Expanding the Rule Set of DNA Circuitry with Associative Toehold Activation

ArticleinJournal of the American Chemical Society 134(1):263-71 · November 2011with27 Reads
DOI: 10.1021/ja206690a · Source: PubMed
  • 30.05 · University Carlos III de Madrid
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.
    • "Synthetic DNA must often be purified before downstream use [13]. Applications include toehold-mediated strand exchange [16] reaction or hairpin opening [17]. Synthesis errors such as single base eliminations and branch impurities can hurt reaction performance. "
    [Show abstract] [Hide abstract] ABSTRACT: A set of instruments and specialized equipment is necessary to equip a laboratory to work with DNA. Reducing the barrier to entry for DNA manipulation should enable and encourage new labs to enter the field. We present three examples of open source/DIY technology with significantly reduced costs relative to commercial equipment. This includes a gel scanner, a horizontal PAGE gel mold, and a homogenizer for generating DNA-coated particles. The overall cost savings obtained by using open source/DIY equipment was between 50 and 90%.
    Full-text · Article · Aug 2015
    • "However, the choice of length and sequence should be flexible as long as hybridization probability and thermodynamic stability under assay conditions remains high. The presence of two bulged thymidine bases at the three-way junction of associative DNA toeholds is known to accelerate strand displacement substantially, especially in reactions mediated by reversible toehold binding (Chen, 2012). Although the eight-nucleotide-trigger toehold is expected to bind reversibly (as is evident from the operation of multiple turnover RNA CHA circuits using eight-nucleotide toeholds; Bhadra & Ellington, 2014a), we have found it unnecessary to include bulged thymidines for stabilization of the RNA three-way junctions. "
    [Show abstract] [Hide abstract] ABSTRACT: We describe design parameters for the synthesis and analytical application of a label-free RNA molecular beacon, termed Spinach.ST. The RNA aptamer Spinach fluoresces upon binding the small-molecule fluorophore DFHBI ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one). Spinach has been reengineered by extending its 5'- and 3'-ends to create Spinach.ST, which is predicted to fold into an inactive conformation that fails to bind DHFBI. Hybridization of a trigger oligonucleotide to a designed toehold on Spinach.ST initiates toehold-mediated strand displacement and restores the DFHBI-binding, fluorescence-enhancing conformation of Spinach. The versatile Spinach.ST sensor can detect DNA or RNA trigger sequences and can readily distinguish single-nucleotide mismatches in the trigger toehold. Primer design techniques are described that augment amplicons produced by enzymatic amplification with Spinach.ST triggers. Interaction between these triggers and Spinach.ST molecular beacons leads to the real-time, sequence-specific quantitation of these amplicons. The use of Spinach.ST with isothermal amplification reactions such as nucleic acid sequence-based amplification (NASBA) may enable point-of-care applications. The same design principles could also be used to adapt Spinach reporters to the assay of nonnucleic acid analytes in trans. © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Jan 2015
    • "Upon amplification , domain D * will pair with the domain D segment generated from the template, forming a hairpin and bringing domains 5 * and 6 * close to one another for activation of Spinach. It has previously been shown that " associative toehold activation, " wherein the toehold and branch migration domains become connected via the hybridization of auxiliary domains, allows strand displacement across a threeway junction (Chen 2012). We have now for the first time successfully applied a similar concept to the cotranscriptional formation of RNA associative trigger sequences for the realtime sequence-specific detection of enzymatically generated RNA amplicons. "
    [Show abstract] [Hide abstract] ABSTRACT: We have re-engineered the fluorescent RNA aptamer Spinach to be activated in a sequence-dependent manner. The original Spinach aptamer was extended at its 5'- and 3'-ends to create Spinach.ST, which is predicted to fold into an inactive conformation and thus prevent association with the small molecule fluorophore DFHBI. Hybridization of a specific trigger oligonucleotide to a designed toehold leads to toehold-initiated strand displacement and refolds Spinach into the active, fluorophore-binding conformation. Spinach.ST not only specifically detects its target oligonucleotide but can discriminate readily against single-nucleotide mismatches. RNA amplicons produced during nucleic acid sequence-based amplification (NASBA) of DNA or RNA targets could be specifically detected and reported in real-time by conformational activation of Spinach.ST generated by in vitro transcription. In order to adapt any target sequence to detection by a Spinach reporter we used a primer design technique that brings together otherwise distal toehold sequences via hairpin formation. The same techniques could potentially be used to adapt common Spinach reporters to non-nucleic acid analytes, rather than by making fusions between aptamers and Spinach.
    Article · Jun 2014
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