FET: Medium: Massively parallel DNA computation using DNA array synthesis, next generation sequencing and nanopore sensing

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Computing with molecular components can enable innovations in a range of areas from health diagnostics and therapies to information technology. For example, molecular circuits that can process the information encoded in the levels and sequences of cellular molecules can be at the heart of embedded controllers for biomedical applications. However, the ability to build molecular controllers lags far behind the ability to engineer embedded control circuits for electromechanical devices. This project aims to develop new methods of writing and reading DNA circuits that will make it possible to scale up circuit complexity by at least an order of magnitude over the current state of the art and thus bring molecular controllers closer to practical applications. Furthermore, this project will accelerate training of students and professionals in the burgeoning field of molecular information systems. This area of research inherently increases the diversity of people, perspectives, and backgrounds in computing by bringing together the historically separated fields of computer science and biology. To increase participation of underrepresented persons in molecular programming research, the team of researchers will host summer interns from Rainier Scholars, an organization that supports students of color from low-income backgrounds in achieving academic success.This project introduces technological innovations that will increase the scalability of molecular circuitry. PIs will do this by taking advantage of massively parallel DNA synthesis technology and coupling it to high throughput readout methods, including next generation sequencing and nanopore sensing. These advances are significant because the scaling up of DNA circuitry is currently limited in two main ways: A first limitation is that the vast majority of DNA gates and similar computational elements are assembled from individually column-synthesized oligonucleotides because this technology provides low synthesis error rate and control over the concentration of individual strands. However, the cost of ordering individually synthesized oligos cannot scale. Array synthesized oligos provide a promising alternative because cost per oligo is orders of magnitude lower. But so far, array-synthesized oligos have not been used for molecular programming applications because of the lower synthesis quality, variation in concentration between oligos and the low yield of each individual oligo in the pool. A second limitation comes from the use of fluorescence-based reporters for reading out the results of a computation. That is, due to spectral overlap between fluorophores, only a very small number of outputs or variables can be monitored in a given computation (i.e. as many as there are independent fluorescence channels, typically no more than 4 and only a limited number of computations can be performed in parallel (i.e. as many as there are separate reaction chambers, typically no more than 96). Next generation DNA sequencing and nanopore sensing methods could theoretically be used to read out hundreds to millions of DNA sequences or barcodes in parallel. However, so far, DNA sequencing and nanopore sensing have not been widely used to read out DNA computations because current gate architectures are not compatible with these read out methods. This project directly addresses these limitations to the current state of the art.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.