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Integrated extraction-preservation strategies for RNA using bio-based ionic liquids Maria J. Quental, Augusto Pedro, Patrícia Pereira, Mukesh Sharma, João A. Queiroz, Joao A.P. Coutinho, Fani Sousa, and Mara G. Freire ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b00688 • Publication Date (Web): 26 Apr 2019 Downloaded from http://pubs.acs.org on April 27, 2019

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ACS Sustainable Chemistry & Engineering

Integrated extraction-preservation strategies for RNA using bio-based ionic liquids

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Maria J. Quental†, Augusto Q. Pedro†, Patrícia Pereira§, Mukesh Sharma†, João A. Queiroz‡,

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João A.P. Coutinho†, Fani Sousa‡* and Mara G. Freire†*

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†CICECO

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193 Aveiro, Portugal.

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§ITQB-NOVA

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NOVA de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.

- Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-

- Institute of Biological Chemistry and Chemical Technology, Universidade

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‡CICS-UBI

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Covilhã, Portugal.

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*E-mail address: [email protected].

- Health Sciences Research Centre, Universidade da Beira Interior, 6201-506

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KEYWORDS. Ribonucleic acid, amino-acid-based ionic liquids, aqueous biphasic

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systems, integrated extraction-preservation process.

ACS Paragon Plus Environment

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to the IL-rich phase without compromising its integrity and stability. Based on these

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results, the integrated extraction-preservation process for RNA is finally demonstrated.

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RNA is initially extracted from the bacterial lysate sample using ABS, after which the IL-

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rich phase can be used as the preservation medium of RNA up to its use. RNA can be

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then recovered from the IL-rich phase by ethanol precipitation, and the ABS phase-

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forming components recovered and reused. Although improvements in the purity level

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of RNA are still required, the approach here reported represents a step forward in the

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development of sustainable processes to overcome the critical demand of high-

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quality/high-purity RNA to be used as biotherapeutics.

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INTRODUCTION

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Ribonucleic acid (RNA) is an essential biopolymer with various biological functions,

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such as in coding, decoding, regulation, and expression of genes.1 Accordingly, nucleic

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acids are powerful biological tools in several fields2, ranging from fundamental to

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applied research. For instance, RNA is a target of study in: (1) the quantification of


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transcripts related to protein levels resulting from the expression of target genes, which

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are essential to develop drugs that regulate gene expression3; (2) structural studies

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using biophysical methods, to further understand RNA features and functions4; and (3)

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the development of RNA-based therapeutics.5 In all these biological-based applications,

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the methods and solvents used for extracting and purifying RNA from various types of

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cells or other biological matrices, and respective preservation, are critical issues.3

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RNA research studies and RNA-based therapeutics applications require large amounts

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of high-purity/high-quality RNA.6 Accordingly, the RNA synthesis by chemical,

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enzymatic and, more recently, by recombinant technologies (using prokaryotic hosts

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such as Escherichia coli and Rhodovulum Sulfidophilum) has become an indispensable

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tool.6 Up to date, two methods for isolating RNA prevail, namely the highly used

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extraction method based on phenol and chloroform7 and solid-phase extraction (SPE)

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using silica-based materials.8,9 In addition to these recovery protocols, other purification

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steps

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electrophoresis13 are applied to remove process-associated impurities.14

including

chromatography10-12

and

denaturing


polyacrylamide

gel

4

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In RNA bioprocessing, besides high purity, other parameters are essential, namely the

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biopolymer integrity and biological activity. Both features are particularly relevant for

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their use as biotherapeutics, which should fulfil the requirements of regulatory

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authorities.10-12 RNA is a highly labile polymer with inherent chemical instability and can

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suffer rapid degradation by nucleases.1 Therefore, RNA is normally preserved in high-

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purity water or sterile phosphate buffered saline (PBS) aqueous solutions under

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refrigeration at temperatures below 4 °C or at -80 °C for short- and long-term

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applications, respectively.1

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Based on the described drawbacks, the development of effective and sustainable

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recovery processes and the identification of new preservation media are of paramount

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relevance, where ionic liquids (ILs) can play a role in both approaches. Some published

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works in the past few years described the strong affinity between ILs and different

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biomolecules, showing that ILs are solvents with a high potential for biomolecules

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preservation and extraction15, including nucleic acids.16 However, in most of these

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studies, no extractions from real complex samples have been performed, and DNA is


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usually the target. This preference is mainly due to the conditions required to work with

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RNA, as consequence of its poor stability (short half-life) when compared to DNA and

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ubiquitous presence of ribonucleases (RNases).1

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Amongst the few reports comprising ILs and RNA, Mazid et al.17 proved that aqueous

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solutions of cholinium dihydrogen phosphate ([Ch][DHP]) are able to prolong the shelf

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life of siRNA up to three months in presence of RNase A. Mamajanov et al.18 evaluated

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the stability of a duplex RNA, which adopts a A-form helix in several aqueous buffers.

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The authors showed that the RNA melting temperature in aqueous solutions of

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cholinium chloride is lower than in other salt aqueous solutions.18 Fister et al.19

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successfully used imidazolium-based ILs to disintegrate virus particles and isolate RNA.

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In summary, these works dealing with ILs and RNA are focused on two strategies: (i) to

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use ILs as alternative solvents to maintain or increase the stability and integrity of RNA;

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or (ii) to use ILs as alternative media to isolation and recover the target nucleic acid.

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The exploration of integrated systems, i.e. by using the same solvent in both the

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recovery and purification steps and as preservation media, was not attempted hitherto.


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ILs can be used to form liquid-liquid systems to be used in separation processes, in

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which aqueous biphasic systems (ABS) are a promising alternative when dealing with

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biologically active products.20 Albeit ABS have been described as biocompatible

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separation systems due to their high water content, most of the studied IL-based ABS

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are

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biomacromolecules. This fact prompted the research on ILs based on natural

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feedstocks; for instance21, biocompatible ILs formed by the cholinium cation and anions

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derived from essential amino acids were reported in 2005 by Ohno and co-workers.22

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After this pioneering work, an increasing number of reports focusing on their

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properties23-25 have emerged. However, cholinium amino-acid-based ILs (AA-ILs) have

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been scarcely applied in the development of separation processes. AA-ILs have been

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combined with inorganic salts (K3PO4 and K2CO3)26 and polypropylene glycol27 to form

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IL-based ABS, where a comprehensive characterization of the respective phase

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diagrams was performed. In two of these works it was evaluated the extraction capacity

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of ABS for two model proteins (bovine serum albumin and trypsin27) and for

formed

by

imidazolium-based

ILs20,

which


may

be

deleterious

for

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phenylalanine enantiomers.28 Still, the performance of these systems was not appraised

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in the separation of target compounds from real matrices.

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As result of the high affinity between amino acids and RNA29, amino-acid-based affinity

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chromatography is highly used to purify this biopolymer.10-12 In addition, amino acids

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may have an important role in RNA loop recognition through a base-flipping process.29

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This well-known preferential affinity, together with the fact that in general amino-acid-

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based ILs are non-toxic, biodegradable and biocompatible30, motivated us to investigate

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the use of aqueous solutions of amino-acid-based ILs as alternative solvents to stabilize

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and protect RNA from nucleases coupled to their use in the formation of ABS,

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envisioning the development of integrated preservation-extraction strategies. In affinity

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chromatography, hydrophilic amino acids (e.g. arginine) are usually used as ligands to

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improve the RNA biorecognition.10-12 Accordingly, we selected two hydrophilic amino

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acids (arginine and lysine) and two hydrophobic amino acids (aspartic and glutamic

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acids) from the aliphatic amino acids group to be used as IL anions. AA-ILs composed

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of the cholinium cation combined with the anions L-lysinate, L-argininate, L-glutamate


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(Mili-Q system, Milipore/Waters) was treated with 0.05 % diethyl pyrocarbonate (DEPC,

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Sigma-Aldrich (USA)). All reagents used for RNA extraction and fluorescein

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isothiocyanate isomer 1 (FITC) were acquired from Sigma Aldrich. Acetonitrile from

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Sigma Aldrich (USA), 99 wt %, and methanol from VWR (USA), HPLC grade, were

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used.

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Small RNA biosynthesis and isolation. A cell culture of E. coli

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plasmid pBHSR1-RM containing the sequence of human pre-miR-29b was used for the

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small RNA (sRNA) production, namely transfer RNA (tRNA), pre-miR-29b and 6S RNA.

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Growth was carried out in a rotary shaker at 37 °C and 250 rpm using shake-flasks of 1

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L, containing 0.25 L of Terrific Broth medium (12 g/L Tryptone, 24 g/L Yeast extract, 4

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mL/L glycerol, 0.017 M KH2PO4 and 0.072 M K2HPO4) and supplemented with 30

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µg/mL of kanamycin. Cell growth was evaluated by measuring the optical density at 600

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nm of the cell culture, and suspended at the beginning of the logarithmic decline phase

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(OD600 after ca. 8 h), as previously described31. The cultivated cells of E. coli

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were recovered by centrifugation at 4500 × g and 4 °C for 20 min, and the pellets stored


D L strain modified with

D L

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at -20 °C. The recovery of sRNA was carried out according to the acid guanidinium

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thiocyanate-phenol-chloroform extraction method described by Chomczynski et al.32,

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with some modifications as described. Bacterial pellets (100 mL) were resuspended in 5

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mL of denaturing cell lysis solutions (4 M guanidinium thiocyanate; 0.025 M sodium

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citrate, pH 7.0; 0.5 % (w/v) N-laurosylsarcosine and 0.1 M O"! *

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perform the lysis. After incubating on ice for 10 min, cellular debris, genomic DNA and

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proteins were precipitated by mixing 5 mL of water-saturated phenol and 0.5 mL of 2 M

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sodium acetate (pH 4.0). The sRNA isolation was achieved by adding 1 mL of

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chloroform/isoamyl alcohol (49:1, v:v), and by vigorously mixing until two immiscible

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phases were obtained. The upper aqueous phase, which contains mostly RNA, was

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recovered and concentrated by the addition of 5 mL of ice-cold isopropanol. Precipitated

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molecules were recovered by centrifugation at 10,000 × g for 20 min at 4 °C, and

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resuspended in 1.5 mL. sRNA molecules were concentrated again with 1.5 mL of ice-

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cold isopropanol. After centrifugation for 10 min at 10,000 × g (4 °C), the RNA pellet

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was washed with 7.5 mL of 75 % ethanol and incubated at room temperature for 10

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min, followed by a 5 min centrifugation at 10,000 × g (4 °C). The air-dried RNA pellet


%(

(&

7 to

12

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was solubilized in 1 mL of 0.05 % DEPC-treated water. Finally, the 260 and 280 nm

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absorbance of the samples were measured using a Nanodrop spectrophotometer to

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assess the RNA quantity, and agarose gel electrophoresis was performed to assess the

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RNA purity.

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Aqueous solutions of ionic liquids as stabilization media of RNA. RNA was dissolved in

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aqueous solutions of different AA-ILs, at 20 wt %, and the mixtures incubated at room

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temperature, (25 ± 1 °C), for 1 h and 15 days. The period of 15 days was selected since

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it is considered an average time between extraction and use of RNA samples, being in

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agreement with our previous experience33 and with the study performed by Mathay et

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al.34 The shorter incubation period of 1 h was set to mimic typical incubation periods

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often performed in different laboratory protocols involving RNAs, while allowing to

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observe the effect of the IL in the stability of the biomolecule at two different periods.

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RNA was precipitated by adding 2 volumes of pure alcohols (butanol in the case of

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[Ch][Arg], and ethanol for the remaining), and further incubated for 2 h at -80 °C, until it

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was recovered by centrifugation at 15,000 × g for 30 min at 4 °C. Nucleic acids pellets


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were air-dried for 10 min at room temperature and solubilized in 0.05 % DEPC-treated

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water.

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Agarose gel electrophoresis. The integrity and identification of sRNAs recovered after

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incubation with ILs were evaluated using agarose gel electrophoresis. 20 Q6 of sRNA

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corresponding to ca. 48 µg was analyzed by horizontal electrophoresis using 0.8 % of

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agarose gel (Hoefer, USA), and carried out at 110 V for 30 min with TAE buffer (40 mM

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Tris base, 20 mM acetic acid and 1 mM EDTA, pH 8.0). The bands corresponding to

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sRNA molecules were visualized in the gel using the UVItec FireReader system

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(UVItec, UK) after staining with 0.01 % GreenSafe Premium (NZYtech, Portugal).

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Circular Dichroism (CD) spectroscopy. CD experiments were performed in a Jasco J-

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815 spectropolarimeter (Jasco, USA), using a Peltier-type temperature control system

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(model CDF-426S/15). CD spectra were acquired at a constant temperature of 25 °C

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using a scanning speed of 10 nm/min, with a response time of 1 s over wavelengths

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ranging from 210 to 320 nm. The recording bandwidth was of 1 nm with a step size of 1

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nm using a quartz cell with an optical path length of 1 mm. Three scans were averaged


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per spectrum to improve the signal to-noise ratio and the spectra were smoothed by

214

using the noise-reducing option in the operating software of the instrument. CD melting

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experiments were performed in the temperature range from 10 to 110 °C, with a heating

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rate of 1 °C/min, by monitoring the ellipticity at 265 nm. Data were converted into

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fraction folded (S) as detailed in the literature.35 Data points where then fitted to a

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Boltzmann distribution (OriginPro 2015) and the melting temperatures (Tm) were

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determined from the two-state transition model used, using the first derivative method.36

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Zeta potential. The zeta potential of RNA samples in presence and absence of [Ch][AA]-

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ILs were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS

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particle analyzer (Malvern Instruments, UK), equipped with a He-Ne laser, at 25 °C.

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These measurements were performed in disposable capillary cells and computed using

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the Henry’s [F(Ka) 1.5] and Smoluchowsky models. All data were treated with the

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Zetasizer software v 7.03 and the analyses were held in triplicate with an average of 30

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measurements per sample. RNA was incubated with IL (20 wt %) aqueous solutions

227

during 1 h at room temperature (25 ± 1 °C), at a total RNA concentration of 200 µg/mL.


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The values given represent the average of three independent samples and respective

229

standard deviation.

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Cell viability. The cellular viability of RNA in AA-ILs aqueous solutions was achieved by

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the MTS assay (shown as percentage of control (%)) using the Cell Titer 96® AQueous

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Non-Radioactive Cell Proliferation Assay (Promega), according to the manufacturer’s

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instructions. Human fibroblasts and cervical cancer cells (HeLa) were cultured at 37 °C,

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in a humidified atmosphere containing 5 % CO2. Three different passages of each cell

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line were seeded at a density of 1×104 cells per mL in a 96-well plate; after 24 h, the cell

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culture medium was replaced by serum-free culture medium. After 12 h, the different

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formulations of ILs with RNA (10 µL of a mixture containing 200 µg/mL RNA and RNA in

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20 wt % ILs aqueous solutions) were added to the cells and transfection was carried out

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for 24 h. The culture medium was exchanged by fresh medium and a mixture of

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MTS/phenazine metasulfate (PMS) was added to each well, and cells were incubated

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for 2 h at 37 °C in a humidified atmosphere containing 5 % CO2, protected from light.

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Following incubation, absorbance measurements of the soluble brown formazan


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produced by MTS were performed in a microplate reader (Bio-Rad) at 490 nm. Cells

244

incubated with absolute ethanol were used as positive control for cytotoxicity, while

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untreated cells were used as negative controls. The values given correspond to the

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average percentage values relative to untreated cells of 3 independent experiments and

247

respective standard deviation.

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Extraction of RNA using AA-IL-based ABS and phase-forming components recyclability.

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Aiming to address the possibility of using AA-IL-based ABS as alternative extraction and

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purification strategies of RNA, the respective ABS phase diagrams (water + AA-IL +

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PPG 400), comprising the binodal curve, tie-lines (TLs) and respective tie-line length

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(TLL), were determined by the cloud point titration method at 25 °C and atmospheric

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pressure, as described in the literature.37 Aqueous solutions of each IL in the range from

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50 to 70 wt % and aqueous solutions of PPG 400 from 50 to 80 wt % were

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gravimetrically prepared and used for the determination of the binodal curves. The

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dropwise addition of each aqueous solution of IL to the PPG 400 aqueous solution was

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carried out until the detection of a cloudy (biphasic) solution, followed by the dropwise


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addition of distilled water until a complete limpid solution (monophasic region) is

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obtained. This procedure was carried out under constant stirring, and the ternary

260

system compositions were determined by weight quantification of all components ( 10-4

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g) using an analytical balance Mettler Toledo Excellence XS205 DualRange. Each ABS

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phase was identified by conductivity (experimental procedure and data are given in the

263

Supporting Information).

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The ternary mixtures used in the extraction experiments of pure RNA were

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gravimetrically prepared at the following mixture composition: 20 wt % of PPG 400 + 20

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wt % of AA-IL ([Ch][Glu] or [Ch][Asp]) + 60 wt % of DEPC-treated water with 400 µg/mL

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RNA. This fixed mixture composition was selected considering the biphasic region of all

268

the studied systems. Furthermore, this mixture point is close to the binodal curves,

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allowing to have a minimum amount of IL and maximum amount of water in each ABS,

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while keeping in mind the development of a sustainable method. The TLs for this

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mixture point in each ABS, which give the composition of each phase, were determined

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according to the method proposed by Merchuk et al.38. The compositions of the top and


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bottom phases and the overall system compositions were determined by the lever-arm

274

rule and mass balance approaches, as described in the literature.38 Further information

275

is given in the Supporting Information. ABS with a total weight of 1 g were used. All

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mixtures were carefully stirred and centrifuged for 5 min at 25 °C (5000 × g), followed by

277

the separation of the two phases. RNA was recovered from both phases by alcohol

278

precipitation, as described before. The partitioning of nucleic acids in AA-IL-based ABS

279

was ascertained by agarose gel electrophoresis.

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After demonstrating the favourable RNA extraction (with pure RNA samples) to the IL-

281

rich phase, new experiments were carried out envisaging the purification of RNA directly

282

from real matrices, in this work with a bacterial lysate sample comprising RNA, genomic

283

DNA, and some proteins and other impurities. Detailed experimental information on the

284

sample preparation and composition is given in the Supporting Information. The ABS

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chosen are composed of 20 wt % of PPG 400, 20 wt % of AA-IL and 60 wt % of lysate.

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Each mixture composition was weighed, mixed and centrifuged for 5 min (5000 × g).

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RNA evaluation was carried out as described before.


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With the final goal of developing sustainable separation processes, the recovery and

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reusability of the ABS phase-forming components was finally ascertained. To this end,

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the recovery of RNA from the IL-rich phase was firstly performed by ethanol

291

precipitation, as described above. Then, the volatile solvents present in the IL-rich

292

phase were evaporated under vacuum at room temperature, up to constant weight,

293

leaving only the IL and PPG 400 as the non-volatile species. The recovered PPG 400

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and IL were then reused (two more times) in the ABS formation, and their impact on the

295

integrity of RNA after a new extraction step was assessed by agarose gel

296

electrophoresis. The recovered phase-forming components aqueous solutions were

297

also evaluated in terms of their feasibility for keeping the RNA stability and integrity

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toward the development of integrated extraction-preservation processes.

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RESULTS AND DISCUSSION

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All ILs used, namely [Ch][Lys], [Ch][Arg], [Ch][Glu] and [Ch][Asp], were synthesized in

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this work. Their chemical structure and details on their characterization and purity are

302

given in the Supporting Information (cf. Figures S2-S5). These ILs were designed and


20

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synthesized based on the specific affinity between amino acids and RNA and their

304

previous use on amino-acid-based affinity chromatography for RNA purification10-12,

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coupled to the fact that amino-acid-based ILs may present low toxicity and low

306

environmental impact.30

307

Integrity, stability and cytotoxicity of RNA in presence of aqueous solutions of ILs. Initial

308

studies on the integrity of RNA at 25 °C in presence of aqueous solutions of [Ch][Lys],

309

[Ch][Arg], [Ch][Glu] and [Ch][Asp] at 20 wt % were carried out by agarose gel

310

electrophoresis in order to address the potential of the synthesized ILs to act as

311

alternative preservation media. Since we aim to apply ABS as a simultaneous extraction

312

and preservation strategy, the concentration of IL used in the preservation stage should

313

be in accordance with the IL content required to form two-phase systems. Higher

314

concentrations of IL were not investigated since they compromise the sustainability of

315

the process (by increasing both the environmental footprint and cost of the process).

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The results obtained are depicted in Figure 1, where a well-defined band with a low

317

molecular weight is shown, corresponding to the recombinant RNA fraction recovered


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from E. coli. To avoid interferences of ILs in the electrophoretic run, alcohol-induced

319

precipitation of RNA was applied in all samples. Different alcohols (propan-2-ol, ethanol,

320

propanol, and butanol) were tested in what concerns to their ability to precipitate RNA,

321

and distinct behaviours were observed (shown in Figure S6 in the Supporting

322

Information). Although discussed here with a different purpose, the identification of

323

appropriate solvents for the induced precipitation of RNA is valuable when considering

324

the RNA and ILs recovery proposed below.

325

The results shown in Figure 1 correspond to RNA precipitated with the best identified

326

alcohols, namely butanol for the [Ch][Arg] aqueous solution and ethanol for the

327

remaining ILs aqueous solutions. The first lane of the agarose gel corresponds to sRNA

328

recovered from E. coli, including a well-defined band with a low molecular weight, and in

329

agreement with a previous work.39 Depending on the original media, either ethanol or

330

isopropanol have been reported for the precipitation of nucleic acids.40 Ethanol and

331

butanol are however more benign and less hazardous alcohols than isopropanol41;

332

therefore, the efficient recovery of RNA from ILs aqueous solutions with these alcohols


22

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ellipticity peak at 265 nm; however, the investigated ILs interfere with the CD spectrum

344

of RNA at lower wavelengths (cf. the representative CD spectrum of RNA in presence of

345

[Ch][Arg] in Figure S7 in the Supporting Information). The CD melting temperatures (Tm)

346

of RNA were thus determined by addressing the changes in the RNA ellipticity at 265

347

nm in the temperature range from 10 to 110 °C. The obtained results corresponding to 1

348

h of incubation with each AA-ILs are given in Table 1 (the respective melting curves are

349

depicted in Figure S8 in the Supporting Information). Tm is defined as the temperature at

350

which half of the molecules of a double-stranded species become single-stranded,

351

where higher Tm values are an indicator of a higher RNA thermostability.43 The Tm of

352

RNA dissolved in high-purity water is 45.8 °C, but it is lower in presence of [Ch][Lys]

353

and [Ch][Arg] aqueous solutions (39.9 °C and 43.3 °C, respectively). Contrarily, a

354

remarkable enhancement of ca. 15 °C (60.0 °C and 60.3 °C, respectively) in the Tm of

355

RNA was observed in presence of aqueous solutions of [Ch][Glu] and [Ch][Asp],

356

indicating that the thermal stability of RNA during 1 h of incubation in AA-ILs is

357

significantly enhanced with these two ILs. In addition to the high stabilizing effect and

358

preservation of RNA afforded by the studied ILs for short-term periods, the results


24

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359

shown in Table 1 also demonstrate the potential of these two ILs as preservation agents

360

even after 15 days of incubation. These results are in agreement with a previous work17,

361

showing an increase of the RNA stability with buffered aqueous solutions of [Ch][DHP],

362

although a lower enhancement, ca. 8.5 °C, was achieved.

363

Overall, this set of results carried out with pure RNA demonstrate that RNA is more

364

stable in aqueous solutions of [Ch][Glu] and [Ch][Asp] than in 0.05 % DEPC-treated

365

water, both for short-term and medium-term storage periods (1 h and 15 days). Thus, it

366

seems that the anionic counterpart of ILs has a significant influence on the RNA

367

structure and stability, which is discussed in more detail below.

368


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Page 26 of 53

370 371

Table 1. Melting temperature of RNA in 0.05 % DEPC-treated water and in AA-ILs (20

372

wt %) aqueous solutions after 1 h and 15 days of incubation at 25 °C.

Tm / °C

Tm / °C

after 1 h

after 15 days

RNA in 0.05 % DEPC-treated water 45.8 ± 0.5

46.0 ± 0.5

RNA in 20 wt % [Ch][Lys]

39.9 ± 0.5

29.5 ± 2.0

RNA in 20 wt % [Ch][Arg]

43.3 ± 0.7

32.4 ± 5.5

RNA in 20 wt % [Ch][Glu]

60.0 ± 0.3

56.4 ± 0.4

RNA in 20 wt % [Ch][Asp]

60.3 ± 0.3

56.2 ± 0.4

373

374

Aiming at better understanding the mechanisms behind the preservation and enhanced

375

stability promoted by some of the investigated ILs, zeta potential measurements of RNA

376

in pure water and in aqueous solutions of 20 wt % of ILs were carried out. Zeta potential


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377

corresponds to the electrostatic potential generated in an applied electric field due to the

378

attraction between charged species and the oppositely charged electrode, thereby

379

allowing to draw some conclusions on the solution effective charge density. Nucleotides

380

are the building blocks of RNA, which comprise phosphate groups that confer to RNA a

381

negative charge, resulting in a zeta potential of approximately – 40 mV, as shown in

382

Figure 2. In all assays it was observed that when dissolved in aqueous solutions of ILs,

383

RNA displays a less negative value, confirming that all ILs employed in this study

384

interact with RNA. Since less negative zeta potential values are obtained when in

385

presence of ILs, it is plausible to admit that when in aqueous solution the cholinium

386

cation forms an outer shell around RNA, in agreement with previously reported

387

molecular dynamics simulations results.33


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Integrated Extraction-Preservation Strategies for ... - ACS Publications

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