Expanding the rule set of DNA circuitry with associative toehold activation.

Toehold-mediated strand displacement has proven extremely powerful in programming enzyme-free DNA circuits and DNA nanomachines. To achieve multistep, autonomous, and complex behaviors, toeholds must be initially inactivated by hybridizing to inhibitor strands or domains and then relieved from inactivation in a programmed, timed manner. Although powerful and reasonably robust, this strategy has several drawbacks that limit the architecture of DNA circuits. For example, the combination between toeholds and branch migration (BM) domains is 'hard wired' during DNA synthesis thus cannot be created or changed during the execution of DNA circuits. To solve this problem, I propose a strategy called 'associative toehold activation', where the toeholds and BM domains are connected via hybridization of auxiliary domains during the execution of DNA circuits. Bulged thymidines that stabilize DNA three-way junctions substantially accelerate strand displacement reactions in this scheme, allowing fast strand displacement initiated by reversible toehold binding. To demonstrate the versatility of the scheme, I show (1) run-time combination of toeholds and BM domains, (2) run-time recombination of toeholds and BM domains, which results in a novel operation 'toehold switching', and (3) design of a simple conformational self-replicator.

[1]  L. Orgel,et al.  Autocatalytic synthesis of a tetranucleotide analogue , 1987, Nature.

[2]  N. Leontis,et al.  Stability and structure of three-way DNA junctions containing unpaired nucleotides. , 1991, Nucleic acids research.

[3]  D. Sievers,et al.  Self-replication of complementary nucleotide-based oligomers , 1994, Nature.

[4]  F. J. Overmars,et al.  Solution structure of a DNA three-way junction containing two unpaired thymidine bases. Identification of sequence features that decide conformer selection. , 2000, Journal of molecular biology.

[5]  A. Turberfield,et al.  A DNA-fuelled molecular machine made of DNA , 2022 .

[6]  Andrew D. Ellington,et al.  Exponential growth by cross-catalytic cleavage of deoxyribozymogens , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Darko Stefanovic,et al.  A deoxyribozyme-based molecular automaton , 2003, Nature Biotechnology.

[8]  E. Shapiro,et al.  An autonomous molecular computer for logical control of gene expression , 2004, Nature.

[9]  S. Wijmenga,et al.  Global structure of a DNA three-way junction by solution NMR: towards prediction of 3H fold. , 2004, Nucleic acids research.

[10]  Michael Zuker,et al.  DINAMelt web server for nucleic acid melting prediction , 2005, Nucleic Acids Res..

[11]  E. Winfree,et al.  Construction of an in vitro bistable circuit from synthetic transcriptional switches , 2006, Molecular systems biology.

[12]  G. Seelig,et al.  Enzyme-Free Nucleic Acid Logic Circuits , 2022 .

[13]  D. Y. Zhang,et al.  Engineering Entropy-Driven Reactions and Networks Catalyzed by DNA , 2007, Science.

[14]  Harry M. T. Choi,et al.  Programming biomolecular self-assembly pathways , 2008, Nature.

[15]  D. Y. Zhang,et al.  Control of DNA strand displacement kinetics using toehold exchange. , 2009, Journal of the American Chemical Society.

[16]  G. F. Joyce,et al.  Self-Sustained Replication of an RNA Enzyme , 2009, Science.

[17]  Ruojie Sha,et al.  A Bipedal DNA Brownian Motor with Coordinated Legs , 2009, Science.

[18]  Mick F. Tuite,et al.  The prion hypothesis: from biological anomaly to basic regulatory mechanism , 2010, Nature Reviews Molecular Cell Biology.

[19]  G. Seelig,et al.  DNA as a universal substrate for chemical kinetics , 2010, Proceedings of the National Academy of Sciences.

[20]  N. Seeman,et al.  A Proximity-Based Programmable DNA Nanoscale Assembly Line , 2010, Nature.

[21]  N. Seeman.  Nanomaterials based on DNA. , 2010, Annual review of biochemistry.

[22]  E. Winfree,et al.  Synthetic in vitro transcriptional oscillators , 2011, Molecular systems biology.

[23]  Lulu Qian,et al.  Supporting Online Material Materials and Methods Figs. S1 to S6 Tables S1 to S4 References and Notes Scaling up Digital Circuit Computation with Dna Strand Displacement Cascades , 2022 .

[24]  A simple DNA gate motif for synthesizing large-scale circuits , 2011, Journal of The Royal Society Interface.

[25]  Teruo Fujii,et al.  Programming an in vitro DNA oscillator using a molecular networking strategy , 2011, Molecular Systems Biology.

[26]  G. Seelig,et al.  Dynamic DNA nanotechnology using strand-displacement reactions. , 2011, Nature chemistry.

[27]  Jonathan Bath,et al.  Remote toehold: a mechanism for flexible control of DNA hybridization kinetics. , 2011, Journal of the American Chemical Society.