Diversifying Substrates and Reaction Conditions for Polymerase Strand Recycling

Cell-free biosensing
systems are being engineered as
versatile
and programmable diagnostic technologies. A core component of cell-free
biosensors is programmable molecular circuits that improve biosensor
speed, sensitivity, and specificity by performing molecular computations
such as logic evaluation and signal amplification. In previous work,
we developed one such circuit system called Polymerase Strand Recycling
(PSR), which amplifies cell-free molecular circuits by using T7 RNA
polymerase off-target transcription to recycle nucleic acid inputs.
We showed that PSR circuits can be configured to detect RNA target
inputs as well as be interfaced with allosteric transcription factor-based
biosensors to amplify signals and enhance sensitivity. Here we expand
the development of PSR circuit empirical design guidelines to generalize
the platform for detecting a diverse set of microRNA inputs. We show
that PSR circuit function can be enhanced through engineering T7 RNAP,
and we present troubleshooting strategies to optimize PSR circuit
performance.

无细胞生物传感系统目前正被工程化为兼具通用性与可编程性的诊断技术。
无细胞生物传感器的核心组件为可编程分子电路，此类电路可通过执行逻辑评估、信号扩增等分子计算操作，提升生物传感器的响应速度、灵敏度与特异性。
既往研究中，我们开发了一款名为聚合酶链循环（Polymerase Strand Recycling, PSR）的电路系统，该系统借助T7 RNA聚合酶（T7 RNA polymerase）的脱靶转录循环回收核酸输入物，以此实现无细胞分子电路的扩增。
我们证实，PSR电路可被配置为检测RNA靶标输入物，同时可与基于变构转录因子（allosteric transcription factor）的生物传感器对接，以完成信号扩增并提升检测灵敏度。
本研究进一步拓展了PSR电路的经验性设计指南，将该平台推广适配至多样化的微RNA（microRNA）输入检测场景。
我们证实，通过工程化改造T7 RNA聚合酶可增强PSR电路的功能，并提出了用于优化PSR电路性能的故障排查策略。