Application of Magnetic Nanoparticles in RNA and DNA Separation

Over the past few decades, magnetic nanoparticles (MNPs) have been increasingly used to separate and differentiate biomolecules, which is the basis of most current molecular diagnostic procedures. The size, morphology, and dispersion of MNPs endow them with specificity, affinity, and binding capacity for biomolecules.

Application of Magnetic Nanoparticles in DNA Separation

A prerequisite for the application of many molecular methods (such as PCR, real-time PCR, and sequencing) to microbial detection is the isolation of high-quality DNA from complex mixtures. However, many reactions and techniques used in molecular biology will be disturbed by the frequent presence of cells or other contaminants in this complex mixture.

The classic approach to DNA extraction is combined with cell lysis, through a series of sedimentation and centrifugation steps to remove cellular contaminants and extracellular components. However, the process requires the addition of harmful chemicals and is difficult to automate. In the late 1970s, researchers discovered that silica as an adsorbent was ideal for DNA isolation, and it then became the basic raw material for most DNA isolation kits today. This discovery also makes the separation process easy to automate. Today, commercial kits for isolating DNA from a variety of biological samples use silica-coated magnetic particles. Recently, two different types of superparamagnetic nanoparticles have been successfully applied to isolate dinoflagellate DNA from marine samples to collect PCR- ready DNA that can be used to detect venomous species. One of the nanoparticles is coated with silica and has an average particle size of 150 nm, and the other contains an agarose derivative with a diethyl aminoethyl (DEAE) base as the support material. When these materials were applied to downstream PCR reactions, DNA yields, and amplification results were satisfactory, suggesting that they are effective alternatives to commercial kits based on immobilized silica resin and superparamagnetic polymer particles. In addition, related experiments have shown that silica-coated nanoparticles have the best yields for DNA extraction from those samples fixed with Lugol's iodine solution.

Saiyed et al. applied magnetizable solid phase support (MSPS) technology to the biological field and found that the quality and yield of DNA isolated from all samples using magnetic nanoparticles was higher than that of traditional DNA extraction procedures. The yield of DNA was quantified after electrophoresis in a 0.8% agarose gel containing 0.5 μg ml ethidium bromide and visualized by a UV transilluminator using a gel recording system. As can be seen from Figure 1, using magnetic nanoparticles as solid supports, the average yield of DNA recovered from agarose gel was 80% (80±5%), while the average yield of DNA recovered by phenol extraction and spin column method The yield is between 50-60%.

Figure 1: (a) Agarose gel electrophoresis of genomic DNA isolated from human blood cells with magnetic nanoparticles. Lanes: 1 = DNA molecular weight markers (λphage DNA/Hind III digestion); 2-4 = genomic DNA (23 kb) isolated from human blood cells (equal volume loaded from tubes containing isolated DNA); (b) Agarose gel elution Agarose gel electrophoresis of extracted DNA. Lane: 1=DNA molecular weight marker (λphage DNA/Hind III digestion); 2=DNA eluted with magnetic nanoparticles as a solid-phase adsorbent; 3=DNA eluted with phenol extraction; 4=spinned with glass wool Column Elution DNA

For DNA sequencing, a DNA separation procedure called solid-phase reversible immobilization (SPRI) was developed based on the principle that DNA binds to the surface of carboxyl-coated superparamagnetic particles under high salt concentration and PEG. This technique shows the great potential of magnetic particle technology for automation, high sample throughput, and cost reduction. Currently, SPRI has been successfully applied to automated systems that can process thousands of samples per day at low cost. Another application of magnetic particle technology is the development of superparamagnetic nanoparticles-based nanosensors for DNA hybridization experiments. This application can be further refined for the design of biocompatible magnetic nanosensors that can sensitively detect specific mRNAs, proteins, enzyme activities, and pathogens in the low femtomolar range.

Application of Magnetic Nanoparticles in RNA Isolation

Those commercially available RNA isolation kits are generally based on the following two strategies:

1) Homogenization in 4M guanidine thiocyanate and 0.1M β-mercaptoethanol, followed by ethanol precipitation;

2) Fast separation procedure using guanidinium thiocyanate and phenol-chloroform mixture.

However, there are still problems such as long processing time, high sample throughput, and RNA degradation during the extraction process. Magnetic separation technology has shown its potential to improve the performance of RNA isolation, including reducing purification time, RNA A

Degradation, and cost. Typically, the first step is to purify nucleic acids by adding silica-coated magnetic beads directly to the homogenized sample. Subsequent washing steps remove proteins and salts from the beads and bound nucleic acids, followed by the addition of DNase to remove genomic DNA. Finally, the purified RNA is eluted with a low-salt buffer. This RNA is usually of high quality and can be used in real-time RT-PCR and microarrays (Figure 2).

Figure 2: (A) TEM image of carboxyl-wrapped MNPs. (B) Isolation of mRNA from MDA-MB-231 cells using MNPs. Lane 1: RNA isolated with RNeasy Mini Kit; Lane 2: mRNA isolated with 50 μg MNPs; Lane 3: 100 μg MNPs-isolated mRNA; Lane 4: 200 μg MNPs-isolated mRNA. (C) Agarose gel of PCR products showing b-actin amplification. Lane 1: DNA labeling; Lane 2 and Lane 3: b-actin was extracted from mRNA isolated from 200 μg and 100 μg MNPs, respectively.

Isolation of mRNA involves specific complementary hybridization between the poly A sequence of the mRNA and the oligo(dT) sequence covalently attached to a solid support. In this context, magnetic-based separation techniques have also shown the ability to increase yield and reduce purification time and cost. Oligo(dT)-coated magnetic beads are loaded into the lysate samples and Poly(A) mRNA will be captured by these beads during the incubation. Then, the bead-mRNA complexes are washed to discard any unbound molecules. Finally, the mRNA will be eluted or used directly for the next experiment.

References

1.Saiyed, Z. M. , Ramchand, C. N. , & Telang, S. D. . (2008). Isolation of genomic DNA using magnetic nanoparticles as solid-phase support. Journal of Physics: Condensed Matter, 20(20), 204153.10.1088/0953-8984/20/20/204153

2.Magnani, M. , Galluzzi, L. , & Bruce, I. J. . (2006). The use of magnetic nanoparticles in the development of new molecular detection systems. J Nanosci Nanotechnol, 6(8), 2302-2311.https://doi.org/10.1166/jnn.2006.505

3.Sarkar, T. R. , & Irudayaraj, J. . (2008). Carboxyl-coated magnetic nanoparticles for mRNA isolation and extraction of supercoiled plasmid DNA. Analytical Biochemistry, 379(1), 130-132.https://doi.org/10.1016/j.ab.2008.04.016