Educational Note # 1. Applications of Magnetic Bead Technology with Versa Workstation in Genomics & Proteomics

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1 Educational Note # 1 Applications of ic Bead Technology with Versa Workstation in Genomics & Proteomics I. Introduction ic separation technology, using magnetic particles, is a quick and easy method for the sensitive and reliable capture of specific proteins, genetic material and other biomolecules. Initially, the technology started with nano-sized iron oxide particles suspended in carrier liquid called magnetic fluids or ferrofluids. Although referred to as magnetic, many of the particles currently used are super-paramagnetic (can easily be magnetised with an external magnetic field) and immediately redispersed once the magnet is removed (Figure 1). This way it leads to magnetically driven separation techniques. Keeping this versatile technology in view, Aurora Biomed Inc has developed a workstation that is adaptable to magnetic beads based protocols. II. Formats of magnetic beads: The formats of particles that are currently available can be broadly classified as: 1. Unmodified or naked particles 2. ic microspheres/ nanospheres having chemically derived particles with general specificity ligand attachments on the surfaces a. Silica beads b. Agarose beads c. Beads with immobilized carboxylic groups a. Beads with immobilized streptavidin b. Beads with immobilized Protein A c. Beads with immobilized lectins d. Beads with immobilized any other general ligand 3. Chemically derived particles with specific recognition groups a. Beads with immobilized specific oligonucleotides for example poly dt b. Monoclonal and polyclonal antibodies c. Beads with immobilized any other specific ligand

2 III. Advantages 1. The analyte is subjected to very little mechanical stress in comparison to other methods. 2. These methods are non-laborious 3. Very cost-effective 4. Reduced reagent costs 5. Highly scalable to smaller and bigger volumes 6. Provides high throughput 7. Faster operations than other methods 8. High-purity yield of biomolecules 9. Highly amenable to automation 10. Economical in terms of miniaturization thus saving costly reagents and biomaterials Figure 1. A typical example of the paramagnetic beads (1µm in diameter) IV. Applications The isolation of specific molecules based on their interaction with complementary binding partners is emerging as an important technology in many fields of research. These magnetic separation techniques are leading to various applications in the filed of biotechnology, biomedicine and drug discovery. 1. Genomic applications In this post-genomic era, technologies based on magnetic separation are becoming an integral part of the standard biology laboratory. a. High throughput nucleic acid isolations i. DNA RNA isolations: Isolation of DNA is a prerequisite step for many molecular biology techniques. The separation of DNA from the complex mixtures in which they are often found is sometimes necessary before other studies and procedures such as sequencing, amplification, hybridization, detection, etc. The presence of large amounts of cellular or other contaminating materials, such as proteins or RNA, in such complex mixtures often impedes many of the reactions and techniques used in molecular biology. The conventional protocol for extracting DNA involves cell lysis followed by the removal of

3 contaminating cellular components such as proteins, lipids and carbohydrates and, finally, isolating DNA using a series of precipitation and centrifuge separation steps, which are difficult to automate. Improvements in methods for isolating DNA. The discovery in the late 1980s that silica can be used as adsorbent for DNA isolation became the basis for most of the DNA isolation kits currently available. One of these kits involves the isolation of DNA, using silica-coated magnetic particles. A high-throughput genome isolation protocol has been developed, based on solid-phase reversible immobilization (SPRI) chemistry. The SPRI protocol is based on the binding of DNA to the surface of carboxyl-coated paramagnetic particles, under the conditions of a high presence of salt and polyethylene glycol. Similarly, the magnetic bead procedures are adapted for RNA isolations. Figure 2: Showing a 96-ring magnet block on which a 96-well plate fits and the magnetic beads make a ring at the bottom of each well thus leaving the centre for aspiration the liquid along with unbound molecules ii. mrna isolations: A number of methods have been reported for the isolation of total RNA from a variety of cells or tissues. In 1979, a method was developed for the efficient isolation of total RNA by homogenization in a 4mol solution of guanidium thiocyanate containing 0.1mol of 2-mercaptoethanol. Homogenization is followed by the extraction of RNA by ethanol or by ultracentrifugation through caesium chloride. This method was further modified in 1987 to devise a rapid single step isolation procedure for RNA. It involves the extraction of RNA using a mixture of guanidium thiocyanate and phenol-chloroform. Many RNA isolation kits are available based on the above two protocols. All of these methods isolate RNA on the basis of its biochemical properties. In contrast, the biomagnetic separation of messenger RNA (mrna) is based on a specific complementary hybridization between the poly A sequence of isolated mrna and the oligo (d T) sequence covalently linked to the surface of paramagnetic particles. In this method, oligo (dt)-coated magnetic beads are added to crude cell or tissue lysate. During incubation, poly (A) mrna from the lysate is caught onto the surface of oligo (dt)-coated magnetic beads. The beads/mrna complex is then washed magnetically. The mrna thus isolated is either eluted or directly applied for many downstream applications, which include: complementary DNA (cdna) library construction; subtractive hybridization; Northern hybridisation; reverse transcription PCR; and in vitro translation.

4 Other RNAs Oligo dt beads C G CCC U A C G U U G A T T T T mrna RNA Binding A A A A A A C G C G U A G C T T T T A A A A A A C G C G U A G C Other RNAs C G CCC U A C G U U G A mrna RNA Wash T T T T Elution A A A A A A C G C G U A G C Micro well beads Figure 3: Isolation of mrna using poly dt magnetic beads b. DNA/RNA binding proteins: The isolation and characterisation of specific transcripts or proteins can be employed to monitor the progression of disease. Several kits are available in the market that work on the principle of magnetic labeling and the direct isolation of biotinylated molecules such as DNA, RNA or proteins onto streptavidin-coated magnetic beads for example µmacs streptavidin Microbeads (Miltenyi Biotec, Germany). These biotinylated molecules can then be used for the indirect isolation of non-biotinylated target molecules that may interact with them. The procedure involves a complex formation between the biotinylated probe (DNA, RNA or proteins) and the target molecule (i.e. interacting biomolecules of DNA, RNA or protein). Based on the interaction of biotin and streptavidin, the probe-target complex is then separated from the rest of the component by the addition of streptavidincoated magnetic beads. The complex is magnetically isolated and washed to remove non-specifically bound molecules. The non-biotinylated target molecules can be eluted off from the complex with high purity, whereas the magnetically labelled, biotinylated probe remains bound to the column. This technique has the potential for rapid and efficient screening of transcriptional and translational regulatory proteins. Target DNAconjugated magnetic beads have also been used for the rapid screening of DNA-binding peptide ligands from a solid-phase, combinatorial library.

5 2. Proteomics applications Protein isolation and manipulation is often achieved using resins and slurries of molecules as column- or liquid-based solid-phase supports. The conventional chromatography columns are very slow to use, as most rely on gravitational flow and, therefore, binding, washing and elution steps can take many hours to complete for even simple isolations. They also generate significant background contamination in downstream analysis due to non-specific binding and incomplete washing. On the other hand, centrifugation steps used during agarose-based methods are time consuming and can generate greater sample loss due to incomplete separation and hazy delineation of the fluid component. With these solid-phase supports being a hindrance in some downstream analyses, a cleaner and easier-to-use support based on magnetic microparticles have proven their worth. Therefore, magnetic particles are now increasingly used as carriers for binding proteins, enzymes and drugs. Studies have shown that proteins and enzymes can be covalently attached to naked magnetic particles in the presence of carbodiimide. Such immobilization (attachment) procedures for proteins, enzymes or drugs will have a major impact in various areas of medicine and biotechnology. The immobilised biomolecules can be used directly for a bioassay, or as affinity ligands to capture or modify target molecules or cells. On this basis, nitriloacetic acid (Ni- NTA)-tagged magnetic agarose beads have been used for versatile magnetocapture assays using 6xHis-tagged proteins (the proteins which are expressed with recombinant DNA technology as multi-histidine aminoacids attached to the protein of interest. The multihistidine residues have affinity to Desired protein Ni His bead Binding Figure 4: ic isolation of recombinant proteins Protein Washing Desired protein (Positive selection) Elution Nickel and bind with it). The procedure involves the use of metal chelating nitriloacetic acid groups covalently bound to the surface of agarose beads,

6 which contain strong magnetic particles. The beads are pre-charged with nickel, which is ready to capture 6xHis-tagged proteins for sensitive-interaction assays or microscale purification of 6xHis tagged proteins (Figure 3). This technique therefore bridges the gap between purification-scale procedures using Ni-NTA metal chelate affinity chromatography resins and microplatebased assays. 4. Biomedical applications The magnetic bead technology is finding versatile applications in biomedical field where basic working principle is to hook and fish out the desired cell with external magnetic field. a. Cell isolation or rare cell detection: The cells isolations with this methods have become very easy as the magnetic beads need to be attached with specific molecules that have specificity for a specific surface molecule of the cell to be isolated (Figure 5). b. Bacterial isolations: beads coated with Protein G and Tosylactivated-280 dynabeads have also been used to purify Bacillus anthracis protective antigen from a liquid culture. The obtained protein was used in the ELISA to detect the bacterial protective antigen antibodies in human sera collected from immunized individuals. The purification method using paramagnetic beads was very effective. It is fast, easy and may be carried out practically in any laboratory. c. ically guided drug targeting: It has been attempted in order to increase the efficacy and reduce the unpleasant side effects associated with chemotherapy. This method of drug delivery involves the immobilization of drug or radionuclide in biocompatible, magnetic nanoor microspheres. This method of delivery makes chemotherapy more effective by increasing the drug concentration at the tumor site, while limiting the systemic drug concentration. In such operations of mixing the drug with magnetic particles and to recover unbound drug, robotic workstation may play a part at the industrial scale. Specific antibody complex Specific surface antigens bead Undesired cell Binding Un/desired cells (Negative selection) Desired cell (Positive selection) Washing Elution Antibody conjugants employed: Phycoerythrin Biotin Streptavidin FITC (fluorescein isthiocyanate) APC (Allophycocyanin) Figure 5: ic isolation of cells

7 4. Drug discovery applications Now that the human genome is sequenced and about 30,000 genes are annotated, the next step is to identify the function of these individual genes, carrying out genotyping studies for allelic variation and single nucleotide polymorphism (SNP) analysis, ultimately leading to the identification of novel drug targets The modern drug discovery process emphasizes rapid data generation and analysis, in order to identify promising new chemical entities as well as new drug targets early in the development cycle. At every step of the rapidly evolving drug discovery process, dozens of technologies and products are required. But innovations in newer technologies for genomics and proteomics are changing the face of drug discovery. Automation has become essential in allowing researchers to meet the high-throughput demands of today s research environment. The most prominent area in which magnetic separation is applied in drug discovery is sample preparation, which includes highthroughput genome isolation for sequencing and PCR amplification to carry out genotyping, SNP scoring and/or expression profiling. V. Versa Workstation As the above mentioned details on the protocols using magnetic bead involve the steps of shaking the contents, washing, aspirations and dispensing, incubation (plate heating/cooling), reagent cooling etc. The Versa series of workstation are dedicated to carry these operations providing all the necessary conditions. The the versa series are equipped as follows: a. One, two or three arms for liquid handling b. Plate transporter c. Accessories include: (1). ic block (2). Plate shaker (3) Plate Incubator (cooler / heater) (4) Reagent / reservoir cooler (5). Vacuum manifold etc. VI. References: 1. Sucholeiki I, Toledo-Sherman LM, Hosfield CM, Boutilier K, DeSouza LV, Stover DR. Novel magnetic supports for small molecule affinity capture of proteins for use in proteomics. Pharmaceutical Discovery, Aug 1, 2005 : Mol Divers. 2004;8(1): ZARZECKA A, BARTOSZCZE M: Application of Beads for Purifying Bacillus anthracis Protective Antigen. Journal of Veterinary Medicine Series B 2006, 53(8)