Multiple-Catalytic Sensing of Nucleic Acid Sequences by Utilising a DNA-RNA-DNA Chimeric Antisense Probe and RNase H with a Eukaryotic Cell-Free Translation System

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, β2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding.

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Aptazyme-based biosensors using a eukaryotic cell-free translation system

Nucleic Acids Symposium Series ◽

10.1093/nass/nrp131 ◽

2009 ◽

Vol 53 (1) ◽

pp. 261-262

Author(s):

A. Ogawa

Keyword(s):

Eukaryotic Cell ◽

Translation System ◽

Free Translation

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Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system

Journal of Biotechnology ◽

10.1016/j.jbiotec.2012.08.020 ◽

2013 ◽

Vol 164 (2) ◽

pp. 220-231 ◽

Cited By ~ 45

Author(s):

Marlitt Stech ◽

Helmut Merk ◽

Jörg A. Schenk ◽

Walter F.M. Stöcklein ◽

Doreen A. Wüstenhagen ◽

...

Keyword(s):

Eukaryotic Cell ◽

Antibody Fragments ◽

Translation System ◽

Free Translation

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A Cucumis sativus cell-free translation system: preparation, optimization and sensitivity to some antibiotics and ribosome-inactivating proteins

Physiologia Plantarum ◽

10.1034/j.1399-3054.1993.880403.x ◽

1993 ◽

Vol 88 (4) ◽

pp. 549-556 ◽

Cited By ~ 1

Author(s):

M. A. Rojo ◽

F. J. Arias ◽

R. Iglesias ◽

J. M. Ferreras ◽

R. Munoz ◽

...

Keyword(s):

Cucumis Sativus ◽

Translation System ◽

Ribosome Inactivating Proteins ◽

Free Translation

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Gold-Aptamer-Nanoconstructs Engineered to Detect Conserved Enteroviral Nucleic Acid Sequences

10.26434/chemrxiv.8312324.v1 ◽

2019 ◽

Author(s):

Veeren Chauhan ◽

Mohamed M Elsutohy ◽

C Patrick McClure ◽

Will Irving ◽

Neil Roddis ◽

...

Keyword(s):

Nucleic Acid ◽

In Silico ◽

Point Of Care ◽

Lateral Flow ◽

Nucleic Acid Sequence ◽

In Silico Screening ◽

Lateral Flow Assays ◽

Life Threatening ◽

Software And Hardware ◽

Nucleic Acid Sequences

Enteroviruses are a ubiquitous mammalian pathogen that can produce mild to life-threatening disease. Bearing this in mind, we have developed a rapid, accurate and economical point-of-care biosensor that can detect a nucleic acid sequences conserved amongst 96% of all known enteroviruses. The biosensor harnesses the physicochemical properties of gold nanoparticles and aptamers to provide colourimetric, spectroscopic and lateral flow-based identification of an exclusive enteroviral RNA sequence (23 bases), which was identified through in silico screening. Aptamers were designed to demonstrate specific complementarity towards the target enteroviral RNA to produce aggregated gold-aptamer nanoconstructs. Conserved target enteroviral nucleic acid sequence (≥ 1x10-7 M, ≥1.4×10-14 g/mL), initiates gold-aptamer-nanoconstructs disaggregation and a signal transduction mechanism, producing a colourimetric and spectroscopic blueshift (544 nm (purple) > 524 nm (red)). Furthermore, lateral-flow-assays that utilise gold-aptamer-nanoconstructs were unaffected by contaminating human genomic DNA, demonstrated rapid detection of conserved target enteroviral nucleic acid sequence (< 60 s) and could be interpreted with a bespoke software and hardware electronic interface. We anticipate our methodology will translate in-silico screening of nucleic acid databases to a tangible enteroviral desktop detector, which could be readily translated to related organisms. This will pave-the-way forward in the clinical evaluation of disease and complement existing strategies at overcoming antimicrobial resistance.

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