Date of Graduation
Doctor of Philosophy in Physics (PhD)
Second Committee Member
Third Committee Member
Fourth Committee Member
Biological sciences, Binding sites, Nanopore, RNAP
In this dissertation, the binding positions of RNAP holoenzyme on a λ DNA are characterized using an apparatus that integrates a Solid State Nanopore with a Tuning Fork based Force sensing probe (SSN-TFFSP). The SSN-TFFSP system combines the measurement of ionic current through a solid-state nanopore with a DNA tethered probe tip. The position of the tip is sensed by a tuning fork force sensor and is controlled with a nanopositioning system. With this apparatus, translocation speed of DNA through solid state nanopores has been brought down to 100 μs/base. Such a controlled movement of DNA through a solid state nanopore can provide enough temporal resolution to determine the individual binding site of a RNAP on a λ DNA. Three signals measured simultaneously from this apparatus were: ionic current through a nanopore, tip position, and tip vibrational amplitude. These signals were measured when the probe tip was approaching towards the nanopore and was being lifted away from the pore. The λ DNA+ RNAP complex tethered to the probe tip can be captured by the electric field near a nanopore. The nanopore current signal measured during the capture of RNAP bound λ DNA provides new insights to the dynamics of λ DNA+RNAPcomplex molecules inside the nanopore. The binding positions of RNAP on a λ DNA are measured directly from the tip position signal corresponding to the distinct current drop within λ DNA current blockage level. The resolution limit of this apparatus is estimated to be 100 nm or 300 bp for RNAP binding sites. The resolution limit was further compared with the free translocation data set of λ DNA+RNAPcomplex through the solid state nanopore.
Kaur, Harpreet, "Characterization of the RNAP Binding Sites on a λ DNA using a Solid State Nanopore Combined with a Tuning Fork Force Sensing Probe Tip" (2017). Theses and Dissertations. 1900.