The chemical synthesis of DNA oligonucleotides and their assembly into synthons, genes, circuits, and even entire genomes by gene synthesis methods has become an enabling technology for modern ...molecular biology and enables the design, build, test, learn, and repeat cycle underpinning innovations in synthetic biology. In this perspective, we briefly review the techniques and technologies that enable the synthesis of DNA oligonucleotides and their assembly into larger DNA constructs with a focus on recent advancements that have sought to reduce synthesis cost and increase sequence fidelity. The development of lower-cost methods to produce high-quality synthetic DNA will allow for the exploration of larger biological hypotheses by lowering the cost of use and help to close the DNA read-write cost gap.
While the field of DNA computing and molecular programming was engendered in large measure as a curiosity-driven exercise, it has taken on increasing importance for analytical applications. This is ...in large measure because of the modularity of DNA circuitry, which can serve as a programmable intermediate between inputs and outputs. These qualities may make nucleic acid circuits useful for making decisions relevant to diagnostic applications. This is especially true given that nucleic acid circuits can potentially directly interact with and be triggered by diagnostic nucleic acids and other analytes. Chemists are, by and large, unaware of many of these advances, and this Account provides a means of touching on what might seem to be an arcane field. We begin by explaining nucleic acid amplification reactions that can lead to signal amplification, such as catalytic hairpin assembly (CHA) and the hybridization chain reaction (HCR). In these circuits, a single-stranded input acts on kinetically trapped substrates via exposed toeholds and strand exchange reactions, refolding the substrates and allowing them to interact with one another. As multiple duplexes (CHA) or concatemers of increasing length (HCR) are generated, there are opportunities to couple these outputs to different analytical modalities, including transduction to fluorescent, electrochemical, and colorimetric signals. Because both amplification and transduction are at their root dependent on the programmability of Waston–Crick base pairing, nucleic acid circuits can be much more readily tuned and adapted to new applications than can many other biomolecular amplifiers. As an example, robust methods for real-time monitoring of isothermal amplification reactions have been developed recently. Beyond amplification, nucleic acid circuits can include logic gates and thresholding components that allow them to be used for analysis and decision making. Scalable and complex DNA circuits (seesaw gates) capable of carrying out operations such as taking square roots or implementing neural networks capable of learning have now been constructed. Into the future, we can expect that molecular circuitry will be designed to make decisions on the fly that reconfigure diagnostic devices or lead to new treatment options.
Signal amplification is a key component of molecular detection. Enzyme-free signal amplification is especially appealing for the development of low-cost, point-of-care diagnostics. It has been ...previously shown that enzyme-free DNA circuits with signal-amplification capacity can be designed using a mechanism called 'catalyzed hairpin assembly'. However, it is unclear whether the efficiency and modularity of such circuits is suitable for multiple analytical applications. We have therefore designed and characterized a simplified DNA circuit based on catalyzed hairpin assembly, and applied it to multiple different analytical formats, including fluorescent, colorimetric, and electrochemical and signaling. By optimizing the design of previous hairpin-based catalytic assemblies we found that our circuit has almost zero background and a high catalytic efficiency, with a k(cat) value above 1 min(-1). The inherent modularity of the circuit allowed us to readily adapt our circuit to detect both RNA and small molecule analytes. Overall, these data demonstrate that catalyzed hairpin assembly is suitable for analyte detection and signal amplification in a 'plug-and-play' fashion.
Plastic waste poses an ecological challenge
and enzymatic degradation offers one, potentially green and scalable, route for polyesters waste recycling
. Poly(ethylene terephthalate) (PET) accounts ...for 12% of global solid waste
, and a circular carbon economy for PET is theoretically attainable through rapid enzymatic depolymerization followed by repolymerization or conversion/valorization into other products
. Application of PET hydrolases, however, has been hampered by their lack of robustness to pH and temperature ranges, slow reaction rates and inability to directly use untreated postconsumer plastics
. Here, we use a structure-based, machine learning algorithm to engineer a robust and active PET hydrolase. Our mutant and scaffold combination (FAST-PETase: functional, active, stable and tolerant PETase) contains five mutations compared to wild-type PETase (N233K/R224Q/S121E from prediction and D186H/R280A from scaffold) and shows superior PET-hydrolytic activity relative to both wild-type and engineered alternatives
between 30 and 50 °C and a range of pH levels. We demonstrate that untreated, postconsumer-PET from 51 different thermoformed products can all be almost completely degraded by FAST-PETase in 1 week. FAST-PETase can also depolymerize untreated, amorphous portions of a commercial water bottle and an entire thermally pretreated water bottle at 50 ºC. Finally, we demonstrate a closed-loop PET recycling process by using FAST-PETase and resynthesizing PET from the recovered monomers. Collectively, our results demonstrate a viable route for enzymatic plastic recycling at the industrial scale.
The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported ...infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.
Aptamers continue to receive interest as potential therapeutic agents for the treatment of diseases, including cancer. In order to determine whether aptamers might eventually prove to be as useful as ...other clinical biopolymers, such as antibodies, we selected aptamers against an important clinical target, human epidermal growth factor receptor (hEGFR). The initial selection yielded only a single clone that could bind to hEGFR, but further mutation and optimization yielded a family of tight-binding aptamers. One of the selected aptamers, E07, bound tightly to the wild-type receptor (K(d) = 2.4 nM). This aptamer can compete with EGF for binding, binds to a novel epitope on EGFR, and also binds a deletion mutant, EGFRvIII, that is commonly found in breast and lung cancers, and especially in grade IV glioblastoma multiforme, a cancer which has for the most part proved unresponsive to current therapies. The aptamer binds to cells expressing EGFR, blocks receptor autophosphorylation, and prevents proliferation of tumor cells in three-dimensional matrices. In short, the aptamer is a promising candidate for further development as an anti-tumor therapeutic. In addition, Aptamer E07 is readily internalized into EGFR-expressing cells, raising the possibility that it might be used to escort other anti-tumor or contrast agents.
Honey bees are essential pollinators threatened by colony losses linked to the spread of parasites and pathogens. Here, we report a new approach for manipulating bee gene expression and protecting ...bee health. We engineered a symbiotic bee gut bacterium,
, to induce eukaryotic RNA interference (RNAi) immune responses. We show that engineered
can stably recolonize bees and produce double-stranded RNA to activate RNAi and repress host gene expression, thereby altering bee physiology, behavior, and growth. We used this approach to improve bee survival after a viral challenge, and we show that engineered
can kill parasitic
mites by triggering the mite RNAi response. This symbiont-mediated RNAi approach is a tool for studying bee functional genomics and potentially for safeguarding bee health.
Loop-mediated isothermal amplification (LAMP) is an extremely powerful tool for the detection of nucleic acids with high sensitivity and specificity. However, LAMP shows optimal performance at around ...65 °C, which limits applications in point-of-care-testing (POCT). Here, we have developed a version of LAMP that uses phosphorothioated primers (PS-LAMP) to enable more efficient hairpin formation and extension at the termini of growing concatamers, and that therefore works at much lower temperatures. By including additional factors such as chaotropes (urea) and single-stranded DNA binding protein (SSB), the sensitivities and selectivities for amplicon detection with PS-LAMP at 40 °C were comparable with a regular LAMP reaction at 65 °C.
Stacking nonenzymatic circuits for high signal gain Chen, Xi; Briggs, Neima; McLain, Jeremy R. ...
Proceedings of the National Academy of Sciences - PNAS,
04/2013, Letnik:
110, Številka:
14
Journal Article
Recenzirano
Odprti dostop
Signal amplification schemes that do not rely on protein enzymes show great potential in areas as abstruse as DNA computation and as applied as point-of-care molecular diagnostics. Toehold-mediated ...strand displacement, a programmable form of dynamic DNA hybridization, can be used to design powerful amplification cascades that can achieve polynomial or exponential amplification of input signals. However, experimental implementation of such amplification cascades has been severely hindered by circuit leakage due to catalyst-independent side reactions. In this study, we systematically analyzed the origins, characteristics, and outcomes of circuit leakage in amplification cascades and devised unique methods to obtain high-quality DNA circuits that exhibit minimal leakage. We successfully implemented a two-layer cascade that yielded 7,000-fold signal amplification and a two-stage, four-layer cascade that yielded upward of 600,000-fold signal amplification. Implementation of these unique methods and design principles should greatly empower molecular programming in general and DNA-based molecular diagnostics in particular.
Catalytic hairpin assembly (CHA) has previously proven useful as a transduction and amplification method for nucleic acid detection. However, the two hairpin substrates in a CHA circuit can ...potentially react non‐specifically even in the absence of a single‐stranded catalyst, and this non‐specific background degrades the signal‐to‐noise ratio. The introduction of mismatched base pairs that impede uncatalyzed strand exchange reactions led to a significant decrease of the background signal, while only partially damping the signal in the presence of a catalyst. Various types and lengths of mismatches were assayed by fluorimetry, and in many instances, our MismatCHA designs yielded 100‐fold increased signal‐to‐background ratios compared to a ratio of 4:1 with the perfectly matched substrates. These observations could be of general utility for the design of non‐enzymatic nucleic acid circuits.
Catalytic hairpin assembly (CHA) is a useful amplification method for the detection of nucleic acids. CHA circuits, however, have been shown to execute non‐specifically even in the absence of catalyst, which can make quantitation of lower input concentrations difficult. By introducing two mismatched bases into a specific domain on the circuit, the signal‐to‐background ratio can be improved from less than 10:1 to over 100:1.
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