As an emerging technology, genetically-encoded molecular recorders have garnered significant attention for their ability to record otherwise inaccessible biological data at cellular resolution. An attractive application is recording neural activity at cellular resolution across the entire brain. Molecular recorders to date however are unsuitable for this task due to time-integrated encoding or low temporal resolution. These limitations could be overcome by engineered DNA polymerases which would be uniquely capable of encoding information at high resolution directly to DNA sequence. This would be accomplished by engineering a polymerase to transduce neural activity (e.g. Ca2+ transient concentrations) into changes in DNA replication fidelity over time. Here, we address some of the engineering and design obstacles to realizing a polymerase-based molecular recorder for nuerobiological applications. The most significant challenge is the difficulty of designing novel calcium allostery. We have generated a library of all possible positions at which Calmodulin can be inserted into E. coli Pol I (&gt;500). Using a selection for DNA replication activity, we have identified over 20 sites in Pol I that support the insertion of calmodulin. By characterizing the calcium-dependent shift in the fidelity of each candidate, we aim to identify and demonstrate a molecular recorder with unprecedented temporal resolution, which will be an essential proof of concept for molecular neurorecording.

Engineering a calcium-responsive molecular recorder from E. coli DNA polymerase I

2019 Spring National Meeting

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