Sensing The Binding Sites Of Rnap Holoenzyme On Lambda Dna Attached To A Probe Tip With Solid State Nanopores

We report our study to detect the binding sites of RNAP Holoenzyme on λ DNA molecules with an apparatus that integrates a Solid State Nanopore (SSN) with a Tuning Fork based Force sensing Probe tip (SSN-TFFSP). Using this apparatus, we can control the DNA translocation rate to be slower than u003e100µs/base or u003c1nm/ms in silicon nitride nanopores, a rate that which is 1000 times slower than free DNA translocation through solid state nanopores. This slow DNA translocation rate could provide sufficient temporal resolution to determine the individual RNAP binding sites on along λ DNA molecule. We show the results of free and controlled translocation of λ DNA with and without bound RNAP through solid state nanopores. λ DNA has two promoter and three pseudo promoter sites for RNAP Holoenzyme to bind. To prevent the separation of RNAP proteins from a λ DNA molecule in the high electric field inside a nanopore, we covalently bound the RNAP Holoenzyme to the λ DNA by formaldehyde crosslinking. We also covalently coupled streptavidin proteins to the gold plated tip by thiol binding. In order to bind DNA to the streptavidin coated tip, a biotinylated primer was ligated to the one end of λ DNA molecules. The accuracy of determining the binding sites of RNAP, as well as other DNA binding proteins will be estimated and discussed.