Analysis of macerated ticks using Boolean logic gating colorimetric isothermal nucleic acid assays for Lyme Borrelia and Ixodes scapularis ticks.

Lyme disease, one of the most common tickborne diseases, has been rapidly spreading in parallel with the expansion of the range of its tick vector. Better tick surveillance efforts are needed to accurately estimate disease risk and to guide public health and clinical management. We have developed two multiplex loop-mediated isothermal amplification (LAMP) reactions coupled with oligonucleotide strand displacement (OSD) probes to identify the tick host, Ixodes scapularis, and the Lyme disease pathogen, Borrelia burgdorferi, they carry. In each multiplex LAMP-OSD assay the co-presence of two target sequences is computed at the DNA level by linking the two corresponding amplicons and detecting the co-product on colorimetric lateral flow dipsticks. In tests with synthetic DNA, the co-presence of as few as four copies of input DNA could be detected, without producing spurious signals. Most importantly, though, the LAMP-OSD assay is amenable to being carried out directly with macerated tick samples, without any sample preparation. In such field conditions, assays performed robustly and demonstrated 97-100% sensitivity and 100% specificity with both field-collected and lab-raised artificially infected ticks. Such easy-to-use, arthropod and pathogen-specific assays would be well suited to field and near patient use without relying on complex instrumentation or infrastructure.

Improved Bst DNA Polymerase Variants Derived via a Machine Learning Approach.

The DNA polymerase I from Geobacillus stearothermophilus (also known as Bst DNAP) is widely used in isothermal amplification reactions, where its strand displacement ability is prized. More robust versions of this enzyme should be enabled for diagnostic applications, especially for carrying out higher temperature reactions that might proceed more quickly. To this end, we appended a short fusion domain from the actin-binding protein villin that improved both stability and purification of the enzyme. In parallel, we have developed a machine learning algorithm that assesses the relative fit of individual amino acids to their chemical microenvironments at any position in a protein and applied this algorithm to predict sequence substitutions in Bst DNAP. The top predicted variants had greatly improved thermotolerance (heating prior to assay), and upon combination, the mutations showed additive thermostability, with denaturation temperatures up to 2.5 °C higher than the parental enzyme. The increased thermostability of the enzyme allowed faster loop-mediated isothermal amplification assays to be carried out at 73 °C, where both Bst DNAP and its improved commercial counterpart Bst 2.0 are inactivated. Overall, this is one of the first examples of the application of machine learning approaches to the thermostabilization of an enzyme.

Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting.

Loop-mediated isothermal amplification (LAMP) has proven to be easier to implement than PCR for point-of-care diagnostic tests. However, the underlying mechanism of LAMP is complicated and the kinetics of the major steps in LAMP have not been fully elucidated, which prevents rational improvements in assay development. Here we present our work to characterize the kinetics of the elementary steps in LAMP and show that: (i) strand invasion / initiation is the rate-limiting step in the LAMP reaction; (ii) the loop primer plays an important role in accelerating the rate of initiation and does not function solely during the exponential amplification phase and (iii) strand displacement synthesis by Bst-LF polymerase is relatively fast (125 nt/s) and processive on both linear and hairpin templates, although with some interruptions on high GC content templates. Building on these data, we were able to develop a kinetic model that relates the individual kinetic experiments to the bulk LAMP reaction. The assays developed here provide important insights into the mechanism of LAMP, and the overall model should be crucial in engineering more sensitive and faster LAMP reactions. The kinetic methods we employ should likely prove useful with other isothermal DNA amplification methods.

Developing predictive hybridization models for phosphorothioate oligonucleotides using high-resolution melting.

The ability to predict nucleic acid hybridization energies has been greatly enabling for many applications, but predictive models require painstaking experimentation, which may limit expansion to non-natural nucleic acid analogues and chemistries. We have assessed the utility of dye-based, high-resolution melting (HRM) as an alternative to UV-Vis determinations of hyperchromicity in order to more quickly acquire parameters for duplex stability prediction. The HRM-derived model for phosphodiester (PO) DNA can make comparable predictions to previously established models. Using HRM, it proved possible to develop predictive models for DNA duplexes containing phosphorothioate (PS) linkages, and we found that hybridization stability could be predicted as a function of sequence and backbone composition for a variety of duplexes, including PS:PS, PS:PO, and partially modified backbones. Individual phosphorothioate modifications destabilize helices by around 0.12 kcal/mol on average. Finally, we applied these models to the design of a catalytic hairpin assembly circuit, an enzyme-free amplification method used for nucleic acid-based molecular detection. Changes in PS circuit behavior were consistent with model predictions, further supporting the addition of HRM modeling and parameters for PS oligonucleotides to the rational design of nucleic acid hybridization.

Preparation and Use of Cellular Reagents: A Low-resource Molecular Biology Reagent Platform.

Protein reagents are indispensable for most molecular and synthetic biology procedures. Most conventional protocols rely on highly purified protein reagents that require considerable expertise, time, and infrastructure to produce. In consequence, most proteins are acquired from commercial sources, reagent expense is often high, and accessibility may be hampered by shipping delays, customs barriers, geopolitical constraints, and the need for a constant cold chain. Such limitations to the widespread availability of protein reagents, in turn, limit the expansion and adoption of molecular biology methods in research, education, and technology development and application. Here, we describe protocols for producing a low-resource and locally sustainable reagent delivery system, termed "cellular reagents," in which bacteria engineered to overexpress proteins of interest are dried and can then be used directly as reagent packets in numerous molecular biology reactions, without the need for protein purification or a constant cold chain. As an example of their application, we describe the execution of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) using cellular reagents, detailing how to replace pure protein reagents with optimal amounts of rehydrated cellular reagents. We additionally describe a do-it-yourself fluorescence visualization device for using these cellular reagents in common molecular biology applications. The methods presented in this article can be used for low-cost, on-site production of commonly used molecular biology reagents (including DNA and RNA polymerases, reverse transcriptases, and ligases) with minimal instrumentation and expertise, and without the need for protein purification. Consequently, these methods should generally make molecular biology reagents more affordable and accessible. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cellular reagents Alternate Protocol 1: Preparation of lyophilized cellular reagents Alternate Protocol 2: Evaluation of bacterial culture growth via comparison to McFarland turbidity standards Support Protocol 1: SDS-PAGE for protein expression analysis of cellular reagents Basic Protocol 2: Using Taq DNA polymerase cellular reagents for PCR Basic Protocol 3: Using Br512 DNA polymerase cellular reagents for loop-mediated isothermal amplification (LAMP) Support Protocol 2: Building a fluorescence visualization device.

Charge Engineering Improves the Performance of Bst DNA Polymerase Fusions.

The charge states of proteins can greatly influence their stabilities and interactions with substrates, and the addition of multiple charges (supercharging) has been shown to be a successful approach for engineering protein stability and function. The addition of a fast-folding fusion domain to the Bacillus stearothermophilus DNA polymerase improved its functionality in isothermal amplification assays, and further charge engineering of this domain has increased both protein stability and diagnostics performance. When combined with mutations that stabilize the core of the protein, the charge-engineered fusion domain leads to the ability to carry out loop-mediated isothermal amplification (LAMP) at temperatures up to 74° C or in the presence of high concentrations of urea, with detection times under 10 min. Adding both positive and negative charges to the fusion domain led to changes in the relative reverse transcriptase and DNA polymerase activities of the polymerase. Overall, the development of a modular fusion domain whose charged surface can be modified at will should prove to be of use in the engineering of other polymerases and, in general, may prove useful for protein stabilization.

Producing molecular biology reagents without purification.

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.

Minimizing Leakage in Stacked Strand Exchange Amplification Circuits.

Signal amplification is ubiquitous in biology and engineering. Protein enzymes, such as DNA polymerases, can routinely achieve >106-fold signal increase, making them powerful tools for signal enhancement. Considerable signal amplification can also be achieved using nonenzymatic, cascaded nucleic acid strand exchange reactions. However, the practical application of such kinetically trapped circuits has so far proven difficult due to uncatalyzed leakage of the cascade. We now demonstrate that strategically positioned mismatches between circuit components can reduce unprogrammed hybridization reactions and therefore greatly diminish leakage. In consequence, we were able to synthesize a three-layer catalytic hairpin assembly cascade that could operate in a single tube and that yielded 3.7 × 104-fold signal amplification in only 4 h, a greatly improved performance relative to previous cascades. This advance should facilitate the implementation of nonenzymatic signal amplification in molecular diagnostics, as well as inform the design of a wide variety of increasingly intricate nucleic acid computation circuits.

High-Surety Isothermal Amplification and Detection of SARS-CoV-2.

Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva.IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes.

One-Enzyme Reverse Transcription qPCR Using Taq DNA Polymerase.

Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCRs. We demonstrate the utility of Taq-alone RT-qPCRs by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/µL of input viral genomic RNA.

Comparison of media and standards for SARS-CoV-2 RT-qPCR without prior RNA preparation.

Since the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there have been demands on the testing infrastructure that have strained testing capacity. As a simplification of method, we confirm the efficacy of RNA extraction-free RT-qPCR and saline as an alternative patient sample storage buffer. In addition, amongst potential reagent shortages, it has sometimes been difficult to obtain inactivated viral particles. We have therefore also characterized armored SARS-CoV-2 RNA from Asuragen as an alternative diagnostic standard to ATCC genomic SARS-CoV-2 RNA and heat inactivated virions and provide guidelines for its use in RT-qPCR.

Dynamic Programming of a DNA Walker Controlled by Protons.

We have now constructed a four-legged DNA walker based on toehold exchange reactions whose movement is controlled by alternating pH changes. A well-characterized, pH-responsive CG-C+ triplex DNA was embedded into a tetrameric catalytic hairpin assembly (CHA) walker. The proton-controlled walker could autonomously move on otherwise unprogrammed microparticles surface, and the walking rate and steps of walking were efficiently controlled by pH. The starting and stopping of the walker, and its association and dissociation from the microparticles, could also be dynamically controlled by pH. The simple, programmable, and robust nature of this proton-controlled walker now provides the impetus for the development of a wide variety of more practical nanomachines.

Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay.

We have developed a generalizable "smart molecular diagnostic" capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR logic gate (gate output is true if either one or more gate inputs is true) signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our methodology by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with crudely macerated ZIKV-infected Aedes aegypti mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use.

Cellular reagents for diagnostics and synthetic biology.

We have found that the overproduction of enzymes in bacteria followed by their lyophilization leads to 'cellular reagents' that can be directly used to carry out numerous molecular biology reactions. We demonstrate the use of cellular reagents in a variety of molecular diagnostics, such as TaqMan qPCR with no diminution in sensitivity, and in synthetic biology cornerstones such as the Gibson assembly of DNA fragments, where new plasmids can be constructed solely based on adding cellular reagents. Cellular reagents have significantly reduced complexity and cost of production, storage and implementation, features that should facilitate accessibility and use in resource-poor conditions.

Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone.

Manipulation of natural mosquito populations using the endosymbiotic bacteria Wolbachia is being investigated as a novel strategy to reduce the burden of mosquito-borne viruses. To evaluate the efficacy of these interventions, it will be critical to determine Wolbachia infection frequencies in Aedes aegypti mosquito populations. However, current diagnostic tools are not well-suited to fit this need. Morphological methods cannot identify Wolbachia, immunoassays often suffer from low sensitivity and poor throughput, while PCR and spectroscopy require complex instruments and technical expertise, which restrict their use to centralized laboratories. To address this unmet need, we have used loop-mediated isothermal amplification (LAMP) and oligonucleotide strand displacement (OSD) probes to create a one-pot sample-to-answer nucleic acid diagnostic platform for vector and symbiont surveillance. LAMP-OSD assays can directly amplify target nucleic acids from macerated mosquitoes without requiring nucleic acid purification and yield specific single endpoint yes/no fluorescence signals that are observable to eye or by cellphone camera. We demonstrate cellphone-imaged LAMP-OSD tests for two targets, the Aedes aegypti cytochrome oxidase I (coi) gene and the Wolbachia surface protein (wsp) gene, and show a limit of detection of 4 and 40 target DNA copies, respectively. In a blinded test of 90 field-caught mosquitoes, the coi LAMP-OSD assay demonstrated 98% specificity and 97% sensitivity in identifying Ae. aegypti mosquitoes even after 3 weeks of storage without desiccant at 37°C. Similarly, the wsp LAMP-OSD assay readily identified the wAlbB Wolbachia strain in field-collected Aedes albopictus mosquitoes without generating any false positive signals. Modest technology requirements, minimal execution steps, simple binary readout, and robust accuracy make the LAMP-OSD-to-cellphone assay platform well suited for field vector surveillance in austere or resource-limited conditions.

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.

Strand Displacement Probes Combined with Isothermal Nucleic Acid Amplification for Instrument-Free Detection from Complex Samples.

Sensitive and specific detection of pathogens via nucleic acid amplification is currently constrained to laboratory settings and portable equipment with costly fluorescent detectors. Nucleic acid-detecting lateral flow immunoassay strips (LFIAs) offer a low-cost visual transduction strategy at points of need. Unfortunately, these LFIAs frequently detect amplification byproducts that can yield spurious results which can only be deciphered through statistical analysis. We integrated customizable strand displacement probes into standard loop mediated isothermal amplification (LAMP) assays to prevent byproduct capture on commercial LFIAs. We find that combining strand displacement with LAMP (SD-LAMP) yields LFIA test band intensities that can be unequivocally interpreted by human subjects without additional instrumentation, thereby alleviating the need for a portable reader's analysis. Using SD-LAMP, we capture target amplicons on commercially available LFIAs from as few as 3.5 Vibrio cholerae and 2 750 Escherichia coli bacteria without false positive or false negative interpretation. Moreover, we demonstrate that LFIA capture of SD-LAMP products remain specific even in the presence of complex sample matrixes, providing a significant step toward reliable instrument-free pathogen detection outside of laboratories.

Portable platform for rapid in-field identification of human fecal pollution in water.

Human fecal contamination of water is a public health risk. However, inadequate testing solutions frustrate timely, actionable monitoring. Bacterial culture-based methods are simple but typically cannot distinguish fecal host source. PCR assays can identify host sources but require expertise and infrastructure. To bridge this gap we have developed a field-ready nucleic acid diagnostic platform and rapid sample preparation methods that enable on-site identification of human fecal contamination within 80 min of sampling. Our platform relies on loop-mediated isothermal amplification (LAMP) of human-associated Bacteroides HF183 genetic markers from crude samples. Oligonucleotide strand exchange (OSD) probes reduce false positives by sequence specifically transducing LAMP amplicons into visible fluorescence that can be photographed by unmodified smartphones. Our assay can detect as few as 17 copies/ml of human-associated HF183 targets in sewage-contaminated water without cross-reaction with canine or feline feces. It performs robustly with a variety of environmental water sources and with raw sewage. We have also developed lyophilized assays and inexpensive 3D-printed devices to minimize cost and facilitate field application.

Amplicon Competition Enables End-Point Quantitation of Nucleic Acids Following Isothermal Amplification.

It is inherently difficult to quantitate nucleic acid analytes with most isothermal amplification assays. We developed loop-mediated isothermal amplification (LAMP) reactions in which competition between defined numbers of "false" and "true" amplicons leads to order of magnitude quantitation by a single endpoint determination. These thresholded LAMP reactions were successfully used to directly and quantitatively estimate the numbers of nucleic acids in complex biospecimens, including directly from cells and in sewage, with the values obtained closely correlating with qPCR quantitations. Thresholded LAMP reactions are amenable to endpoint readout by cell phone, unlike other methods that require continuous monitoring, and should therefore prove extremely useful in developing one-pot reactions for point-of-care diagnostics without needing sophisticated material or informatics infrastructure.

Strand-Exchange Nucleic Acid Circuitry with Enhanced Thermo-and Structure- Buffering Abilities Turns Gene Diagnostics Ultra-Reliable and Environmental Compatible.

Catalytic hairpin assembly (CHA) is one of the most promising nucleic acid amplification circuits based on toehold-mediated strand exchange reactions. But its performance is usually ruined by fluctuated environmental temperatures or unexpected self-structures existing in most real-world targets. Here we present an amide-assistant mechanism that successfully reduces the prevalence of these problems for CHA and maximizes its thermo- and structure- buffering abilities. Such an organic amide-promoted CHA (shortened as OHT-CHA) can unprecedentedly amplify through 4 °C to 60 °C without rebuilding sequences or concerning target complexity. We are then for the first time able to employ it as a direct and universal signal booster for loop mediated isothermal reaction (LAMP). LAMP is one of the most promising point-of-care (POC) gene amplifiers, but has been hard to detect precisely due to structured products and haunted off-target amplicons. OHT-CHA guarantees a significant and reliable signal for LAMP reaction amplified from as little as 10-19 M virus gene. And one single set of OHT-CHA is qualified to any detection requirement, either in real-time at LAMP running temperature (~60 °C), or at end-point on a POC photon counter only holding environmental temperatures fluctuating between 4 °C to 42 °C.