Developing a DNA Polymerase-Based Biosensor for Neural Recording

Progress in neural recording is critical to better understand the brain and to evolve treatments for brain-related illnesses. Further advances in mapping brain activity are limited by the low spatiotemporal resolution of current technologies. Our objective is to create a molecular recording device that combines the rapid writing capability of DNA polymerases with the robust information storage capability of DNA to generate dense spatiotemporal data on neural activity. Such a recording device, combined with the
advent of highly parallel next generation sequencing technologies, has the potential to simultaneously monitor neurons at time scales matching mental activity1-3.
Ca2+ fluctuations on the millisecond timescale are indicative of neuron firing. We aim to engineer a DNA polymerase-based biosensor that responds to high calcium by
writing errors into DNA. By coupling the nucleotide misincorporation rate of a DNA polymerase with levels of Ca2+ in the environment we can read out the relative Ca+2 concentration at a particular time by sequencing the copied DNA1-3.
As a first step toward building a Ca2+ biosensor, we are interested in understanding the metal cation-polymerase fidelity landscape to determine strategies for
engineering DNA polymerase recorders. In order to screen metal-dependent fidelity
properties of a multitude of DNA polymerases, we have developed a novel medium- throughput method for rapidly identifying changes in DNA polymerase error rate across a wide spectrum of metal cation and DNA sequence context conditions. Unlike former methods for determining polymerase fidelity, which rely on bacterial transformations4 or gel electrophoresis5, this in vitro technique is scalable (100 conditions/day) and allows
for standardization of fidelity changes. Additionally, since DNA polymerase properties are difficult to study in their full complexity, this screen will allow us to decouple the singular and combinatorial effects of environment and sequence context on inducing replication errors.
Using this screen, we will identify polymerase candidates, environmental
conditions and DNA template sequences that facilitate recording metal cation time course information via changes in fidelity. We expect these results to catalyze synthetic biology efforts toward a high-performance metal cation sensing and storage system based on incorrect nucleotides being incorporated into DNA. This first demonstration of a divalent metal recording platform will inform future development of a Ca2+-specific biosensor for neural recording applications.
Works Cited
1 Kording, K. P. Of toasters and molecular ticker tapes. PLoS Comput Biol 7, e1002291, doi:10.1371/journal.pcbi.1002291 (2011).
2 Zamft, B. M. et al. Measuring cation dependent DNA polymerase fidelity landscapes by deep sequencing. PLoS One 7, e43876,
doi:10.1371/journal.pone.0043876 (2012).
3 Glaser, J. I. et al. Statistical analysis of molecular signal recording. PLoS Comput

Biol 9, e1003145, doi:10.1371/journal.pcbi.1003145 (2013).

4 Bebenek, K. & Kunkel, T. A. Analyzing fidelity of DNA polymerases. Methods

Enzymol 262, 217-232 (1995).

5 Creighton, S., Bloom, L. B. & Goodman, M. F. Gel fidelity assay measuring
nucleotide misinsertion, exonucleolytic proofreading, and lesion bypass efficiencies. Methods Enzymol 262, 232-256 (1995).