Nanopore current transduction analysis of protein binding to non-terminal and terminal DNA regions: analysis of transcription factor binding, retroviral DNA terminus dynamics, and retroviral integrase-DNA binding.

Background: Synthetic transcription factors (STFs) promise to offer a powerful new therapeutic against Cancer, AIDS, and genetic disease. Currently, 10% of drugs are of this type, including salicylate and tamoxifen. STFs that can appropriately target (and release) their transcription factor binding sites on native genomic DNA provide a means to directly influence cellular mRNA production. An effective mechanism for screening amongst transcription factor (TF) candidates would itself be highly valued, and such may be possible with nanopore cheminformatics methods.

Results: It is hypothesized that binding targets on channel-captured molecules, that are well away from the channel-captured region, can be monitored insofar as their binding status, or history, is concerned. The first set of experiments we perform to explore this "transduction" hypothesis involve non-terminal dsDNA binding to protein (DNA TATA box receptor binding to TBP), where we show new experimental results and application of a new cheminformatics data analysis method. In the second series of experiments to explore the transduction hypothesis we examine terminal (blunt-ended) dsDNA binding to protein. We show experimental results before and after introduction of HIV's DNA integrase to a solution of bifunctional "Y" shaped aptamers that have an HIV consensus terminus exposed for interaction.

Conclusion: X-ray crystallographic studies have guided our understanding of DNA structure for almost a century. It is still difficult, however, to translate the sequence-directed curvature information obtained through these tools to actual systems found in solution. With a nanopore detector the sequence-dependent conformation kinetics of DNA, especially at the DNA terminus, can be studied in a new way while still in solution and on a single molecule basis.