By coupling biological signals of interest to DNA sequence modifications, molecular recorders enable nano-scale data acquisition and storage. Such systems, often based on the activity of Cas9-directed mutagenesis or sequence acquisition, have succeeded in capturing information on cellular process on the order of hours to days, but recording faster events remains challenging due to the multipart cascades linking signals of interest to changes in DNA. To improve the temporal resolution of molecular recording, we developed a method employing the DNA synthesis activity of terminal deoxynucleotidyl transferase (TdT). We showed that TdT exhibits altered nucleotide selectivity in response to environmental cations, enabling post hoc determination of the reaction conditions based on DNA sequence alone. By characterizing changes in the average base composition along strands of synthesized DNA, we demonstrated that the time of cation fluctuations in vitro can be accurately estimated to within 1 minute over a 60-minute period. Subsequently, we have explored cellular implementations of TdT-based recording system. We show that the sequence composition of DNA synthesized by TdT at genomic cleavage sites stores information about cellular environments and that user-specified data can be robustly encoded and stored in barcoded cellular populations.

Cellular data recording and storage by in situ DNA synthesis

ACS Spring 2022

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