A modular DNA signal translator for the controlled release of a protein by an aptamer

Owing to the intimate linkage of sequence and structure in nucleic acids, DNA is an extremely attractive molecule for the development of molecular devices, in particular when a combination of information processing and chemomechanical tasks is desired. Many of the previously demonstrated devices are driven by hybridization between DNA ‘effector’ strands and specific recognition sequences on the device. For applications it is of great interest to link several of such molecular devices together within artificial reaction cascades. Often it will not be possible to choose DNA sequences freely, e.g. when functional nucleic acids such as aptamers are used. In such cases translation of an arbitrary ‘input’ sequence into a desired effector sequence may be required. Here we demonstrate a molecular ‘translator’ for information encoded in DNA and show how it can be used to control the release of a protein by an aptamer using an arbitrarily chosen DNA input strand. The function of the translator is based on branch migration and the action of the endonuclease FokI. The modular design of the translator facilitates the adaptation of the device to various input or output sequences

Nanopore Force Spectroscopy of Aptamer–Ligand Complexes

AbstractThe stability of aptamer–ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans–cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant Kd ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer–target interactions in this case, the stability of the ligand-free aptamer—containing G-quadruplexes—is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar Kd were identified

Diversity in the dynamical behaviour of a compartmentalized programmable biochemical oscillator

In vitro compartmentalization of biochemical reaction networks is a crucial step towards engineering artificial cell-scale devices and systems. At this scale the dynamics of molecular systems becomes stochastic, which introduces several engineering challenges and opportunities. Here we study a programmable transcriptional oscillator system that is compartmentalized into microemulsion droplets with volumes between 33 fl and 16 pl. Simultaneous measurement of large populations of droplets reveals major variations in the amplitude, frequency and damping of the oscillations. Variability increases for smaller droplets and depends on the operating point of the oscillator. Rather than reflecting the stochastic kinetics of the chemical reaction network itself, the variability can be attributed to the statistical variation of reactant concentrations created during their partitioning into droplets. We anticipate that robustness to partitioning variability will be a critical challenge for engineering cell-scale systems, and that highly parallel time-series acquisition from microemulsion droplets will become a key tool for characterization of stochastic circuit function.1172sciescopu

Structural DNA nanotechnology: from bases to bricks, from structure to function.

ABSTRACT The two fields of structural DNA nanotechnology and functional nucleic acids have been independently coevolving, with the former seeking to arrange and bring about movement of nucleic acid modules precisely and with control in space and the latter producing modules with incredible diversity in effective recognition and function. Here, we track the key developments in structural DNA nanotechnology that reveal a current trend that is seeing the integration of functional nucleic acid modules into their architectures to access a range of new functions. This contribution will seek to provide a perspective for the field of structural DNA nanotechnology where the integration of such functional modules on precisely controlled architectures can uncover phenomena of interest to physical chemists. D NA has proven to be a powerful material for construction on the nanoscale on the basis of the following properties: (i) the availability of automated synthetic methods and continually dropping costs, (ii) chemical robustness that confers stability on the resultant architectures and their subsequent ability to be functional under a variety of environmental conditions, (iii) the uniformly rodlike nature of the DNA double helix irrespective of its primary sequence, (iv) the specificity of Watson-Crick base pairing, which functions as an easily engineerable, site-specific, molecular-scale glue applicable to any DNA double helix, (v) the periodic nature of the DNA double helix and the predictable nature of sequence-specific thermal stability, both of which predispose it to computational methods to design and fabricate superarchitectures, (vi) the availability of well-characterized biochemical and molecular biological methods to cut, copy, and covalently link B-DNA double helices sequencespecifically, which allows manipulation of the construction material, (vii) the modular nature of the DNA scaffold that allows fabrication of architectures that are complex in terms of both structure and function when multiple modules are appended to each other, and (viii) single-stranded DNA sequences, called functional nucleic acids, which can fold and offer three-dimensional cavities suited to bind with great specificity a range of molecular entities with diverse function. In 1982, Ned Seeman proposed that DNA, which until then had been thought of as a linear polymer, could be used to make branched architectures by using stable artificial junctions with helical DNA limbs radiating from a central node. 1 These structures were analogous to metastable naturally occurring DNA motifs, such as the replication fork and Holliday junction. "It appears to be possible to generate covalently joined...networks of nucleic acids which are periodic in connectivity and perhaps in space." 1 This marked the origin of structural DNA nanotechnology that seeks to create defined architectures on the nanoscale using sequences of DNA that self-assemble into rigid rods that are, in turn, connected to form superarchitectures of precise dimensions. In 1999, it was shown that DNA could switch between two forms (the B-form and the Z-form), and this motion could be transduced along a DNA architecture, making it undergo a twisting motion. 2 Thus began a complementary aspect of structural DNA nanotechnology, of bringing about defined molecular-scale movements of DNA architectures triggered by the addition of input stimuli that are chemical, photonic, thermal, or electrical in nature. Functional nucleic acids are obtained from a test tube evolution method called SELEX independently conceptualized by the Szostak and Gold groups. 3,4 It uses molecular biology tools to pick out from a library of ∼10 15 different DNA (or RNA) sequences, a subset of sequences based on a given selection criterion and amplify them. 5 When subjected to the same selection criterion repeatedly with progressively higher stringencies, it is possible to progressively enrich from the library, a pool of DNA (or RNA) sequences with a specific functionality. If the selection criterion is the recognition of a target molecule, then selected single-stranded DNA (ssDNA) sequences are capable of binding to the target with high specificity and affinity. Thus, SELEX has yielded DNA sequences that can bind a huge variety of chemical entities ranging from small molecules to proteins, peptides, transition-stat

Periodic Operation of a Dynamic DNA Origami Structure Utilizing the Hydrophilic–Hydrophobic Phase‐Transition of Stimulus‐Sensitive Polypeptides

Dynamic DNA nanodevices are designed to perform structure‐encoded motion actuated by a variety of different physicochemical stimuli. In this context, hybrid devices utilizing other components than DNA have the potential to considerably expand the library of functionalities. Here, the reversible reconfiguration of a DNA origami structure using the stimulus sensitivity of elastin‐like polypeptides is reported. To this end, a rectangular sheet made using the DNA origami technique is functionalized with these peptides and by applying changes in salt concentration the hydrophilic–hydrophobic phase transition of these peptides actuate the folding of the structure. The on‐demand and reversible switching of the rectangle is driven by externally imposed temperature oscillations and appears at specific transition temperatures. Using transmission electron microscopy, it is shown that the structure exhibits distinct conformational states with different occupation probabilities, which are dependent on structure‐intrinsic parameters such as the local number and the arrangement of the peptides on the rectangle. It is also shown through ensemble fluorescence resonance energy transfer spectroscopy that the transition temperature and thus the thermodynamics of the rectangle‐peptide system depends on the stimuli salt concentration and temperature, as well as on the intrinsic parameters

Diversity in the dynamical behaviour of a compartmentalized programmable biochemical oscillator

In vitro compartmentalization of biochemical reaction networks is a crucial step towards engineering artificial cell-scale devices and systems. At this scale the dynamics of molecular systems becomes stochastic, which introduces several engineering challenges and opportunities. Here we study a programmable transcriptional oscillator system that is compartmentalized into microemulsion droplets with volumes between 33 fl and 16 pl. Simultaneous measurement of large populations of droplets reveals major variations in the amplitude, frequency and damping of the oscillations. Variability increases for smaller droplets and depends on the operating point of the oscillator. Rather than reflecting the stochastic kinetics of the chemical reaction network itself, the variability can be attributed to the statistical variation of reactant concentrations created during their partitioning into droplets. We anticipate that robustness to partitioning variability will be a critical challenge for engineering cell-scale systems, and that highly parallel time-series acquisition from microemulsion droplets will become a key tool for characterization of stochastic circuit functio

Optoelectronic properties of a photosystem I - carbon nanotube hybrid system

The photoconductance properties of photosystem I (PSI) covalently bound to carbon nanotubes (CNTs) are measured. We demonstrate that the PSI forms active electronic junctions with the CNTs enabling control of the CNTs photoconductance by the PSI. In order to electrically contact the photoactive proteins, a cysteine mutant is generated at one end of the PSI by genetic engineering. The CNTs are covalently bound to this reactive group using carbodiimide chemistry. We detect an enhanced photoconductance signal of the hybrid material at photon wavelengths resonant to the absorption maxima of the PSI compared to nonresonant wavelengths. The measurements prove that it is feasible to integrate photosynthetic proteins into optoelectronic circuits at the nanoscale

A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a

Point-of-care testing (POCT) in low-resource settings requires tools that can operate independently of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably reaches a lower limit of detection (LOD) of. 6.8 nM fluorescein, which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs with an LOD of 3.7 nM using a Cas13a-based fluorescence assay. Cas13a is an RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device may be used in educational settings, through providing low cost instrumentation for quantitative assays or as a platform to integrate hardware, software and biochemistry concepts in the future