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

利用分子成分进行计算可以使从健康诊断和治疗到信息技术等一系列领域的创新成为可能。例如，可以处理细胞分子水平和序列中编码的信息的分子电路可以成为生物医学应用的嵌入式控制器的核心。然而，构建分子控制器的能力远远落后于为机电设备设计嵌入式控制电路的能力。该项目旨在开发新的DNA电路写入和读取方法，这将使电路复杂性在目前的技术水平上至少提高一个数量级，从而使分子控制器更接近实际应用。此外，该项目将加速培养新兴分子信息系统领域的学生和专业人员。这一研究领域通过将历史上分离的计算机科学和生物学领域结合在一起，固有地增加了计算领域的人员、观点和背景的多样性。为了增加未被充分代表的人在分子编程研究中的参与，研究团队将接待来自雷尼尔学者的暑期实习生，雷尼尔学者是一个支持低收入背景的有色人种学生取得学术成功的组织。该项目引入了技术创新，将增加分子电路的可扩展性。pi将利用大规模并行DNA合成技术，并将其与高通量读出方法相结合，包括下一代测序和纳米孔传感。这些进步是重要的，因为DNA电路的扩展目前主要受到两方面的限制：第一个限制是绝大多数DNA门和类似的计算元件是由单个柱合成的寡核苷酸组装而成的，因为这种技术提供了低合成错误率和对单个链浓度的控制。然而，单独合成寡聚物的排序成本无法扩展。阵列合成低聚物提供了一个很有前途的选择，因为每个低聚物的成本要低几个数量级。但到目前为止，阵列合成的低聚物还没有被用于分子编程应用，因为合成质量较低，低聚物之间的浓度变化，以及池中每个低聚物的产率低。第二个限制来自于使用基于荧光的报告来读出计算结果。也就是说，由于荧光团之间的光谱重叠，在给定的计算中只能监测非常少量的输出或变量(即，有多少个独立的荧光通道，通常不超过4个；只有有限数量的计算可以并行执行（即，有多少个独立的反应室，通常不超过96个）。从理论上讲，下一代DNA测序和纳米孔传感方法可用于并行读取数亿个DNA序列或条形码。然而，到目前为止，DNA测序和纳米孔传感尚未广泛应用于DNA读出计算，因为目前的门结构与这些读出方法不兼容。这个项目直接解决了当前艺术状态的这些限制。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.