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

Patrícia Pereira,Márcia C Neves,João C Martins,Augusto Q Pedro,Fani Sousa,Inês Rodrigues,Mara G Freire

doi:10.3390/life11101090

Patrícia Pereira, Márcia C Neves + Show 5 more

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https://doi.org/10.3390/life11101090

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Abstract

High quality nucleic acids (with high integrity, purity, and biological activity) have become indispensable products of modern society, both in molecular diagnosis and to be used as biopharmaceuticals. As the current methods available for the extraction and purification of nucleic acids are laborious, time-consuming, and usually rely on the use of hazardous chemicals, there is an unmet need towards the development of more sustainable and cost-effective technologies for nucleic acids purification. Accordingly, this study addresses the preparation and evaluation of silica-based materials chemically modified with chloride-based ionic liquids (supported ionic liquids, SILs) as potential materials to effectively isolate RNAs. The investigated chloride-based SILs comprise the following cations: 1-methyl-3-propylimidazolium, triethylpropylammonium, dimethylbutylpropylammonium, and trioctylpropylammonium. All SILs were synthesized by us and characterized by solid-state 13C Nuclear Magnetic Resonance (NMR), Scanning Electron Microscopy (SEM), elemental analysis, and zeta potential measurements, confirming the successful covalent attachment of each IL cation with no relevant changes in the morphology of materials. Their innovative application as chromatographic supports for the isolation of recombinant RNA was then evaluated. Adsorption kinetics of transfer RNA (tRNA) on the modified silica-based materials were investigated at 25 °C. Irrespective to the immobilized IL, the adsorption experimental data are better described by a pseudo first-order model, and maximum tRNA binding capacities of circa 16 µmol of tRNA/g of material were achieved with silica modified with 1-methyl-3-propylimidazolium chloride and dimethylbutylpropylammonium chloride. Furthermore, the multimodal character displayed by SILs was explored towards the purification of tRNA from Escherichia coli lysates, which in addition to tRNA contain ribosomal RNA and genomic DNA. The best performance on the tRNA isolation was achieved with SILs comprising 1-methyl-3-propylimidazolium chloride and dimethylbutylpropylammonium chloride. Overall, the IL modified silica-based materials represent a more efficient, sustainable, and cost-effective technology for the purification of bacterial RNAs, paving the way for their use in the purification of distinct biomolecules or nucleic acids from other sources.

Highlights

Biopharmaceuticals make up about one-third of drugs currently in development and refer to pharmaceutical substances derived from biological sources with clinical efficacy
Silica was chosen as the stationary phase due to its remarkable properties, namely a high surface area, high thermal and mechanical stabilities, and low-cost [34]
Aiming for the isolation of transfer RNA (tRNA) from bacterial lysates, silica-based materials functionalized with ionic liquids (ILs) were prepared by the modification of chloropropyl silica to obtain the following supported ionic liquids (SILs): [Si][C3C1im]Cl, [Si][N3222]Cl, [Si][N3114]Cl and [Si][N3888]Cl

Summary

Introduction

Biopharmaceuticals make up about one-third of drugs currently in development and refer to pharmaceutical substances derived from biological sources with clinical efficacy. In parallel with developments in biotechnology and clinical studies, the importance of obtaining pure RNA samples has greatly increased once it is a critical step affecting many techniques such as reverse transcriptase polymerase chain reaction, cDNA library construction, Northern blot, and microarrays analysis and their application as biopharmaceuticals [4,5]. Significant advances in biopharmaceuticals purification technologies allowed the development of novel and improved strategies [6]; considering the strict quality criteria (identity—should be confirmed after manufacturing; purity—testing for bacterial endotoxins and process/product associated impurities; safety —tests for endotoxins, bacterial and fungal sterility or bioburden; stability—to establish shelf-life and appropriate storage conditions; potency—in vitro, in vivo tests, or both and based on individual product attributes) that must be fulfilled envisioning therapeutic applications [7,8], there is an unmet need to surpass technical challenges while envisaging products that fulfill the guidelines of regulatory agencies. The main drawbacks are usually related to the capacity of handling the increasing concentration of products in the crude feedstock, the ability to reach a high purity degree, and the possibility to integrate primary isolation and purification steps, while guaranteeing that the structure and biological activity of these bioproducts are preserved [9,10]

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