(191cl) Methods for Development and Characterization of DNA Polymerase Based Bio-Recorders

Biosensors engineered to respond to custom input functions are invaluable tools for reporting changes in the physiological states of cells. Such biosensors are genetically encoded, making their delivery and induction precisely controllable. Taking biosensing a step further, we intend to develop a DNA polymerase-based in vivo cation bio-recorder. Such a nano-scale device would have an altered error rate when the concentration of a cation of interest changes in its environment. We are currently using neural firing as the ideal system where we would employ such a genetically encoded bio-recorder, using it to write the temporal trace of neural activity as error patterns onto DNA molecules. This would enable us to uncover neural interactions with single neuron precision. Along those lines, here we report methods we have established for the development and characterization of such a device. We first used rational design to make the processivity of the distributive Y-family Sulfolobus acidocaldarius DNA polymerase, Dbh, suitable for a neural recording application. We then developed methods to characterize the fidelity of the engineered Dbh under neural firing conditions and compared it with the fidelity of the wild-type Dbh. We intend to heavily rely on the tools established here to reach the final goal of an engineered DNA polymerase with kinetic parameters (error rate and processivity) fitting their bio-recording application in a neural environment.