Development of small molecule biosensors by coupling the recognition of the bacterial allosteric transcription factor with isothermal strand displacement amplification

2018 ◽  
Vol 54 (38) ◽  
pp. 4774-4777 ◽  
Author(s):  
Yongpeng Yao ◽  
Shanshan Li ◽  
Jiaqian Cao ◽  
Weiwei Liu ◽  
Keqiang Fan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
Strand Displacement ◽  
Strand Displacement Amplification

We demonstrate a novel small molecule biosensing strategy by coupling the recognition of aTF with SDA reaction in vitro.

scholarly journals Detection of Mycobacterium tuberculosis DNA with thermophilic strand displacement amplification and fluorescence polarization

1996 ◽  
Vol 42 (10) ◽  
pp. 1604-1608 ◽  
Author(s):  
G T Walker ◽  
C P Linn
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Restriction Enzyme ◽  
Fluorescence Polarization ◽  
Target Sequence ◽  
Strand Displacement ◽  
Insertion Element ◽  
Strand Displacement Amplification ◽  
Simple Protocol ◽  
Polymerase Binding

Abstract Strand displacement amplification (SDA) is an isothermal, in vitro method for diagnostics that amplifies a target DNA sequence by using a restriction enzyme and DNA polymerase. We have combined a new thermophilic form of SDA that involves restriction enzyme BsoBI and polymerase exo-Bca with fluorescence polarization for detection of Mycobacterium tuberculosis DNA by using the IS6110 insertion element as the target sequence. A 5'-fluorescein-labeled oligodeoxynucleotide detector probe hybridizes to the amplified product as it rises in concentration during SDA, and the single- to double-stranded conversion is monitored through an increase in fluorescence polarization. The associated change in polarization upon amplification of the target sequence is enhanced by specific polymerase binding to the double-stranded detector probe. Fewer than 10 M. tuberculosis genomes can be amplified and detected with an extremely simple protocol that takes only 20 min and uses relatively simple instrumentation and reagents, all of which can be purchased off-the-shelf.

scholarly journals Detection of Viable Mycobacterium tuberculosis by Reverse Transcriptase-Strand Displacement Amplification of mRNA

1999 ◽  
Vol 37 (3) ◽  
pp. 518-523 ◽  
Author(s):  
T. J. Hellyer ◽  
L. E. DesJardin ◽  
L. Teixeira ◽  
M. D. Perkins ◽  
M. D. Cave ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Reverse Transcriptase ◽  
Therapeutic Efficacy ◽  
Sputum Sample ◽  
Drug Susceptibility ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
Amplification System

Numerous assays have been described for the detection of DNA and rRNA sequences that are specific for the Mycobacterium tuberculosis complex. Although beneficial to initial diagnosis, such assays have proven unsuitable for monitoring therapeutic efficacy owing to the persistence of these nucleic acid targets long after conversion of smears and cultures to negative. However, prokaryotic mRNA has a typical half-life of only a few minutes and we have previously shown that the presence of mRNA is a good indicator of bacterial viability. The purpose of the present study was to develop a novel reverse transcriptase-strand displacement amplification system for the detection of M. tuberculosis α-antigen (85B protein) mRNA and to demonstrate the use of this assay in assessing chemotherapeutic efficacy in patients with pulmonary tuberculosis. The assay was applied to sequential, noninduced sputum specimens collected from four patients: 10 of 11 samples (91%) collected prior to the start of therapy were positive for alpha-antigen mRNA, compared with 1 of 8 (13%), 2 of 8 (25%), 2 of 8 (25%), and 0 of 8 collected on days 2, 4, 7, and 14 of treatment, respectively. In contrast, 39 of 44 samples (89%) collected on or before day 14 were positive for α-antigen DNA. The loss of detectable mRNA corresponded to a rapid drop over the first 4 days of treatment in the number of viable organisms present in each sputum sample, equivalent to a mean fall of 0.43 log10 CFU/ml/day. Analysis of mRNA is a potentially useful method for monitoring therapeutic efficacy and for rapid in vitro determination of drug susceptibility.

scholarly journals In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity

2015 ◽  
Vol 4 (10) ◽  
pp. 1144-1150 ◽  
Author(s):  
Laura Martini ◽  
Adam J. Meyer ◽  
Jared W. Ellefson ◽  
John N. Milligan ◽  
Michele Forlin ◽  
...  
Keyword(s):  
Small Molecule ◽  
In Vitro Selection ◽  
Strand Displacement ◽  
Selection For

Rational design of allosterically regulated toehold mediated strand displacement circuit for sensitive and on-site detection of small molecule metabolites

The Analyst ◽  
2021 ◽  
Author(s):  
Haosi Lin ◽  
Alan F. Rodríguez-Serrano ◽  
I-Ming Hsing
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
Health Monitoring ◽  
Rational Design ◽  
Rapid Diagnosis ◽  
Strand Displacement ◽  
Small Molecule Detection

Development of small molecule biosensor enables rapid and de-centralized small molecule detection that meets the demand of routine health monitoring and rapid diagnosis. Among them, allosteric transcription factor (aTF)-based biosensors...

scholarly journals Small Molecule Inhibitors of LcrF, a Yersinia pseudotuberculosis Transcription Factor, Attenuate Virulence and Limit Infection in a Murine Pneumonia Model

2010 ◽  
Vol 78 (11) ◽  
pp. 4683-4690 ◽  
Author(s):  
Lynne K. Garrity-Ryan ◽  
Oak K. Kim ◽  
Joan-Miquel Balada-Llasat ◽  
Victoria J. Bartlett ◽  
Atul K. Verma ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
Type Iii Secretion ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Secretion System ◽  
Bacterial Burden ◽  
Small Molecule Library ◽  
Null Mutants

ABSTRACT LcrF (VirF), a transcription factor in the multiple adaptational response (MAR) family, regulates expression of the Yersinia type III secretion system (T3SS). Yersinia pseudotuberculosis lcrF-null mutants showed attenuated virulence in tissue culture and animal models of infection. Targeting of LcrF offers a novel, antivirulence strategy for preventing Yersinia infection. A small molecule library was screened for inhibition of LcrF-DNA binding in an in vitro assay. All of the compounds lacked intrinsic antibacterial activity and did not demonstrate toxicity against mammalian cells. A subset of these compounds inhibited T3SS-dependent cytotoxicity of Y. pseudotuberculosis toward macrophages in vitro. In a murine model of Y. pseudotuberculosis pneumonia, two compounds significantly reduced the bacterial burden in the lungs and afforded a dramatic survival advantage. The MAR family of transcription factors is well conserved, with members playing central roles in pathogenesis across bacterial genera; thus, the inhibitors could have broad applicability.

scholarly journals Efficient generation of lower induced Motor Neurons by coupling Ngn2 expression with developmental cues

2022 ◽  
Author(s):  
Francesco Limone ◽  
Jana M. Mitchell ◽  
Irune Guerra San Juan ◽  
Janell L.M. Smith ◽  
Kavya Raghunathan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
Motor Neurons ◽  
High Yield ◽  
Directed Differentiation ◽  
Gfp Reporter ◽  
Functional Profiling ◽  
Synaptic Markers ◽  
Bona Fide

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modelling and drug discovery, especially when access to primary tissue is limited, such as in the brain. Current neuronal differentiation approaches use either small molecules for directed differentiation or transcription-factor-mediated programming. In this study we coupled the overexpression of the neuralising transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced Motor Neurons (liMoNes). We showed that this approach induced activation of the motor neuron (MN) specific transcription factor Hb9/MNX1, using an Hb9::GFP-reporter line, with up to 95% of cells becoming Hb9::GFP+. These cells acquired and maintained expression of canonical early and mature MN markers. Molecular and functional profiling revealed that liMoNes resembled bona fide hPSC-derived MN differentiated by conventional small molecule patterning. liMoNes exhibited spontaneous electrical activity, expressed synaptic markers and formed contacts with muscle cells in vitro. Pooled, multiplex single-cell RNA sequencing on 50 cell lines revealed multiple anatomically distinct MN subtypes of cervical and brachial, limb-innervating MNs in reproducible quantities. We conclude that combining small molecule patterning with Ngn2 can facilitate the high-yield, robust and reproducible production of multiple disease-relevant MN subtypes, which is fundamental in the path to propel forward our knowledge of motoneuron biology and its disruption in disease.

scholarly journals A Curcumin Derivative Activates TFEB and Protects Against Parkinsonian Neurotoxicity in Vitro

2020 ◽  
Vol 21 (4) ◽  
pp. 1515 ◽  
Author(s):  
Ziying Wang ◽  
Chuanbin Yang ◽  
Jia Liu ◽  
Benjamin Chun-Kit Tong ◽  
Zhou Zhu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Animal Models ◽  
Small Molecule ◽  
Therapeutic Target ◽  
Cell Models ◽  
Neuroprotective Effects ◽  
Lysosome Biogenesis ◽  
Transcription Factor Eb ◽  
Curcumin Derivative

TFEB (transcription factor EB), which is a master regulator of autophagy and lysosome biogenesis, is considered to be a new therapeutic target for Parkinson’s disease (PD). However, only several small-molecule TFEB activators have been discovered and their neuroprotective effects in PD are unclear. In this study, a curcumin derivative, named E4, was identified as a potent TFEB activator. Compound E4 promoted the translocation of TFEB from cytoplasm into nucleus, accompanied by enhanced autophagy and lysosomal biogenesis. Moreover, TFEB knockdown effectively attenuated E4-induced autophagy and lysosomal biogenesis. Mechanistically, E4-induced TFEB activation is mainly through AKT-MTORC1 inhibition. In the PD cell models, E4 promoted the degradation of α-synuclein and protected against the cytotoxicity of MPP+ (1-methyl-4-phenylpyridinium ion) in neuronal cells. Overall, the TFEB activator E4 deserves further study in animal models of neurodegenerative diseases, including PD.

A simple and sensitive detection of small molecule–protein interactions based on terminal protection-mediated exponential strand displacement amplification

The Analyst ◽  
2018 ◽  
Vol 143 (9) ◽  
pp. 2023-2028 ◽  
Author(s):  
Chang Yeol Lee ◽  
Hyo Yong Kim ◽  
Soeun Kim ◽  
Ki Soo Park ◽  
Hyun Gyu Park
Keyword(s):  
Small Molecule ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Sensitive Detection ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
New Strategy

A new strategy to detect a small molecule–protein interaction was devised based on terminal protection-mediated exponential strand displacement amplification (eSDA).

scholarly journals Disruption of IRElα through its Kinase Domain Attenuates Multiple Myeloma

2018 ◽  
Author(s):  
Jonathan M Harnoss ◽  
Adrien Le Thomas ◽  
Scot A Marsters ◽  
David A Lawrence ◽  
Min Lu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Transcription Factor ◽  
Small Molecule ◽  
Therapeutic Target ◽  
Plasma Cells ◽  
Critical Role ◽  
Kinase Domain ◽  
Kinase Inhibition

AbstractMultiple myeloma (MM) arises from malignant immunoglobulin-secreting plasma cells and remains an incurable, often lethal disease despite recent therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may coopt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Here we show that genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice, and augmented efficacy of two well-established frontline antimyeloma agents, bortezomib or lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the ER-associated degradation machinery, as well as cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138− cells from bone marrow of newly diagnosed MM patients or patients whose disease relapsed after 1 - 4 lines of treatment in both US- and EU-based cohorts. IRE1α inhibition preserved survival and glucose-induced insulin secretion by pancreatic microislets. Together, these results establish a strong therapeutic rationale for targeting IRE1α with kinase-based small-molecule inhibitors in MM.Significance statementMultiple myeloma (MM) is a lethal malignancy of plasma cells. MM cells have an expanded endoplasmic reticulum (ER) that is constantly under stress due to immunoglobulin hyperproduction. The ER-resident sensor IRE1α mitigates ER stress by expanding the ER’s protein-folding capacity while supporting proteasomal degradation of misfolded ER proteins. IRE1α elaborates these functions by deploying its cytoplasmic kinase-RNase module to activate the transcription factor XBP1s. The validity of IRE1α as a potential therapeutic target in MM has been questioned. Using genetic and pharmacologic disruption in vitro and in vivo, we demonstrate that the IRE1α-XBP1s pathway plays a critical role in MM growth. We further show that IRE1α’s kinase domain is an effective and safe potential small-molecule target for MM therapy.

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