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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.

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.

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.

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.

We report two isothermal nucleic acid amplification assays for detection of monkeypox virus (MPXV) clades 2 and 3 that include the strains responsible for the current global outbreak of monkeypox. The assays use loop-mediated isothermal amplification (LAMP) to amplify two distinct sequences in the MPXV genome. Readout specificity is ensured by oligonucleotide strand displacement (OSD) probes integrated in one-pot LAMP-OSD reactions. OSD probes undergo toehold-mediated strand displacement hybridization to LAMP amplicon loop sequences derived from MPXV clades 2 and 3 resulting in fluorescence readable both in realtime and visually at endpoint. We also perform both assays on two different portable devices, the GeneTiger and the miniPCR, to exemplify compatibility with minimum infrastructure point-of-care (POC) testing in clinical and at-home settings. Both assays could readily detect single digit copies of MPXV synthetic double stranded DNA templates within 30 min.

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.

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.

DNA polymerase fromGeobacillus stearothermophilus, BstDNA polymerase (BstDNAP), is a versatile enzyme with robust strand-displacing activity that enables loop-mediated isothermal amplification (LAMP). Despite its exclusive usage in LAMP assay, its properties remain open to improvement. Here, we describe logical redesign ofBstDNAP by using multimodal application of several independent and orthogonal rational engineering methods such as domain addition, supercharging, and machine learning predictions of amino acid substitutions. The resulting Br512g3 enzyme is not only thermostable and extremely robust but it also displays improved reverse transcription activity and the ability to carry out ultrafast LAMP at 74 °C. Our study illustrates a new enzyme engineering strategy as well as contributes a novel engineered strand displacing DNA polymerase of high value to diagnostics and other fields.

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-qPCR reactions. We demonstrate the utility of Taq-alone RT-qPCR reactions 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.

Despite the fact that strand-displacing activity is of great utility for a variety of applications, including isothermal amplification assays, there are relatively few strand-displacing DNA polymerases. In particular, the thermotolerant DNA polymerase from Geobacillus stearothermophilus (previously Bacillus stearothermophilus), Bst DNA polymerase (Bst DNAP), is used in a variety of assays, including loop-mediated isothermal amplification. However, despite its wide use, its properties remain open to improvement, as has been demonstrated by a variety of engineering efforts, including the identification of point mutations that impact its robustness, strand-displacement capabilities, and nascent reverse transcriptase activity.Interestingly, a strategy that has been commonly used to alter the capabilities of DNA polymerases, the addition of additional DNA- or RNA-binding domains, has yet to be applied to Bst DNAP. To this end, we now show that by adding fusion domains the performance of Bst DNAP in isothermal amplification assays, including its nascent RT activity, can be greatly improved. The impact of these improvements on the development of LAMP assays for the detection of SARS-CoV-2 is fully explored.

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.

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-qPCR reactions. We demonstrate the utility of Taq-alone RT-qPCR reactions 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.

Isothermal nucleic acid amplification tests (iNAT), such as loop-mediated isothermal amplification (LAMP), are good alternatives to polymerase chain reaction (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 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. Internally redundant assays targeting different regions of the target sequence can help to reduce such false negatives. Here we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on non-sequence-specific readout into assays that can be visually read using sequence-specific fluorogenic oligonucleotide strand exchange (OSD) probes. We evaluate one-pot operation of both individual and multiplex LAMP-OSD assays and demonstrate detection of SARS-CoV-2 virions in crude human saliva.

Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed a RT-qPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase / DNA polymerase (RTX)1. Here we show that RTX performs comparably to the other assays sanctioned by the CDC and validated in kit format. We demonstrate two modes of RTX use – (i) dye-based RT-qPCR assays that require only RTX polymerase, and (ii) TaqMan RT-qPCR assays that use a combination of RTX and Taq DNA polymerases (as the RTX exonuclease does not degrade a TaqMan probe). We also provide straightforward recipes for the purification of this alternative reagent. We anticipate that in low resource or point-of-need settings researchers could obtain the available constructs from Addgene or our lab and begin to develop their own assays, within whatever regulatory framework exists for them.We lay out protocols for dye-based and TaqMan probe-based assays, in order to best compare with ‘gold standard’ reagents. These protocols should form the basis of further modifications that can simplify the assay to the use of overexpressing cells themselves as reagents.Developing dye-based and TaqMan probe-based RT-qPCR assays with RTX

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.

We extend the concept of DNA proximity ligation from a single readout per oligonucleotide pair to multiple reversible, iterative ligations re-using the same oligonucleotide molecules. Using iterative proximity ligation (IPL), we can in principle capture multiple ligation events between each oligonucleotide and its various neighbors and thus recover a far richer knowledge about their relative positions than single, irreversible ligation events. IPL would thus act to sample and record local molecular neighborhoods. By integrating a unique DNA barcode into each participating oligonucleotide, we can catalog the individual ligation events and thus capture the positional information contained therein in a high throughput manner using next-generation DNA sequencing. We propose that by interpreting IPL sequencing results in the context of graph theory and by applying spring layout algorithms, we can recover geometric patterns of objects labeled by DNA. Using simulations, we demonstrate that we can in principle recover letter patterns photolithographed onto slide surfaces using only IPL sequencing data, illustrating how our technique maps complex spatial configurations into DNA sequences and then – using only this sequence information – recovers them. We complement our theoretical work with an experimental proof-of-concept of iterative proximity ligation on an oligonucleotide population.

We have developed a generalizable ‘smart molecular diagnostic’ capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our one-pot 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 gate signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our platform 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 20 virions / µl being directly detected in human saliva within 90 minutes, and 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.

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.

Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 10(9) copies from less than 100 copies of template DNA within an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains analytically challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (rpoB) and the melanoma-related biomarker BRAF. OSD allowed the Yes/No detection of rpoB in a complex mixture such as synthetic sputum and also demonstrated single nucleotide specificity in Yes/No detection of a mutant BRAF allele (V600E) in the presence of 20-fold more of the wild-type gene. Real-time detection of different genes in multiplex LAMP reactions also proved possible. The development of simple, readily designed, modular equivalents of TaqMan probes for isothermal amplification reactions should generally improve the applicability of these reactions and may eventually assist with the development of point-of-care tests.

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.

It is inherently difficult to quantitate nucleic acid analytes with most isothermal amplification assays. We have now developed loop-mediated isothermal amplification (LAMP) reactions in which competition between defined numbers of 'false' and 'true' amplicons leads to order of magnitude quantitation via a single end-point 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 via 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 determinations without any sophisticated material or informatics infrastructure.

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 an enzyme-free amplification method that has previously proven useful in amplifying and transducing signals at the terminus of nucleic acid amplification reactions. Here, for the first time, we engineered CHA to be thermostable from 37 to 60 °C and in consequence have generalized its application to the real-time detection of isothermal amplification reactions. CHA circuits were designed and optimized for both high- and low-temperature rolling circle amplification (RCA) and strand displacement amplification (SDA). The resulting circuits not only increased the specificity of detection but also improved the sensitivity by as much as 25- to 10000-fold over comparable real-time detection methods. These methods have been condensed into a set of general rules for the design of thermostable CHA circuits with high signals and low noise.

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.

We have developed a self-reporting polymerase chain reaction (PCR) system for visual colorimetric gene detection and distinction of single nucleotide polymorphisms (SNPs). Amplification is performed using target-specific primers modified with a 5′-end tail that is complementary to a G-quadruplex deoxyribozyme-forming sequence. At end-point, G-quadruplexes are forced to fold from PCR-generated duplex DNA and then are used to colorimetrically report the successful occurrence of PCR by assaying their peroxidase activity using a chromogenic substrate. Furthermore, primer design considerations for the G-quadruplex-generating PCR system have allowed us to visually distinguish SNPs associated with Mycobacterium tuberculosis drug resistance alleles.

Type B leukemogenic virus is a variant of mouse mammary tumor virus (MMTV) that causes thymic lymphomas rather than mammary tumors in mice. We demonstrate that conversion of a mammotropic MMTV to a T-cell-tropic virus requires two alterations in the long terminal repeat: (i) acquisition of a T-cell-specific enhancer and (ii) loss of transcriptional repression through deletion of negative regulatory elements (NREs) or by suppression of NRE activity after appropriate positioning of the enhancer.

Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20–100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device.

We describe design parameters for the synthesis and analytical application of a label-free RNA molecular beacon, termed Spinach.ST. The RNA aptamer Spinach fluoresces upon binding the small-molecule fluorophore DFHBI ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one). Spinach has been reengineered by extending its 5′- and 3′-ends to create Spinach.ST, which is predicted to fold into an inactive conformation that fails to bind DHFBI. Hybridization of a trigger oligonucleotide to a designed toehold on Spinach.ST initiates toehold-mediated strand displacement and restores the DFHBI-binding, fluorescence-enhancing conformation of Spinach. The versatile Spinach.ST sensor can detect DNA or RNA trigger sequences and can readily distinguish single-nucleotide mismatches in the trigger toehold. Primer design techniques are described that augment amplicons produced by enzymatic amplification with Spinach.ST triggers. Interaction between these triggers and Spinach.ST molecular beacons leads to the real-time, sequence-specific quantitation of these amplicons. The use of Spinach.ST with isothermal amplification reactions such as nucleic acid sequence-based amplification (NASBA) may enable point-of-care applications. The same design principles could also be used to adapt Spinach reporters to the assay of nonnucleic acid analytes in trans.

We have re-engineered the fluorescent RNA aptamer Spinach to be activated in a sequence-dependent manner. The original Spinach aptamer was extended at its 5′- and 3′-ends to create Spinach.ST, which is predicted to fold into an inactive conformation and thus prevent association with the small molecule fluorophore DFHBI. Hybridization of a specific trigger oligonucleotide to a designed toehold leads to toehold-initiated strand displacement and refolds Spinach into the active, fluorophore-binding conformation. Spinach.ST not only specifically detects its target oligonucleotide but can discriminate readily against single-nucleotide mismatches. RNA amplicons produced during nucleic acid sequence-based amplification (NASBA) of DNA or RNA targets could be specifically detected and reported in real-time by conformational activation of Spinach.ST generated by in vitro transcription. In order to adapt any target sequence to detection by a Spinach reporter we used a primer design technique that brings together otherwise distal toehold sequences via hairpin formation. The same techniques could potentially be used to adapt common Spinach reporters to non-nucleic acid analytes, rather than by making fusions between aptamers and Spinach.

We report the full reconstruction of ${\mathrm{\ensuremath{\chi}}}_{\mathit{c}}$ mesons through the decay chain ${\mathrm{\ensuremath{\chi}}}_{\mathit{c}}$\ensuremath{\rightarrow}J/\ensuremath{\psi} \ensuremath{\gamma}, J/\ensuremath{\psi}\ensuremath{\rightarrow}${\mathrm{\ensuremath{\mu}}}^{+}$${\mathrm{\ensuremath{\mu}}}^{\mathrm{\ensuremath{-}}}$, using data obtained at the Collider Detector at Fermilab in 2.6\ifmmode\pm\else\textpm\fi{}0.2 ${\mathrm{pb}}^{\mathrm{\ensuremath{-}}1}$ of p\ifmmode\bar\else\textasciimacron\fi{}p collisions at \ensuremath{\surd}s =1.8 TeV. This exclusive ${\mathrm{\ensuremath{\chi}}}_{\mathit{c}}$ sample is used to measure the ${\mathrm{\ensuremath{\chi}}}_{\mathit{c}}$-meson production cross section times branching fractions. We obtain \ensuremath{\sigma}\ifmmode\times\else\texttimes\fi{}B=3.2\ifmmode\pm\else\textpm\fi{}0.4(stat${)}_{\mathrm{\ensuremath{-}}1.1}^{+1.2}$(syst) nb for ${\mathrm{\ensuremath{\chi}}}_{\mathit{c}}$ mesons decaying to J/\ensuremath{\psi} with ${\mathit{p}}_{\mathit{T}}$g6.0 GeV/c and pseudorapidity \ensuremath{\Vert}\ensuremath{\eta}\ensuremath{\Vert}0.5. From this and the inclusive J/\ensuremath{\psi} cross section we calculate the inclusive b-quark cross section to be 12.0\ifmmode\pm\else\textpm\fi{}4.5 \ensuremath{\mu}b for ${\mathit{p}}_{\mathit{T}}^{\mathit{b}}$g8.5 GeV/c and \ensuremath{\Vert}${\mathit{y}}^{\mathit{b}}$\ensuremath{\Vert}1.

We have searched for the $b$-flavored baryon decay ${\ensuremath{\Lambda}}_{b}\ensuremath{\rightarrow}\frac{J}{\ensuremath{\psi}}{\ensuremath{\Lambda}}^{0}$ in $p\overline{p}$ collisions at $\sqrt{s}=1.8$ TeV with the Collider Detector at Fermilab (CDF). In the $\frac{J}{\ensuremath{\psi}}{\ensuremath{\Lambda}}^{0}$ invariant-mass distribution we observe a flat distribution and a small number of events, consistent with no signal. Using the $b$-quark cross section measured by the CDF we put an upper limit on the $b\ensuremath{\rightarrow}{\ensuremath{\Lambda}}_{b}$ production fraction times the branching ratio $F({\ensuremath{\Lambda}}_{b})B({\ensuremath{\Lambda}}_{b}\ensuremath{\rightarrow}\frac{J}{\ensuremath{\psi}}{\ensuremath{\Lambda}}^{0})$ of 0.50\ifmmode\times\else\texttimes\fi{}${10}^{\ensuremath{-}3}$ at 90% confidence level.

Endogenous retroviruses (ERVs) abound in avian and mammalian genomes, including humans, as a result of germline infections by exogenous retroviruses. Most ERVs are defective for production of infectious virus. The defectiveness of ERVs is generally inversely correlated with the length of their residence in the host germline. These ERVs affect retrovirus-induced disease in a number of ways, including manipulation of the immune response, inhibition or facilitation of entry or other steps of virus replication, or as participants in the generation of infectious pathogenic viruses. Ancient ERVs likely have neutral or beneficial roles for the hosts that carry them. However, multiple examples show that additional pathogenic retroviruses will continue to emerge using ERVs as a source of genetic diversity.

Inclusive [ital J]/[psi] and [psi](2[ital S]) production has been studied in [ital [bar p]p] collisions at [radical][ital s] =1.8 TeV using 2.6[plus minus]0.2 pb[sup [minus]1] of data taken with the Collider Detector at Fermilab. The products of production cross section times branching fraction were measured as functions of [ital P][sub [ital T]] for [ital J]/[psi][r arrow][mu][sup +][mu][sup [minus]] and [psi](2[ital S])[r arrow][mu][sup +][mu][sup [minus]]. In the kinematic range [ital P][sub [ital T]][gt]6 GeV/[ital c] and [vert bar][eta][vert bar][le]0.5 we get [sigma]([ital [bar p]p][r arrow][ital J]/[psi] [ital X])[ital B]([ital J]/[psi][r arrow][mu][sup +][mu][sup [minus]]) =6.88[plus minus]0.23(stat)[sub [minus]1.08][sup +0.93](syst) nb, and [sigma]([ital [bar p]p][r arrow][psi](2[ital S])[ital X])[ital B]([psi](2[ital S])[r arrow][mu][sup +][mu][sup [minus]]) =0.232[plus minus]0.051(stat)[sub [minus]0.032][sup +0.029](syst)nb. From these values we calculate the inclusive [ital b]-quark production cross section.

We search for the rare decay ${\mathit{W}}^{\ifmmode\pm\else\textpm\fi{}}$\ensuremath{\rightarrow}\ensuremath{\gamma}${\mathrm{\ensuremath{\pi}}}^{\ifmmode\pm\else\textpm\fi{}}$ in 4.2 ${\mathrm{pb}}^{\mathrm{\ensuremath{-}}1}$ of pp\ifmmode\bar\else\textasciimacron\fi{} collisions recorded with the Collider Detector at Fermilab. At the 95% confidence level, we find an upper limit on the partial decay width to be \ensuremath{\Gamma}(${\mathit{W}}^{\ifmmode\pm\else\textpm\fi{}}$\ensuremath{\rightarrow}\ensuremath{\gamma}${\mathrm{\ensuremath{\pi}}}^{\ifmmode\pm\else\textpm\fi{}}$)/\ensuremath{\Gamma}(${\mathit{W}}^{\ifmmode\pm\else\textpm\fi{}}$\ensuremath{\rightarrow}${\mathit{e}}^{\ifmmode\pm\else\textpm\fi{}}$\ensuremath{\nu})\ensuremath{\le}7.5\ifmmode\times\else\texttimes\fi{}${10}^{\mathrm{\ensuremath{-}}3}$.

The ${\mathit{B}}^{0}$ B\ifmmode\bar\else\textasciimacron\fi{} $^{0}$ average mixing parameter \ensuremath{\chi} has been extracted from e\ensuremath{\mu} and ee events produced in pp\ifmmode\bar\else\textasciimacron\fi{} collisions at \ensuremath{\surd}s =1.8 TeV. In a sample of 900 e\ensuremath{\mu} events, the like-sign to opposite-sign charge ratio R is measured to be 0.556\ifmmode\pm\else\textpm\fi{}0.048(stat${)}_{\mathrm{\ensuremath{-}}0.042}^{+0.035}$(syst). In the absence of mixing, the expected value of R would be 0.23\ifmmode\pm\else\textpm\fi{}0.06. The corresponding number for 212 ee events is 0.573\ifmmode\pm\else\textpm\fi{}0.116(stat)\ifmmode\pm\else\textpm\fi{}0.047(syst) with an expected nonmixing value of 0.24\ifmmode\pm\else\textpm\fi{}0.07. The observed excess in R leads to a combined determination of \ensuremath{\chi}=0.176\ifmmode\pm\else\textpm\fi{}0.031(stat+syst) \ifmmode\pm\else\textpm\fi{}0.032 (model), where the last uncertainty is due to Monte Carlo modeling.

An analysis of the forward-backward asymmetry in {ital Z}{sup 0} decays using data from the Collider Detector at Fermilab at {radical}{ital s} =1.8 TeV yields {ital A}{sub {ital F}{ital B}}=(5.2{plus minus}5.9(stat){plus minus}0.4(syst))% and sin{sup 2}{bar {theta}}{sub {ital W}} =0.228{sub {minus}0.015}{sup +0.017}(stat){plus minus}0.002(syst).

Using the Collider Detector at Fermilab, the {ital W}-boson differential cross section {ital d}{sigma}/{ital dP}{sub {ital T}} is measured using {ital W}{r arrow}{ital e}{nu} events in proton-antiproton collisions at {radical}{ital s} =1.8 TeV. A next-to-leading-order theoretical calculation agrees well with the data. The cross section ({sigma}) for {ital P}{sub {ital T}}{gt}50 GeV/{ital c} is measured to be 423{plus minus}58(stat){plus minus}108(syst) pb.

The absence of endogenous mouse mammary tumor viruses (MMTVs) in the congenic mouse strain, BALB/ Mtv -null, restricts the early steps of exogenous C3H MMTV infection, preventing the superantigen (Sag) response and mammary tumorigenesis. Here we demonstrate that BALB/ Mtv -null mice also resist tumor induction by FM MMTV, which encodes a stronger Sag compared to C3H MMTV. In contrast to infections with C3H MMTV, Mtv -null mice show FM-MMTV Sag-specific responses comparable to those observed in susceptible BALB/c mice. Neither virus shows significant replication in the spleen or mammary gland. Thus, Mtv -null mice restrict MMTV replication and mammary tumorigenesis even after a robust Sag response.

Mouse mammary tumor virus (MMTV) is a slow-acting oncogenic retrovirus that causes mammary tumors in mice after a long period (6 to 9 months) (1, 29). An MMTV variant, type B leukemogenic virus (TBLV), has been demonstrated to cause thymic lymphomas with a short latency period (1.5 to 2 months) when injected intrathymically into neonatal mice (2). Compared to MMTV, TBLV has a deletion of 443 nucleotides and a substitution of 124 nucleotides in the U3 region of the long terminal repeat (LTR) (3). This deletion removes a negative regulatory element (NRE) present in the MMTV LTR (17-19, 23, 36, 37), which has been implicated in tissue-specific MMTV expression (6, 30). The substitution creates a triplication of a 62-bp element comprised of sequences flanking the deletion (3) (Fig. ​(Fig.1A1A). FIG. 1. Construction and characterization of hybrid MMTV-TBLV proviral clones. (A) Diagram of parental and hybrid MMTV clones. The pHYB-MTV plasmid contains a hybrid between Mtv1 and C3H MMTV. A white box indicates the region upstream of the EcoRI site that is ... The LTR deletion and substitution also truncate the open reading frame (ORF) encompassing the U3 region of the LTR (Fig. ​(Fig.1A).1A). This ORF encodes a 37-kDa, type II transmembrane glycoprotein known as superantigen (Sag) that functions in the milk-borne transmission of MMTV (1, 7, 9, 10, 16) by the stimulation of T cells through the T-cell receptor (TCR) (33, 35). The truncated Sag protein encoded in the TBLV LTR lacks the entire C-terminal region that interacts with the TCR, yet it retains the transmembrane region, a part of the extracellular region that includes the five glycosylation sites, a single proteolytic processing site, and the major histocompatibility complex class II protein binding motif (12, 20). This study shows that the truncated TBLV Sag protein is dispensable for virally induced T-cell lymphomas.

The ability to detect and monitor single nucleotide polymorphisms (SNPs) in biological samples is an enabling research and clinical tool. We have developed a surprising, inexpensive primer design method that provides exquisite discrimination between SNPs. The field of DNA computation is largely reliant on using so-called toeholds to initiate strand displacement reactions, leading to the execution of kinetically trapped circuits. We have now similarly found that the short toehold sequence to a target of interest can initiate both strand displacement within the hairpin and extension of the primer by a polymerase, both of which will further stabilize the primer:template complex. However, if the short toehold does not bind, neither of these events can readily occur and thus amplification should not occur. Toehold hairpin primers were used to detect drug resistance alleles in two genes, rpoB and katG, in the Mycobacterium tuberculosis genome, and ten alleles in the Escherichia coli genome. During real-time PCR, the primers discriminate between mismatched templates with Cq delays that are frequently so large that the presence or absence of mismatches is essentially a ‘yes/no’ answer.