Date of Graduation

5-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Physics (PhD)

Degree Level

Graduate

Department

Physics

Advisor/Mentor

Jiali Li

Committee Member

Surendra Singh

Second Committee Member

David Mcnabb

Third Committee Member

Paul Thibado

Fourth Committee Member

Woodrow Shew

Keywords

Biological sciences, Binding sites, Nanopore, RNAP

Abstract

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.

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