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

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 ...

University of Washington

We study the negative correction to the quantized value 2e(2)/h of the conductance of a quantum point contact due to the backscattering of electrons by acoustic phonons. The correction shows activated temperature dependence and also gives rise to a zero-bias anomaly in conductance. Our results are in qualitative agreement with recent experiments studying the 0.7 feature in the conductance of quantum point contacts.

Electron-phonon scattering in quantum point contacts.

We study the resistivity of a quantum wire caused by backscattering of electrons by acoustic phonons. In the presence of Coulomb interactions, backscattering is strongly enhanced at low temperatures due to Luttinger liquid effects. Information about the strength of the interactions can be obtained from a measurement of the temperature dependence of the resistivity.

Phonon-induced resistivity of electron liquids in quantum wires.

Practically all of life's molecular processes, from chemical synthesis to replication, involve enzymes that carry out their functions through the catalytic transformation of metastable fuels into waste products. Catalytic control of reaction rates will prove to be as useful and ubiquitous in nucleic-acid-based engineering as it is in biology. Here we report a metastable DNA "fuel" and a corresponding DNA "catalyst" that improve upon the original hybridization-based catalyst system (Turberfield et al. Phys. Rev. Lett. 90, 118102-1-118102-4) by more than 2 orders of magnitude. This is achieved by identifying and purifying a fuel with a kinetically trapped metastable configuration consisting of a "kissing loop" stabilized by flanking helical domains; the catalyst strand acts by opening a helical domain and allowing the complex to relax to its ground state by a multistep pathway. The improved fuel/catalyst system shows a roughly 5000-fold acceleration of the uncatalyzed reaction, with each catalyst molecule capable of turning over in excess of 40 substrates. With k(cat)/K(M) approximately 10(7)/M/min, comparable to many protein enzymes and ribozymes, this fuel system becomes a viable component enabling future DNA-based synthetic molecular machines and logic circuits. As an example, we designed and characterized a signal amplifier based on the fuel-catalyst system. The amplifier uses a single strand of DNA as input and releases a second strand with unrelated sequence as output. A single input strand can catalytically trigger the release of more than 10 output strands.

Catalyzed relaxation of a metastable DNA fuel.

Biological organisms perform complex information processing and control tasks using sophisticated biochemical circuits, yet the engineering of such circuits remains ineffective compared with that of electronic circuits. To systematically create complex yet reliable circuits, electrical engineers use digital logic, wherein gates and subcircuits are composed modularly and signal restoration prevents signal degradation. We report the design and experimental implementation of DNA-based digital logic circuits. We demonstrate AND, OR, and NOT gates, signal restoration, amplification, feedback, and cascading. Gate design and circuit construction is modular. The gates use single-stranded nucleic acids as inputs and outputs, and the mechanism relies exclusively on sequence recognition and strand displacement. Biological nucleic acids such as microRNAs can serve as inputs, suggesting applications in biotechnology and bioengineering.

Enzyme-free nucleic acid logic circuits.

Molecular programming aims to systematically engineer molecular and chemical systems of autonomous function and ever-increasing complexity. A key goal is to develop embedded control circuitry within a chemical system to direct molecular events. Here we show that systems of DNA molecules can be constructed that closely approximate the dynamic behavior of arbitrary systems of coupled chemical reactions. By using strand displacement reactions as a primitive, we construct reaction cascades with effectively unimolecular and bimolecular kinetics. Our construction allows individual reactions to be coupled in arbitrary ways such that reactants can participate in multiple reactions simultaneously, reproducing the desired dynamical properties. Thus arbitrary systems of chemical equations can be compiled into real chemical systems. We illustrate our method on the Lotka-Volterra oscillator, a limit-cycle oscillator, a chaotic system, and systems implementing feedback digital logic and algorithmic behavior.

DNA as a universal substrate for chemical kinetics.

The specificity and predictability of Watson-Crick base pairing make DNA a powerful and versatile material for engineering at the nanoscale. This has enabled the construction of a diverse and rapidly growing set of DNA nanostructures and nanodevices through the programmed hybridization of complementary strands. Although it had initially focused on the self-assembly of static structures, DNA nanotechnology is now also becoming increasingly attractive for engineering systems with interesting dynamic properties. Various devices, including circuits, catalytic amplifiers, autonomous molecular motors and reconfigurable nanostructures, have recently been rationally designed to use DNA strand-displacement reactions, in which two strands with partial or full complementarity hybridize, displacing in the process one or more pre-hybridized strands. This mechanism allows for the kinetic control of reaction pathways. Here, we review DNA strand-displacement-based devices, and look at how this relatively simple mechanism can lead to a surprising diversity of dynamic behaviour.

Dynamic DNA nanotechnology using strand-displacement reactions.

Small variations in nucleic acid sequences can have far-reaching phenotypic consequences. Reliably distinguishing closely related sequences is therefore important for research and clinical applications. Here, we demonstrate that conditionally fluorescent DNA probes are capable of distinguishing variations of a single base in a stretch of target DNA. These probes use a novel programmable mechanism in which each single nucleotide polymorphism generates two thermodynamically destabilizing mismatch bubbles rather than the single mismatch formed during typical hybridization-based assays. Up to a 12,000-fold excess of a target that contains a single nucleotide polymorphism is required to generate the same fluorescence as one equivalent of the intended target, and detection works reliably over a wide range of conditions. Using these probes we detected point mutations in a 198 base-pair subsequence of the Escherichia coli rpoB gene. That our probes are constructed from multiple oligonucleotides circumvents synthesis limitations and enables long continuous DNA sequences to be probed.

Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA.

Biological organisms use complex molecular networks to navigate their environment and regulate their internal state. The development of synthetic systems with similar capabilities could lead to applications such as smart therapeutics or fabrication methods based on self-organization. To achieve this, molecular control circuits need to be engineered to perform integrated sensing, computation and actuation. Here we report a DNA-based technology for implementing the computational core of such controllers. We use the formalism of chemical reaction networks as a 'programming language' and our DNA architecture can, in principle, implement any behaviour that can be mathematically expressed as such. Unlike logic circuits, our formulation naturally allows complex signal processing of intrinsically analogue biological and chemical inputs. Controller components can be derived from biologically synthesized (plasmid) DNA, which reduces errors associated with chemically synthesized DNA. We implement several building-block reaction types and then combine them into a network that realizes, at the molecular level, an algorithm used in distributed control systems for achieving consensus between multiple agents.

Programmable chemical controllers made from DNA.

Achieving precise control of mammalian transgene expression has remained a long-standing, and increasingly urgent, challenge in biomedical science. Despite much work, single-cell methods have consistently revealed that mammalian gene expression levels remain susceptible to fluctuations (noise) and external perturbations. Here, we show that precise control of protein synthesis can be realized using a single-gene microRNA (miRNA)-based feed-forward loop (sgFFL). This minimal autoregulatory gene circuit consists of an intronic miRNA that targets its own transcript. In response to a step-like increase in transcription rate, the network generated a transient protein expression pulse before returning to a lower steady state level, thus exhibiting adaptation. Critically, the steady state protein levels were independent of the size of the stimulus, demonstrating that this simple network architecture effectively buffered protein production against changes in transcription. The single-gene network architecture was also effective in buffering against transcriptional noise, leading to reduced cell-to-cell variability in protein synthesis. Noise was up to 5-fold lower for a sgFFL than for an unregulated control gene with equal mean protein levels. The noise buffering capability varied predictably with the strength of the miRNA-target interaction. Together, these results suggest that the sgFFL single-gene motif provides a general and broadly applicable platform for robust gene expression in synthetic and natural gene circuits.

MicroRNA-based single-gene circuits buffer protein synthesis rates against perturbations.

The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology - applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems - lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

DNA nanotechnology from the test tube to the cell.

Most human transcripts are alternatively spliced, and many disease-causing mutations affect RNA splicing. Toward better modeling the sequence determinants of alternative splicing, we measured the splicing patterns of over two million (M) synthetic mini-genes, which include degenerate subsequences totaling over 100 M bases of variation. The massive size of these training data allowed us to improve upon current models of splicing, as well as to gain new mechanistic insights. Our results show that the vast majority of hexamer sequence motifs measurably influence splice site selection when positioned within alternative exons, with multiple motifs acting additively rather than cooperatively. Intriguingly, motifs that enhance (suppress) exon inclusion in alternative 5' splicing also enhance (suppress) exon inclusion in alternative 3' or cassette exon splicing, suggesting a universal mechanism for alternative exon recognition. Finally, our empirically trained models are highly predictive of the effects of naturally occurring variants on alternative splicing in vivo.

Article	Year
Electron-phonon scattering in quantum point contacts. Physical review letters\| PubMed ID: 12786092	2003
Phonon-induced resistivity of electron liquids in quantum wires. Physical review letters\| PubMed ID: 15783764	2005
Catalyzed relaxation of a metastable DNA fuel. Journal of the American Chemical Society\| PubMed ID: 16967972	2006
Enzyme-free nucleic acid logic circuits. Science (New York, N.Y.)\| PubMed ID: 17158324	2006
DNA as a universal substrate for chemical kinetics. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 20203007	2010
Dynamic DNA nanotechnology using strand-displacement reactions. Nature chemistry\| PubMed ID: 21258382	2011
Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA. Nature chemistry\| PubMed ID: 23965681	2013
Programmable chemical controllers made from DNA. Nature nanotechnology\| PubMed ID: 24077029	2013
MicroRNA-based single-gene circuits buffer protein synthesis rates against perturbations. ACS synthetic biology\| PubMed ID: 24847681	2014
DNA nanotechnology from the test tube to the cell. Nature nanotechnology\| PubMed ID: 26329111	2015
Learning the Sequence Determinants of Alternative Splicing from Millions of Random Sequences. Cell\| PubMed ID: 26496609	2015

Georg Seelig

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