DNA-PROTEIN NANOTECHNOLOGY: DEVELOPING UNIQUE BIOLOGICAL NANOSTRUCTURES AND BIOLOGICAL TOOLS

摘要

Lac repressor (LacI) when bound to two operator DNA sites loops the intervening DNA sequence, enhancing the efficiency of transcriptional repression. Previous results from footprinting, electrophoretic mobility shift and ring closure experiments on lac operator/A-tract DNA constructs suggest that the LacI C-terminal 4-helix bundle probably acts as a hinge allowing the protein to adopt multiple conformations. One DNA molecule under study, designated "9C14," was proposed to be capable of forming two different geometries, with either a closed (V-shaped) LacI or a more open, extended form of the repressor. Previous bulk Fluorescence Resonance Energy Transfer (FRET) results confirmed the existence of a closed loop form (giving a net 70 % efficiency of transfer) but did not definitively confirm the presence of an open form complex or whether there existed populations with intermediate LacI-DNA geometries. We applied the technique of single molecule FRET to Cy3-Cy5 labeled 9C14-LacI DNA loops freely diffusing in solution in order to further assess looping geometries. Through careful consideration of photophysical bleaching effects, which can affect both single molecule and ensemble FRET measurements, we have shown that our results clearly demonstrate that LacI-9C14 exists strictly as a closed loop exhibiting nearly 100 % energy transfer (ET) efficiency. In a second project, we set out to design and characterize DNA-LacI self-assembled 2D and 3D nanostructures, applying four critical design rules. First, DNA provides us with a building material that is stiff, stable and easily manipulated. Second, we use circular DNA to build our molecules because it adopts a more restricted range of conformations then its linear counterpart. Third, the positioning of curved A-tracts (short runs of (dA) 4-6 (dT)4-6)) at the corners of designed "squares" and "triangles" allows the sides of the "squares" and "triangles" to remain straight, with the majority of the bending localized at the corners of the molecule, making it compact and rigid. Fourth, the formation of three proposed large nanostructures is dependent on the orientation of the lac operator sequence relative to the plane of the DNA.