L

Buckingham, J.

doi:10.1007/978-1-4899-3312-6_12

Cited by 3 publications

(3 citation statements)

References 43 publications

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“…Overall, [Ch][Glu] and [Ch][Asp] can increase the thermal stability of RNA and do not display cytotoxicity onto two human cell lines. However, the major factor affecting the RNA stability, and thereby causing its degradation, is the ubiquitous presence of RNases (active nucleases) . By mimicking conditions often observed in in vitro assays wherein culture media are usually supplemented with fetal bovine serum (FBS) as a source of essential growth factors, we further appraised the protective effect of both [Ch][Glu] and [Ch][Asp] aqueous solutions against FBS-derived ribonucleases.…”

Section: Results and Discussionmentioning

confidence: 99%

Integrated Extraction-Preservation Strategies for RNA Using Biobased Ionic Liquids

Quental

Pedro

Pereira

et al. 2019

ACS Sustainable Chem. Eng.

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The ubiquitous instability of RNA along with issues associated with its purity degree have been preventing its widespread use as low-cost biotherapeutics. On the basis of the well-known capacity of amino acids to specifically interact with RNA when used as chromatographic ligands, a set of amino-acid-based ionic liquids (AA-ILs) was herein investigated, both to act as preservation media and as phase-forming agents of aqueous biphasic systems (ABS). This set of strategies was combined with the goal of developing integrated extraction-preservation platforms. AA-ILs comprising the cholinium cation and anions derived from l-lysine ([Ch][Lys]), l-arginine ([Ch][Arg]), l-glutamic acid ([Ch][Glu]), and dl-aspartic acid ([Ch][Asp]) were studied. It is shown that the stability of RNA is preserved in aqueous solutions of the studied AA-ILs, even in the presence of ribonucleases (RNases). Furthermore, almost all the investigated AA-ILs display no cytotoxicity onto two distinct human cell lines. After identifying the most promising ILs, ABS formed by AA-ILs and polypropylene glycol with a molecular weight of 400 g mol–1 (PPG 400) were investigated as extraction and purification platforms for RNA. Both with pure RNA and bacterial lysate samples, RNA is successfully extracted to the IL-rich phase without compromising its integrity and stability. On the basis of these results, the integrated extraction-preservation process for RNA is finally demonstrated. RNA is initially extracted from the bacterial lysate sample using ABS, after which the IL-rich phase can be used as the preservation medium of RNA up to its use. RNA can be then recovered from the IL-rich phase by ethanol precipitation, and the ABS phase-forming components recovered and reused. Although improvements in the purity level of RNA are still required, the approach here reported represents a step forward in the development of sustainable processes to overcome the critical demand of high-quality/high-purity RNA to be used as biotherapeutics.

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Section: Results and Discussionmentioning

confidence: 99%

Integrated Extraction-Preservation Strategies for RNA Using Biobased Ionic Liquids

Quental

Pedro

Pereira

et al. 2019

ACS Sustainable Chem. Eng.

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“…More than 95% HeLa cells can be arrested in mitosis Problems associated with slow entry (and exit) of mimosine into cells can be minimized by permeabilizing the cells with the bacterial toxin, streptolysin O [29,30]. Such permeabilized cells are fragile, so they were also encapsulated in agarose microbeads (25-150 mm diameter) to protect them during subsequent manipulation.…”

Section: Mimosine Arrests Cells At the G1/s Phase Bordermentioning

confidence: 99%

Mimosine Arrests the Cell Cycle after Cells Enter S-Phase

Hughes

Cook

1996

Experimental Cell Research

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“…Well established approaches are based on, e.g., phenol–chloroform extraction or CsCl/ethidium bromide density gradient centrifugation. These extraction techniques, performed in liquid phases, are time-consuming and associated with the use of toxic, hazardous reagents. , Solid phase extraction (SPE) techniques using mostly glass fiber, silicon dioxide, diatomaceous earth, nitrocellulose, polyamide, or anion exchange resins as solid phases have become the most common method to prepare DNA samples for genetic analysis and other biomedical techniques. − …”

Section: Introductionmentioning

confidence: 99%

Matrix Supported Poly(2-oxazoline)-Based Hydrogels for DNA Catch and Release

et al. 2014

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We describe the synthesis of matrix supported hydrogel structures based on amine containing poly(2-oxazoline)s and their use to bind and release genetic material for potential applications in diagnostics or pathogen detection. Amine containing poly(2-oxazoline)s were synthesized by copolymerization of 2-ethyl-2-oxazoline with a monomer bearing a tert-butyl oxycarbonyl (Boc) protected amine group in the 2-position and subsequent deprotection. The statistical copolymers were used to generate hydrogels and matrix supported hydrogels by cross-linking of a certain fraction of the amine groups with epichlorhydrin. Supported structures were prepared by soaking porous polyethylene (PE) or polypropylene (PP) filter materials in a copolymer/epichlorhydrin solution, which was cross-linked upon heating. Scanning electron microscopy (SEM) of the composites revealed a bead like structure of the gel phase, which could be attributed to a lower critical solution temperature (LCST) behavior of the initial polymer prior to gelation. The dependency of the LCST behavior on the content of amine groups was investigated. Swelling values and the ratio of hydrogel per composite was determined using water sorption analysis. Subsequently, the ability of the systems to absorb and release labeled DNA was tested. Uptake and stimulated release, triggered by changes in pH, temperature, and heparin concentration, were investigated using fluorescence microscopy. Polymerase chain reaction (PCR) proved the successful recovery of the DNA, demonstrating the potential of the presented system for a broad range of molecular biological applications.

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L

Cited by 3 publications

References 43 publications

Integrated Extraction-Preservation Strategies for RNA Using Biobased Ionic Liquids

Integrated Extraction-Preservation Strategies for RNA Using Biobased Ionic Liquids

Mimosine Arrests the Cell Cycle after Cells Enter S-Phase

Matrix Supported Poly(2-oxazoline)-Based Hydrogels for DNA Catch and Release

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