An official website of the United States government
DNA nanotechnology can be leveraged to engineer nanoscale biochemical reactions, and thus, revolutionize biomanufacturing. The programmability is encoded in the interactions between base pairs of the nucleic acids. Functional nanostructures can be envisioned and formed, such as DNA nanostars, whose properties can be fine-tuned by engineering the number of arms or base pairs per arm and can yield synthetic condensate structures, and DNA-based enzymes that exhibit peroxidase-like activity. For example, certain guanine-rich sequences of DNA can fold into a quadruplex structure, bind a hemin co-factor, and catalyze a peroxidation reaction in which the substrate ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) gets oxidized by hydrogen peroxide and results in a colorimetric change. Because ABTS produces a blue-green color change upon oxidation, it can be used to visually observe the peroxidation reaction taking place within the DNA condensates. In this work, peroxidase-mimicking DNAzymes were used to catalyze colorimetric peroxidation within DNA condensate compartments; and toehold-mediated strand displacement (TMSD) was explored as a strategy to program the peroxidation reaction–specifically, by unwinding the G-quadruplex structure, which would effectively turn the reaction “off”. TMSD is a method of designing a single strand of DNA with an additional overhang region, called a toehold, to oust and replace a second strand attached to the toehold-possessing target strand. The presence of complementary toeholds on both the invading strand and the target strand increases the thermodynamic probability of displacing the single DNA strand originally bound to the target. Here, TMSD was adapted for use in ‘turning off’ the DNAzyme-catalyzed peroxidation reaction, either by preventing folding or disrupting the folded structure of the DNAzyme. A displacer strand complementary to the DNAzyme/toehold region was designed and added to the reaction mixture at different time points and concentrations for this purpose. Elucidating mechanisms to unwind the G-quadruplex structure of DNAzymes has promise in treating genetic disorders caused by unregulated G4 formation in the human genome. Furthermore, DNA nanotechnology can be used to compartmentalize, functionalize, and program the release of bioactive molecules in drug delivery strategies and other synthetic biology applications, highlighting the potential of TMSD to program DNA-based bioreactors. This high-impact study, carried out as part of the NSF Future Manufacturing program at Pasadena City College in collaboration with UCLA, UCSB, and Caltech, allowed undergraduate researchers to design and conduct their own experiments within a community college setting after undergoing scientific training by graduate students and postdocs from our collaborators’ institutions. \n\nIt also provided opportunities to communicate the scientific research through writing, poster presentations at national conferences, and teaching in courses and STEM outreach. The student researchers of the PCC nanostar program applied their knowledge in a classroom setting, where they taught other undergraduate students how to conduct aspects of this research in a General, Organic and Biochemistry laboratory course at PCC. This article underscores the importance of creating significant research and teaching opportunities for students as they begin their careers in STEM, impactful mentorship through undergraduate research, and the creativity involved in modern synthetic biology research and in the development of accessible and innovative science lessons.
more »
« less
Toehold clipping: A mechanism for remote control of DNA strand displacement
Abstract The ability to create stimuli-responsive DNA nanostructures has played a prominent role in dynamic DNA nanotechnology. Primary among these is the process of toehold-based strand displacement, where a nucleic acid molecule can act as a trigger to cause conformational changes in custom-designed DNA nanostructures. Here, we add another layer of control to strand displacement reactions through a 'toehold clipping' process. By designing DNA complexes with a photocleavable linker-containing toehold or an RNA toehold, we show that we can use light (UV) or enzyme (ribonuclease) to eliminate the toehold, thus preventing strand displacement reactions. We use molecular dynamics simulations to analyze the structural effects of incorporating a photocleavable linker in DNA complexes. Beyond simple DNA duplexes, we also demonstrate the toehold clipping process in a model DNA nanostructure, by designing a toehold containing double-bundle DNA tetrahedron that disassembles when an invading strand is added, but stays intact after the toehold clipping process even in the presence of the invading strand. This work is an example of combining multiple physical or molecular stimuli to provide additional remote control over DNA nanostructure reconfiguration, advances that hold potential use in biosensing, drug delivery or molecular computation.
more »
« less
- Award ID(s):
- 1651877
- PAR ID:
- 10384469
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 51
- Issue:
- 8
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- p. 4055-4063
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
In molecular programming, the Chemical Reaction Network model is often used to describe real or hypothetical systems. Often, an interesting computational task can be done with a known hypothetical Chemical Reaction Network, but often such networks have no known physical implementation. One of the important breakthroughs in the field was that any Chemical Reaction Network can be physically implemented, approximately, using DNA strand displacement mechanisms. This allows us to treat the Chemical Reaction Network model as a programming language and the implementation schemes as its compiler. This also suggests that it would be useful to optimize the result of such a compilation, and in general to find effective ways to design better DNA strand displacement systems. We discuss DNA strand displacement systems in terms of "motifs", short sequences of elementary DNA strand displacement reactions. We argue that describing such motifs in terms of their inputs and outputs, then building larger systems out of the abstracted motifs, can be an efficient way of designing DNA strand displacement systems. We discuss four previously studied motifs in this abstracted way, and present a new motif based on cooperative 4-way strand exchange. We then show how Chemical Reaction Network implementations can be built out of abstracted motifs, discussing existing implementations as well as presenting two new implementations based on 4-way strand exchange, one of which uses the new cooperative motif. The new implementations both have two desirable properties not found in existing implementations, namely both use only at most 2-stranded DNA complexes for signal and fuel complexes and both are physically reversible. There are reasons to believe that those properties may make them more robust and energy-efficient, but at the expense of using more fuel complexes than existing implementation schemes.more » « less
-
DNA strand displacement cascades have proven to be a uniquely flexible and programmable primitive for constructing molecular logic circuits, smart structures and devices, and for systems with complex autonomously generated dynamics. Limiting their utility, however, strand displacement systems are susceptible to the spurious release of output even in the absence of the proper combination of inputs—so-called leak. A common mechanism for reducing leak involves clamping the ends of helices to prevent fraying, and thereby kinetically blocking the initiation of undesired displacement. Since a clamp must act as the incumbent toehold for toehold exchange, clamps cannot be stronger than a toehold. In this paper we systematize the properties of the simplest of strand displacement cascades (a translator) with toehold-size clamps. Surprisingly, depending on a few basic parameters, we find a rich and diverse landscape for desired and undesired properties and trade-offs between them. Initial experiments demonstrate a significant reduction of leak.more » « less
-
Abstract Artificial biomolecular condensates are emerging as a versatile approach to organize molecular targets and reactions without the need for lipid membranes. Here we ask whether the temporal response of artificial condensates can be controlled via designed chemical reactions. We address this general question by considering a model problem in which a phase separating component participates in reactions that dynamically activate or deactivate its ability to self-attract. Through a theoretical model we illustrate the transient and equilibrium effects of reactions, linking condensate response and reaction parameters. We experimentally realize our model problem using star-shaped DNA motifs known as nanostars to generate condensates, and we take advantage of strand invasion and displacement reactions to kinetically control the capacity of nanostars to interact. We demonstrate reversible dissolution and growth of DNA condensates in the presence of specific DNA inputs, and we characterize the role of toehold domains, nanostar size, and nanostar valency. Our results will support the development of artificial biomolecular condensates that can adapt to environmental changes with prescribed temporal dynamics.more » « less
-
Cascades of DNA strand displacement reactions enable the design of potentially large circuits with complex behaviour. Computational modelling of such systems is desirable to enable rapid design and analysis. In previous work, the expressive power of graph theory was used to enumerate reactions implementing strand displacement across a wide range of complex structures. However, coping with the rich variety of possible graph-based structures required enumeration rules with complicated side-conditions. This paper presents an alternative approach to tackle the problem of enumerating reactions at domain level involving complex structures by integrating with a geometric constraint solving algorithm. The rule sets from previous work are simplified by replacing side-conditions with a general check on the geometric plausibility of structures generated by the enumeration algorithm. This produces a highly general geometric framework for reaction enumeration. Here, we instantiate this framework to solve geometric constraints by a structure sampling approach in which we randomly generate sets of coordinates and check whether they satisfy all the constraints. We demonstrate this system by applying it to examples from the literature where molecular geometry plays an important role, including DNA hairpin and remote toehold reactions. This work therefore enables integration of reaction enumeration and structural modelling.more » « less
