Sanchita Bhadra

Real-time detection of isothermal amplification reactions with thermostable catalytic hairpin assembly.

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.

Mismatches improve the performance of strand-displacement nucleic Acid circuits.

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.

Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

Nucleic acid circuits are finding increasing real-life applications in diagnostics and synthetic biology. Although DNA has been the main operator in most nucleic acid circuits, transcriptionally produced RNA circuits could provide powerful alternatives for reagent production and their use in cells. Towards these goals, we have implemented a particular nucleic acid circuit, catalytic hairpin assembly, using RNA for both information storage and processing. Our results demonstrated that the design principles developed for DNA circuits could be readily translated to engineering RNA circuits that operated with similar kinetics and sensitivities of detection. Not only could purified RNA hairpins perform amplification reactions but RNA hairpins transcribed in vitro also mediated amplification, even without purification. Moreover, we could read the results of the non-enzymatic amplification reactions using a fluorescent RNA aptamer ‘Spinach’ that was engineered to undergo sequence-specific conformational changes. These advances were applied to the end-point and real-time detection of the isothermal strand displacement amplification reaction that produces single-stranded DNAs as part of its amplification cycle. We were also able to readily engineer gate structures with RNA similar to those that have previously formed the basis of DNA circuit computations. Taken together, these results validate an entirely new chemistry for the implementation of nucleic acid circuits.

Robust strand exchange reactions for the sequence-specific, real-time detection of nucleic acid amplicons.

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.

Real-Time Sequence-Validated Loop-Mediated Isothermal Amplification Assays for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

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.

The Type B Leukemogenic Virus Truncated Superantigen Is Dispensable for T-Cell Lymphomagenesis

ABSTRACT Type B leukemogenic virus (TBLV) is a variant of mouse mammary tumor virus (MMTV) that causes T-cell lymphomas in mice. We have constructed a TBLV-MMTV hybrid, pHYB-TBLV, in which 756 bp of the C3H MMTV long terminal repeat (LTR) was replaced with 438 bp of the TBLV LTR. Intraperitoneal injection of pHYB-TBLV transfectants consistently resulted in T-cell lymphomas in 50% of injected weanling BALB/c mice with an average latency period of 5.7 (± 1.5) months. Transfectants of pHYB-TBLV containing a double-frameshift mutation in the truncated superantigen gene (sag) induced T-cell lymphomas with similar incidences, latency periods, and phenotypes, suggesting that cis-acting elements in the TBLV LTR determine disease specificity.

Endogenous MMTV Proviruses Induce Susceptibility to Both Viral and Bacterial Pathogens

Most inbred mice carry germline proviruses of the retrovirus, mouse mammary tumor virus (MMTV) (called Mtvs), which have multiple replication defects. A BALB/c congenic mouse strain lacking all endogenous Mtvs (Mtv-null) was resistant to MMTV oral and intraperitoneal infection and tumorigenesis compared to wild-type BALB/c mice. Infection of Mtv-null mice with an MMTV-related retrovirus, type B leukemogenic virus, also resulted in severely reduced viral loads and failure to induce T-cell lymphomas, indicating that resistance is not dependent on expression of a superantigen (Sag) encoded by exogenous MMTV. Resistance to MMTV in Mtv-null animals was not due to neutralizing antibodies. Further, Mtv-null mice were resistant to rapid mortality induced by intragastric inoculation of the Gram-negative bacterium, Vibrio cholerae, but susceptibility to Salmonella typhimurium was not significantly different from BALB/c mice. Susceptibility to both MMTV and V. cholerae was reconstituted by the presence of any one of three endogenous Mtvs located on different chromosomes and was associated with increased pathogen load. One of these endogenous proviruses is known to encode only Sag. Therefore, Mtv-encoded Sag appears to provide a unique genetic susceptibility to specific viruses and bacteria. Since human endogenous retroviruses also encode Sags, these studies have broad implications for pathogen-induced responses in mice and humans.

A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

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.

Conversion of Mouse Mammary Tumor Virus to a Lymphomagenic Virus

ABSTRACT 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.

BALB/Mtv-Null Mice Responding to Strong Mouse Mammary Tumor Virus Superantigens Restrict Mammary Tumorigenesis

ABSTRACT 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.