Figures 1, 2, 3, 4, 6, 8 and Tables 2, 3, 10, 15, 16 are modified from Ref29. This work was supported by the National Science Foundation (grant NSF-CCF 1117143 and NSF-CCF 1162141 to G.S.). Y.-J. C. was supported by Taiwanese Government Fellowships. S.D.R was supported by the National Science Foundation Graduate Research Fellowship Program (GRFP).

<ol>
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The authors declare no competing financial interests.

This paper describes a method for deriving ndsDNA gates from highly pure plasmid DNA. Moreover, a protocol is presented for characterizing gate performance using a fluorescence kinetics assay. Experimental data shows that the plasmid-derived system outperforms its synthetic counterpart even if the synthetic system is assembled from strands purified using polyacrylamide gel electrophoresis (PAGE). Likely, the improved performance of plasmid-derived gates is primarily due to the very high purity of the biological DNA. Synthetic DNA contains a variety of errors, in particular deletions that result in oligonucleotides of length n-1, and such side products are typically not completely removed in PAGE or high-performance liquid chromatography (HPLC) purification procedures. Similar improvements to the ones reported here were also observed in a previous study of a catalyzed hairpin amplifier that used DNA derived from biological sources21.
However, even the use of plasmid-derived gates cannot completely eliminate errors in the gate performance, for which there are at least two reasons: first over-digestion or lack of cut precision can lead to gates with too many nicks or nicks in the wrong positions. In either case, gates are more likely to participate in undesired reactions. Such problems may be eased by optimizing the amount of enzyme used (see Figure 4). Second, in these experiments, most inputs and auxiliary strands were synthetic DNA and thus contained deletions and mutations. In principle, all single-stranded input and auxiliary strands could also be obtained from phagemid DNA through a nicking enzyme digestion of the pre-encoded m13 viral genome26. Perhaps the circuit performance can be further improved using ssDNA derived from the bacterial genome.
While the use of plasmid-derived gates was found to improve circuit performance, an analysis of the cost and processing times revealed that while the production of plasmid-derived gates is slightly cheaper (Table 15), it takes 2-3 times longer processing time compared to assembly and purification of gates from commercially synthesized oligos (Table 16). The primary costs of plasmid-derived gates are gene synthesis and the use of restriction enzymes. For 300 pmole of gates (enough for 15 reactions at 30 nM), the estimated cost for Join gates is approximately $170 and $200 for Fork gates, the cost difference being due to the use of different nicking enzymes. In contrast, the chemical synthesis of the strands for the same gate costs around $260 including a PAGE purification fee. The primary time cost for plasmid-derived gates is in the cloning procedure, which, just like DNA synthesis, can be outsourced to a gene synthesis company. However, once assembled, plasmid-derived gates have the advantage that the host plasmids can easily be replicated and can be stored in the form of bacterial glycerol stocks. This makes it possible to reuse the gates many times over.
Looking forward, the improved performance of plasmid-derived gates could enable a much larger range of dynamics behaviors than have been experimentally demonstrated so far with DNA CRNs. For example, recent theoretical work47,48 suggested that self-organized spatial patterns at the macro-scale can be realized with DNA CRNs through a reaction diffusion mechanism. The method presented here provides a viable path for constructing the underlying molecular components for such self-patterning DNA materials. Though challenging, developing macro-scale morphologies in a programmable way would have significant implications in areas ranging from biomaterials research to regenerative medicine.

The predictability of Watson-Crick base pairing has allowed dynamic DNA nanotechnology to emerge as a programmable way to design molecular devices with dynamic properties1,2. In particular, DNA strand displacement &#8212; a programmable, competitive hybridization reaction &#8212; has proved to be a powerful mechanism for engineering dynamic DNA systems. In a DNA strand displacement reaction, an incoming oligonucleotide displaces a previously bound &#8220;output&#8221; strand from a complementary binding partner. Multiple such reactions can be chained together into multi-step reaction cascades with a high degree of control over the order and timing of individual reaction steps3. DNA strand displacement cascades have been used to create digital and analog molecular circuits4-7, switchable nanostructures8-10, autonomous molecular motors11-15, and non-covalent catalytic amplifiers13,16-21. Moreover, DNA devices using strand displacement reactions can be simulated and designed for diverse applications using computer-assisted design software22-24.
Currently, chemically synthesized DNA serves as the main material for DNA nanotechnology. However, errors in the DNA synthesis process, and the resulting imperfect oligonucleotides, are believed to limit performance of dynamic DNA devices by causing erroneous side reactions. For example, &#8220;leak&#8221; reactions can result in the release of an output oligonucleotide even in the absence of a reaction trigger. Such effects are most obvious in autocatalytic reaction cascades where even a minimal amount of initial leak will eventually result in the full activation of the cascade19,20. Conversely, reactions often fail to reach the expected level of activation because some components do not trigger even in the presence of the intended input7,25. To make the performance of DNA-based nanodevices comparable to their biological protein-based counterparts, such error modes need to be dramatically reduced.
Bacterial plasmids or other biological DNA could serve as a relatively cheap source of highly pure DNA for nanotechnology applications. Large amounts of DNA can be generated by replication in bacteria and the intrinsic proofreading abilities of living systems ensure the purity of the resulting DNA. In fact, several recent papers have recognized the potential utility of biological DNA for nanotechnology applications21,26-28. However, the fully double-stranded nature of plasmid DNA has so far prohibited its use as a material for making dynamic DNA devices, which typically consist of multiple oligonucleotides and contain both double-stranded and single-stranded domains. In a recent paper29 this issue was addressed and a new DNA gate architecture that consists primarily of nicked double-stranded DNA (ndsDNA) was introduced.
Importantly, systems of ndsDNA gates can be designed that realize the dynamics specified by any formal chemical reaction network (CRN)29. ndsDNA gates could thus be used, in principle, to create dynamical systems that exhibit oscillations and chaos, bistability and memory, Boolean logic or algorithmic behaviors30-38. For example, Ref. 29 demonstrated a three-reaction CRN that provided a molecular implementation of a &#8220;consensus&#8221; protocol, a type of distributed computing algorithm29,39,40. This work first demonstrated a novel use for the CRN formalism as a &#8220;programming language&#8221; for rapidly synthesizing functional molecular systems (Figure 1A).
Here, a detailed protocol for deriving ndsDNA gates from plasmid DNA is provided. First is a review of the sequence design process. Then follows an explanation of how synthetic oligonucleotides containing the gate sequences are cloned into plasmids and sequence verified and amplified via bacterial culture. Next, it is shown how ndsDNA gates can be derived from the plasmids by enzymatic processing (see Figure 2). Finally, a method for testing gate behavior using fluorescence kinetics assays is outlined.
Reaction mechanism 
As an example, the protocol focuses on the catalytic chemical reaction A+B-&#62;B+C. The species A, B, and C (&#8220;signals&#8221;, Figure 1B) all correspond to a different single-stranded DNA molecule. The sequences of these molecules are completely independent and the strands do not react with one another directly. The sequences of all signals have two different functional domains, i.e., subsequences that act together in strand displacement reactions: 1) a short toehold domain (labels ta, tb, tc) that is used for initiation of strand displacement reaction, and 2) a long domain (labels a, b, c) that determines the signal identity.
Interactions between signal strands are mediated by nicked double-stranded DNA (ndsDNA) gate complexes (called JoinAB and ForkBC) and auxiliary single-stranded species (&#60;tr r&#62;, &#60;b tr&#62;, &#60;c tb&#62; and &#60;i tc&#62;). The formal reaction A+B-&#62;B+C is executed through a series of strand displacement reaction steps, where each reaction step exposes a toehold for a subsequent reaction (Figure 1B). In this example, signals A and B are initially free in solution while signal C is bound to the fork gate. At the end of the reaction B and C are in solution. More generally, signals that are bound to a gate are inactive while signals that are free in solution are active, that is, they can participate in a strand displacement reaction as an input. The time course of the reaction is followed using a fluorescent reporter strategy (Figure 1C). In previous work29, it was demonstrated that this reaction mechanism not only realizes the correct stoichiometry but also the kinetics of the target reaction.

<table border="0">
	<tbody>
		<tr>
			<td>Phusion High-Fidelity PCR Master Mix with HF Buffer</td>
			<td>NEB</td>
			<td>M0531S</td>
			<td></td>
		</tr>
		<tr>
			<td>PvuII-HF</td>
			<td>NEB</td>
			<td>R3151L</td>
			<td></td>
		</tr>
		<tr>
			<td>PstI-HF</td>
			<td>NEB</td>
			<td>R3140S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibson Assembly Master Mix</td>
			<td>NEB</td>
			<td>E2611S</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth, Modified</td>
			<td>SIGMA-ALDRICH</td>
			<td>T0918-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAGEN Hispeed Maxi-prep Kit</td>
			<td>QIAGEN</td>
			<td>12662</td>
			<td></td>
		</tr>
		<tr>
			<td>Nb.BsrDI</td>
			<td>NEB</td>
			<td>R0648L</td>
			<td></td>
		</tr>
		<tr>
			<td>Nt.BstNBI</td>
			<td>NEB</td>
			<td>R0607L</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000c</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Double-stranded Genomic Blocks</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Horiba Jobin-Yvon Spex Fluorolog-3 Fluorimeter</td>
			<td>Horiba/Jobin Yvon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synthetic Quartz Cells&#160;</td>
			<td>Starna</td>
			<td>23-5.45-S0G-5</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAGEN Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28706</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Backbones</td>
			<td>BioBrick</td>
			<td>E0240-pSB1A2</td>
			<td>High copy number plasmid with Ampicillin resistance. Sequence can be found from http://parts.igem.org</td>
		</tr>
	</tbody>
</table>

plasmid-derived dna strand displacement gates for implementing chemical reaction networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

Watch this Scientific Journal Video about Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks at JoVE.com

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

plasmid-derived-dna-strand-displacement-gates-for-implementing

Research

JoVE Journal

Biology

14.0K Views.  University of Washington. This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language. 