<i>In Vitro</i> Transcription Networks Based on Hairpin Promoter Switches

<i>In vitro</i> transcription networks are analogs of naturally occurring gene regulatory networks that consist of synthetic DNA templates that are cross-regulated by their own transcripts. This ability to design and execute <i>in vitro</i> transcription networks has allowed bottom-up construction of complex network topologies with predictable dynamic behavior. Here we describe the simplified design of an <i>in vitro</i> transcription network based on single-stranded synthetic DNA hairpin switches that function similar to molecular beacons, <i>via</i> toehold mediated strand displacement. Systematic construction of increasingly larger circuits was achieved by programming interactions between multiple switches through rational sequence design, and the dynamic behavior of networks was accurately predicted using a simple mathematical model. Ultimately, we engineered a cascade of switches that acted as a Boolean complete NAND gate capable of sensing both DNA and RNA inputs. The tools and framework that have been developed makes the execution of <i>in vitro</i> transcription circuits much simpler, which will enable them to more readily serve as testbeds for nucleic acid computations both <i>in vitro</i> and <i>in vivo</i>

Design and Selection of a Synthetic Operon

Cell-free systems are showing increasing promise for biosynthesis of both proteins and small molecules. However, <i>in vitro</i> transcription and translation reactions have so far primarily been used for the production of single proteins. In order to demonstrate the possibilities for coupled reactions, we designed synthetic operons that included different combinations of wild-type or evolved biotin ligases and streptavidins and demonstrated a mechanism for self-selection of operons following expression <i>in vitro</i>. Peptide substrates for biotin ligase were conjugated to the DNA operons and could be modified by a biotin ligase specific for either biotin or desthiobiotin and subsequently captured via a streptavidin specific for either biotin or desthiobiotin

Evolving Orthogonal Suppressor tRNAs To Incorporate Modified Amino Acids

There have been considerable advancements in the incorporation of noncanonical amino acids (ncAA) into proteins over the last two decades. The most widely used method for site-specific incorporation of noncanonical amino acids, amber stop codon suppression, typically employs an orthogonal translation system (OTS) consisting of a heterologous aminoacyl-tRNA synthetase:tRNA pair that can potentially expand an organism’s genetic code. However, the orthogonal machinery sometimes imposes fitness costs on an organism, in part due to mischarging and a lack of specificity. Using compartmentalized partnered replication (CPR) and a newly developed <i>pheS</i> negative selection, we evolved several new orthogonal <i>Methanocaldococcus jannaschii</i> (<i>Mj</i>) tRNA variants tRNAs with increased amber suppression activity, but that also showed up to 3-fold reduction in promiscuous aminoacylation by endogenous aminoacyl-tRNA synthetases (aaRSs). The increased orthogonality of these variants greatly reduced organismal fitness costs associated in part due to tRNA mischarging. Using these methods, we were also able to evolve tRNAs that supported the specific incorporation of 3-halo-tyrosines (3-Cl-Y, 3-Br-Y, and 3-I-Y) in <i>E. coli</i>

Adapting Enzyme-Free DNA Circuits to the Detection of Loop-Mediated Isothermal Amplification Reactions

Loop-mediated isothermal amplification of DNA (LAMP) is a powerful isothermal nucleic acid amplification technique that can accumulate ∼10⁹ copies from less than 10 copies of input template within an hour or two. Unfortunately, while the amplification reactions are extremely powerful, the quantitative detection of LAMP products is still analytically difficult. In this article, to both improve the specificity of LAMP detection and to make direct readout of LAMP amplification simpler and much more reliable, we have developed a nonenzymatic nucleic acid circuit (catalyzed hairpin assembly, CHA) that can both amplify and integrate the specific sequence signals present in LAMP amplicons. Through a hairpin acceptor, one of the four loop products amplified from the LAMP is transduced to an active catalyst ssDNA which can in turn trigger a CHA reaction. After CHA detection, even less than 10 molecules/μL model templates (<b>M13mp18</b>) can produce significant signal, and both nonspecific template and parasitic amplicons cannot bring interference at all. More importantly, to further enhance the specificity, we have designed a dual-CHA circuit that only gave positive responses in presence of two LAMP loops. The AND-GATE detector will act as a simultaneous, specific readout of the LAMP product, rather than of competing and parasitic amplicons

A Simple, Cleated DNA Walker That Hangs on to Surfaces

We designed and demonstrated a single-legged or unipedal walker that has a “cleat” that allows it to persistently associate with a track and make autonomous decisions about movement. The walker is highly processive over long periods of time, as shown by its movement over a microparticle surface suffused with substrate. The simple design can be readily optimized on the basis of simple energetic considerations. The walker can be used for signal amplification and should prove especially valuable for programming amorphous computations within chemical reaction networks

Directed Evolution of a Panel of Orthogonal T7 RNA Polymerase Variants for <i>in Vivo</i> or <i>in Vitro</i> Synthetic Circuitry

T7 RNA polymerase is the foundation of synthetic biological circuitry both <i>in vivo</i> and <i>in vitro</i> due to its robust and specific control of transcription from its cognate promoter. Here we present the directed evolution of a panel of orthogonal T7 RNA polymerase:promoter pairs that each specifically recognizes a synthetic promoter. These newly described pairs can be used to independently control up to six circuits in parallel

DNA Detection Using Origami Paper Analytical Devices

We demonstrate the hybridization-induced fluorescence detection of DNA on an origami-based paper analytical device (<i>o</i>PAD). The paper substrate was patterned by wax printing and controlled heating to construct hydrophilic channels and hydrophobic barriers in a three-dimensional fashion. A competitive assay was developed where the analyte, a single-stranded DNA (ssDNA), and a quencher-labeled ssDNA competed for hybridization with a fluorophore-labeled ssDNA probe. Upon hybridization of the analyte with the fluorophore-labeled ssDNA, a linear response of fluorescence vs analyte concentration was observed with an extrapolated limit of detection <5 nM and a sensitivity relative standard deviation as low as 3%. The <i>o</i>PAD setup was also tested against OR/AND logic gates, proving to be successful in both detection systems

Phosphorothioated Primers Lead to Loop-Mediated Isothermal Amplification at Low Temperatures

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

Probing Spatial Organization of DNA Strands Using Enzyme-Free Hairpin Assembly Circuits

Catalyzed hairpin assembly (CHA) is a robust enzyme-free signal-amplification reaction that has a wide range of potential applications, especially in biosensing. Although most studies of the analytical applications of CHA have focused on the measurement of concentrations of biomolecules, we show here that CHA can also be used to probe the spatial organization of biomolecules such as single-stranded DNA. The basis of such detection is the fact that a DNA structure that brings a toehold and a branch-migration domain into close proximity can catalyze the CHA reaction. We quantitatively studied this phenomenon and applied it to the detection of domain reorganization that occurs during DNA self-assembly processes such as the hybridization chain reaction (HCR). We also show that CHA circuits can be designed to detect certain types of hybridization defects. This principle allowed us to develop a “signal on” assay that can simultaneously respond to multiple types of mutations in a DNA strand in one simple reaction, which is of great interest in genotyping and molecular diagnostics. These findings highlight the potential impacts of DNA circuitry on DNA nanotechnology and provide new tools for further development of these fields

Evolution of a Thermophilic Strand-Displacing Polymerase Using High-Temperature Isothermal Compartmentalized Self-Replication

Strand-displacing polymerases are a crucial component of isothermal amplification (IA) reactions, where the lack of thermal cycling reduces equipment needs and improves the time to answer, especially for point-of-care applications. In order to improve the function of strand-displacing polymerases, we have developed an emulsion-based directed evolution scheme, high-temperature isothermal compartmentalized self-replication (HTI-CSR) that does not rely on thermal cycling. Starting from an algorithm-optimized shuffled library of exonuclease-deficient Family A polymerases from Geobacillus stearothermophilus (Bst LF) and Thermus aquaticus (Klentaq), we have applied HTI-CSR to generate a more thermostable strand-displacing polymerase variant that performs well in loop-mediated isothermal amplification and rolling circle amplification, even after thermal challenges of up to 95 °C that lead to better primer annealing. The new enzyme (v5.9) is also capable of a variety of new reactions, including isothermal hyperbranched rolling circle amplification. The HTI-CSR method should now prove useful for evolving additional beneficial phenotypes in strand-displacing polymerases