drosophila chromosomes. A TISH protocol has been developed for human metaphase spreads (Johnson and Fresco 1999). A biotin and psoralen-conjugated third-strand is hybridized to fixed metaphase chromosomes and cross-linked with psoralen using longwave ultraviolet light. Epifluorescent microscopy with fluorescein dye is used to visualize binding (Figure 14). This procedure should be transferable to Drosophila without major complications. Sections of immature Drosophila eggs can similarly be probed in situ (Ambrosio and Schedl 1984).
We also propose an in vivo competition binding assay against an embryonic Drosophila protein. During the syncytial growth stage of the fly larvae, GAGA protein is required to allow correct chromatin segregation and nuclear division. Mutations in the protein leave it unable to bind to the chromatid, exposing a lethal phenotype. We believe that microinjection of a targeting third-strand for the GAGA binding site into a wild type embryo should generate a phenotype not unlike that of a Trl- mutant. Injected embryos can be fixed and visualized by staining with a chromatin-specific dye. If this experiment should prove successful, then the GAGA-targeted third-strand can be employed to examine the physiological role of the GAGA binding protein.
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| Figure 14. Human metaphase chromosome spread bound by a fluorescein-labeled biotin conjugated third-strand oligonucleotide targeting Chromosome 17. Metaphase chromosomes and whole interphase nuclei are shown. Courtesy of Marion Johnson. |
