“ The very same medium that literally specifies who we are as individuals might also store our art, our culture, and our history as a species.”2
Enzymatic nicking involves using a highly-specific and versatile endonuclease enzyme (Pf Ago) to cut a single bond in the sugar-phosphate backbone (fig.1.a), forming fragments which can be recognised using NGS/nanopore sequencing. Figure 1. outlines the enzymatic nicking process and visualises the writing/reading aspect of nicking -related DNA storage.
The Pf Ago enzyme used by Tabatabaei et al. is a highly accurate artificial restriction enzyme5 with a high turnover rate, enabling one enzyme to produce hundreds of nicks. Normally, Pf Ago cleaves both strands of DNA when it binds to the recognition site. However, this is unnecessary with regards to storage as it requires the presence of two guide DNA (gDNA) molecules in close proximity. Tabatabaei found that under the correct conditions (e.g. buffer and temperature) Pf Ago can target only one of the DNA strands, using a single gDNA, allowing efficient and precise nicking of that strand simultaneously, in under 40 minutes. This greatly reduces the writing latency expressed by other DNA-storage methods. The lack of errors when writing also means that high coverages are not required for accurate data readout, as the reads are easily mapped to the reference. By decreasing the coverage, costs associated with sequencing also decrease, increasing the cost-effectiveness of this technique. To prove that enzymatic nicking worked, the researchers5compressed and converted two files into ASCII format, and retrieved them with 100% accuracy (not possible with synthetic DNA storage). The first was a 0.4KB text file containing Lincoln’s Gettysburg Address (272 words), and the second was a 14KB JPEG image of the Lincoln Memorial. Both were successfully retrieved using NGS, however solid-state nanopore sequencing could also have been used.
An issue with nanopore sequencing is the lack of translocation controls, making the nick reads noisy. To combat this, Tabatabaei et al.used Pf Ago to extend nicks to ‘toehold’ regions, consisting of two nicks placed closely together, forming a short segment of DNA. This is easily recognisable by solid-state nanopores, allowing for quick and clear reads.
Toeholds also allowed Tabatabaei et al. to achieve something synthetic DNA storage methods could not; Bitwise Random Access. This makes performing molecular computations possible, using toeholds as initiation sites to allow the controlled binding/release of DNA strands. This ability was then enhanced by the addition of fluorescent tags which enabled the visual recognition and retrieval of a specific toehold.