Microfluidic platforms for the engineering of continuously-operating, synthetic nucleic acid-based systems

核酸纳米技术是一个多功能的平台，工程分子网络。通过设计具有特定序列的寡核苷酸分子，可以构建具有可预测和可编程反应的复杂合成系统。最通用的网络是基于“链置换”反应，其中碱基配对用于驱动多链“门复合物”中的一条或多条链的置换。这些置换反应的产物可以引发后续反应，从而构建大型网络。传统上，基本的门络合物是通过多步过程产生的，无法在原位实现。单独的链被单独合成，退火以形成多链门，然后这些门被混合以创建产生单个固定输出的网络。传统的基于链位移的网络在没有外部供应以这种方式产生的门的情况下不能连续操作。这一限制禁止了它们在工程化或合成细胞中的应用，在这些细胞中，网络必须使用原位和真实的时间产生的组分连续地响应于它们的环境。我们最近已经证明了直接从转录原位产生多链RNA门分子，利用将折叠的单链RNA转录物转化为多链复合物的自切割RNA核酶基序[Bae等人，https://pubs.acs.org/doi/10.1021/acs.nanolett.0c03629].这些构建体有可能成为连续运行的链置换网络的基础;该项目旨在使用可以维持连续运行网络的微流体“细胞”来探索这种潜力。结果将是一个新的工程平台的核酸纳米技术，也是一个重要的步骤，在将核酸为基础的电路到活细胞的过程中。学生将开始开发微流控平台，适合托管持续活跃的核酸为基础的系统，确定最佳的几何形状和材料的挑战。随后，学生将在构建的微流控芯片中测试越来越复杂的基于核酸的电路。该项目的两个阶段都将涉及计算建模的重要输入;因此，该项目非常适合寻求跨工程，纳米技术和生物学的跨学科挑战的学生。

Nucleic acid nanotechnology is a versatile platform for engineering molecular networks. By designing oligonucleotide molecules with specific sequences, one can build complex synthetic systems with predictable and programmable reactions. The most versatile networks are based on "strand displacement" reactions, in which base pairing is used to drive the replacement of one or more strands in a multi-stranded "gate complex". The outputs of these displacement reactions can trigger subsequent reactions, allowing the construction of large networks.Traditionally, the essential gate complexes are produced via a multi-step process that cannot be realised in situ. Individual strands are separately synthesized, annealed to form multi-stranded gates, and these gates are then mixed to create a network that produces a single fixed output. Traditional strand displacement-based networks cannot operate continuously without an external supply of gates produced in this way. This limitation prohibits their application in engineered or synthetic cells, where networks must continuously respond to their environment using components produced in situ and in real time.We have recently demonstrated in situ production of multi-stranded RNA gate molecules directly from transcription, utilising self-cleaving RNA ribozyme motifs that convert a folded single-stranded RNA transcript into a multi-stranded complex [Bae et al., https://pubs.acs.org/doi/10.1021/acs.nanolett.0c03629]. These constructs have the potential to underlie continuously-operating strand displacement networks; this project aims to explore this potential using microfluidic "cells" that can sustain continuously-operating networks. The result will be a novel engineering platform for nucleic acid nanotechnologists, and also a vital step in the process of incorporating nucleic acid-based circuits into living cells.The student will start by developing microfluidic platforms that are appropriate to hosting continuously active nucleic acid-based systems, identifying optimal geometries and materials for the challenge. Subsequently, the student will test nucleic acid-based circuits of increasing complexity in the microfluidic chips built. Both stages of the project will involve significant input from computational modelling; the project is therefore highly appropriate for students seeking an interdisciplinary challenge straddling engineering, nanotechnology and biology.