Programmable autonomous synthesis of single-stranded DNA


DNA performs diverse functional roles in biology, nanotechnology and biotechnology, but current methods for autonomously synthesizing arbitrary single-stranded DNA are limited. Here, we introduce the concept of primer exchange reaction (PER) cascades, which grow nascent single-stranded DNA with user-specified sequences following prescribed reaction pathways. PER synthesis happens in a programmable, autonomous, in situ and environmentally responsive fashion, providing a platform for engineering molecular circuits and devices with a wide range of sensing, monitoring, recording, signal-processing and actuation capabilities. We experimentally demonstrate a nanodevice that transduces the detection of a trigger RNA into the production of a DNAzyme that degrades an independent RNA substrate, a signal amplifier that conditionally synthesizes long fluorescent strands only in the presence of a particular RNA signal, molecular computing circuits that evaluate logic (AND, OR, NOT) combinations of RNA inputs, and a temporal molecular event recorder that records in the PER transcript the order in which distinct RNA inputs are sequentially detected.

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Figure 1: PER overview.
Figure 2: PER mechanism.
Figure 3: PER cascades.
Figure 4: PER nanodevice for conditional RNA degradation.
Figure 5: Signal amplifier with PER.
Figure 6: Logic computation with PER.
Figure 7: PER temporal molecular event recorder.


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The authors thank W. Shih, J. Kim, X. Chen, N. Hanikel, E. Winfree, B. Beliveau and N. Liu for their discussions and comments. This work was supported by the Office on Naval Research (grants N000141310593, N000141410610, N000141612182 and N000141612410), the National Science Foundation (grants CCF1317291, CMMI1334109 and 1540214), the National Institutes of Health (grant 1R01EB01865901) and the Wyss Institute's Molecular Robotics Initiative. J. Kishi was supported by an NSF graduate research fellowship and T. Schaus was supported by the Jane Coffin Childs Postdoctoral Fellowship.

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J.Y.K. conceived and designed the study, designed and performed the experiments, analysed the data and wrote the manuscript. T.E.S. designed and performed the experiments and analysed the data. N.G. designed and performed the experiments and analysed the data. F.X. designed and performed the experiments and analysed the data. P.Y. conceived and supervised the study, interpreted the data and wrote the manuscript. All authors reviewed, edited and approved the manuscript.

Corresponding author

Correspondence to Peng Yin.

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Competing interests

A provisional US patent has been filed based on this work. P.Y. is co-founder of Ultivue Inc. and NuProbe Global.

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Kishi, J., Schaus, T., Gopalkrishnan, N. et al. Programmable autonomous synthesis of single-stranded DNA. Nature Chem 10, 155–164 (2018).

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