Integrated Extraction-Preservation Strategies for RNA Using Biobased Ionic Liquids

resumo

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 morl (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.

palavras-chave

AQUEOUS BIPHASIC SYSTEMS; SINGLE-STEP METHOD; SECONDARY STRUCTURES; PROTEIN SEPARATION; 2-PHASE SYSTEMS; GOODS BUFFERS; ACID; PURIFICATION; STABILITY; CHROMATOGRAPHY

categoria

Chemistry; Science & Technology - Other Topics; Engineering

autores

Quental, MV; Pedro, AQ; Pereira, P; Sharma, M; Queiroz, JA; Coutinho, JAP; Sousa, F; Freire, MG

Grupos

G1 - Materiais Porosos e Nanossistemas
G4 - Materiais Renováveis e Economia Circular
G5 - Materiais Biomiméticos, Biológicos e Vivos

Projectos

CICECO - Aveiro Institute of Materials (UID/CTM/50011/2019)

Ionic liquid-based supports for pre-miRNAs purification targeting Alzheimer's disease (PUREmiRSILs)

agradecimentos

The authors acknowledge to the CICS-UBI projects supported by FEDER funds through the POCI - COMPETE 2020 (Project POCI-01-0145-FEDER-007491) and National Funds by FCT (Project UID/Multi/00709/2013), and to the project CICECO-Aveiro Institute of Materials, FCT ref. UID/CTM/50011/2019, financed by national funds through the FCT/MCTES. This work was also supported by the project POCI-01-0145-FEDER-029496, cofinanced by FEDER, through POCI -Operational Programme Competitiveness and Internationalization, and National Funds by FCT. The authors acknowledge the financial support from the European Union Framework Programme for Research and Innovation HORIZON 2020, under the TEAMING Grant agreement No 739572 -The Discoveries CTR. P.P. acknowledges a postdoctoral fellowship from a FCT-funded project (PTDC/1399/2014), and M.V.Q, acknowledges the FCT Ph.D. grant SFRH/BD/109765/2015. M.G.F. acknowledges the European Research Council (ERC) for the Starting Grant ERC-2013-StG-337753.