Ionic liquids for biomacromolecule purification: from molecular interactions to practical applications.

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.

Nucleic acids have emerged as powerful biological and nanotechnological tools. In biological and nanotechnological experiments, methods of extracting and purifying nucleic acids from various types of cells and their storage are critical for obtaining reproducible experimental results. In nanotechnological experiments, methods for regulating the conformational polymorphism of nucleic acids and increasing sequence selectivity for base pairing of nucleic acids are important for developing nucleic acid-based nanomaterials. However, dearth of media that foster favourable behaviour of nucleic acids has been a bottleneck for promoting the biology and nanotechnology using the nucleic acids. Ionic liquids (ILs) are solvents that may be potentially used for controlling the properties of the nucleic acids. Here, we review researches regarding the behaviour of nucleic acids in ILs. The efficiency of extraction and purification of nucleic acids from biological samples is increased by IL addition. Moreover, nucleic acids in ILs show long-term stability, which maintains their structures and enhances nuclease resistance. Nucleic acids in ILs can be used directly in polymerase chain reaction and gene expression analysis with high efficiency. Moreover, the stabilities of the nucleic acids for duplex, triplex, and quadruplex (G-quadruplex and i-motif) structures change drastically with IL cation-nucleic acid interactions. Highly sensitive DNA sensors have been developed based on the unique changes in the stability of nucleic acids in ILs. The behaviours of nucleic acids in ILs detailed here should be useful in the design of nucleic acids to use as biological and nanotechnological tools.

Chapter 6 - Extraction and purification of nucleic acids

Evaluation of commercial kits for the extraction and purification of viral nucleic acids from environmental and fecal samples

Improved ionic-liquid-functionalized macroporous supports able to purify nucleic acids in one step.

The interaction between imidazole-based ionic liquids and the mustard gas simulant CEES and its influence on its solubility

The purpose of this work is to develop a rapid, automated system for nucleic acid purification and concentration from environmental and food processing samples. Our current approach involves off-line filtration and cell lysis (ballistic disintegration) functions in appropriate buffers followed by automated nucleic acid capture and purification on renewable affinity matrix microcolumns. Physical cell lysis and renewable affinity microcolumns eliminate the need for toxic organic solvents, enzyme digestions or other time- consuming sample manipulations. Within the renewable affinity microcolumn, we have examined nucleic acid capture and purification efficiency with various microbead matrices (glass, polymer, paramagnetic), surface derivitization (sequence-specific capture oligonucleotides or peptide nucleic acids), and DNA target size and concentration under variable solution conditions and temperatures. Results will be presented comparing automated system performance relative to benchtop procedures for both clean (pure DNA from a laboratory culture) and environmental (soil extract) samples, including results which demonstrate 8 minute purification and elution of low-copy nucleic acid targets from a crude soil extract in a form suitable for PCR or microarray-based detectors. Future research will involve the development of improved affinity reagents and complete system integration, including upstream cell concentration and cell lysis functions and downstream, gene-based detectors. Results of this research will ultimately lead to improved processes and instrumentation for on-line, automated monitors for pathogenic micro-organisms in food, water, air, and soil samples.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Purification of nucleic acids from a low quantity of bacterial cells in minute volume is important in many clinical and biological applications. We developed a novel microfluidic liquid phase nucleic acid purification chip to selectively isolate DNA or RNA from bacterial cells in the range of 5000 down to a single cell in the sample volume of 1 μl or 125 nl, which can be directly put through on-chip quantitative PCR assay. The aqueous phase bacterial lysate was isolated in an array of microwells, after which an immiscible organic (phenol-chloroform) phase was introduced in a headspace channel connecting the microwell array. Continuous flow of the organic phase increases the interfacial contact with the aqueous phase to achieve purification of target nucleic acid through phase partitioning. Significantly enhanced nucleic acid recovery yield, up to 10 fold higher, was achieved using the chip-based liquid phase nucleic acid purification technique compared to that obtained by the conventional column-based solid phase nucleic acid extraction method. One step vacuum-driven microfluidics allowed an on-chip quantitative PCR assay to be carried out in the same microwells within which bacterial nucleic acids were isolated, avoiding sample loss during liquid transfer. Using this nucleic acid purification device set in a two-dimensional (2D) array format of 900 microwells, it was demonstrated for the first time that high-throughput extraction of RNA couple with direct on-chip PCR analysis from single bacterial cells could be achieved. Our microfluidic platform offered a simple and effective solution for nucleic acid preparation, which can be integrated for automated bacterial pathogen detection and high throughput transcriptional profiling.

Nucleic Acid-free Matrix: Regeneration of DNA Binding Columns

Electrospray ionization mass spectrometry with variable collision induced dissociation of the isolated [(cation)(2)anion](+) and/or [(anion)(2)cation](-) ions of imidazolium-, pyridinium-, pyrrolidinium-, and piperidinium-based ionic liquids (ILs) combined with a large set of anions, such as chloride, tetrafluoroborate, hexafluorophosphate, trifluoromethanesulfonate, and bis[(trifluoromethyl)sulfonyl]imide, was used to carry out a systematic and comprehensive study on the ionic liquids relative interaction energies. The results are interpreted in terms of main influences derived from the structural characteristics of both anion and cation. On the basis of quantum chemical calculations, the effect of the anion upon the dissociation energies of the ionic liquid pair, and isolated [(cation)(2)anion](+) and/or [(anion)(2)cation](-) aggregates, were estimated and are in good agreement with the experimental data. Both experimental and computational results indicate an energetic differentiation between the cation and the anion to the ionic pair. Moreover, it was found that the quantum chemical calculations can describe the trend obtained for the electrostatic cation-anion attraction potential. The impact of the cation-anion interaction strengths in the surface tension of ionic liquids is further discussed. The surface tensions dependence on the cation alkyl chain length, and on the anion nature, follows an analogous pattern to that of the relative cation-anion interaction energies determined by mass spectrometry.

Proteinase K (PK) is a proteolytic enzyme that has been widely used in nucleic acid purification, leather production, environmental protection, and other industrial applications. However, this biocatalyst cannot tolerate high temperatures which has severely restricted its wider application. As reported in previous studies, cholinium-based ionic liquids (ILs) have gained tremendous attention serving as a promising media to stabilize and preserve proteins, DNA, and other biomolecules due to their environmentally benign nature and biocompatibility. In this work, we chose 13 different kinds of cholinium-based ILs to examine their effects on the thermal stability and enzymatic activity of PK. We found that biocompatible cholinium-based ions with appropriately chosen anions can greatly improve the thermal stability of PK, whose melting temperature (Tm) is increased from ∼74.4 °C to 87.7 °C. However, the enzymatic activity is slightly reduced in the presence of ILs. Further comparison of our results with other literature findings suggests that kosmotropic anions of cholinium-based ILs are crucial to maintain the thermal stability of proteins. However, to achieve the best performance, the choice of IL anions is protein specific.

Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries.

The use of affinity tagged PNA capture probes offers an efficient means for the purification of nucleic acids by hybridization. Two different approaches are described. A sequence specific method and a generic method. The sequence specific method requires sequence information on the target and synthesis of a dedicated PNA. It can be used to selectively purify the nucleic acid containing the target from non-related nucleic acids and other cellular components. The generic method uses a "universal" triplex forming PNA and requires no sequence information on the target. It can be used in the bulk purification of large nucleic acids.

Connection between dielectric constant and total number of hydrogen-bond groups per cation–anion pair in ionic liquids

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!