Integrated Extraction-Preservation Strategies for RNA Using Biobased Ionic Liquids

Abstract

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.

Efficient Isolation of Bacterial RNAs Using Silica-Based Materials Modified with Ionic Liquids

“…Aiming the isolation of both tRNA and bacterial lysates containing tRNA, rRNA, and gDNA, the corresponding bacterial cells were lysed using the lysis solution reported by Chomczynski and Sacchi [ 32 ] with some modifications as previously described [ 24 ]. Briefly, cell lysis of a bacterial pellet from 100 mL of culture medium was accomplished by successive pipetting the pellet in 5 mL of denaturing cell lysis solution (4 M guanidinium thiocyanate; 0.025 M sodium citrate, pH 7.0; 0.5% ( w / v ) N-laurosylsarcosine and 0.1 M β-mercaptoethanol).…”

“…These solvents, which can be liquid at room temperature, have been applied in distinct fields, ranging from (bio)catalysis to extraction and separation techniques [ 14 , 15 , 16 , 17 , 18 ]. Furthermore, a lot of evidence has shown that ILs maintain the structure and integrity of biologically active biomolecules, such as DNA [ 19 , 20 ], proteins [ 21 , 22 ], and RNA [ 23 , 24 , 25 ]. In this field, our research group previously reported pioneering results on the enhanced stability of bacterial transfer RNAs afforded by aqueous solutions of ILs [ 24 , 25 ].…”

“…Furthermore, a lot of evidence has shown that ILs maintain the structure and integrity of biologically active biomolecules, such as DNA [ 19 , 20 ], proteins [ 21 , 22 ], and RNA [ 23 , 24 , 25 ]. In this field, our research group previously reported pioneering results on the enhanced stability of bacterial transfer RNAs afforded by aqueous solutions of ILs [ 24 , 25 ]. On the other hand, by taking advantage of the chemical diversity shown by ILs, a variety of materials (like silica, carbon nanotubes, polymers) have been chemically modified with ILs, resulting in the known supported ionic liquids (SILs).…”

Valorization of Spent Coffee by Caffeine Extraction Using Aqueous Solutions of Cholinium-Based Ionic Liquids

“…With the aim of converting [Ch][Bic] into a compound naturally present in vegetable or animal food sources, namely [Ch]Cl [30], the extracted solution rich in caffeine (after six successive extractions) was titrated with HCl at 2 M, with simultaneous carbon dioxide (CO 2 ) release. The extract, before and after the addition of HCl, was analyzed by 13 C NMR spectra and FTIR to address the conversion of [Ch][Bic] into [Ch]Cl.…”

“…Their biocompatible nature is usually ascribed to their similar chemical structure to the water-soluble essential nutrient cholinium chloride (also known as vitamin B4), which is found in food obtained from vegetable or animal sources and supports several biological functions [26,27]. The cholinium cation has been combined with a wide variety of organic or inorganic anions, producing ILs with different properties [28][29][30]. In the field of biomass extractions, Ribeiro et al [31] applied aqueous solutions of cholinium-based ILs as potential alternatives to imidazolium-based counterparts for the extraction of polyphenols and saponins from the leaves and aerial parts of Ilex paraguariensis (mate) and Camellia sinensis (tea).…”