Magnetic DNA hybridization properties of oligonucleotide probes attached to superparamagnetic beads and their use in the isolation of poly(A) mRNA from eukaryotic cells

145 GATA 7(6): 145-150, 1990

EMERGING TECHNIQUES

Magnetic DNA Hybridization Properties of Oligonucleotide Probes Attached to Superparamagnetic Beads and Their Use in the Isolation of Poly(A) mRNA From Eukaryotic Cells E R I K H O R N E S and L A R S K O R S N E S Poly(A) messenger RNA is generally purified from total RNA using oligo(dT) cellulose affinity chromatography or centrifugation through spin columns. We present a new method for rapid purification of poly(A) mRNA using oligo(dT) probes attached to superparamagnetic beads. By magnetic separation, washing, and elution, pure m R N A is obtained from living cells within lO minutes. This procedure works for crude RNA preparations or cell lysates that would otherwise clog standard oligo(dT) cellulose column systems. The present method reduces the risk of degradation, is highly eJficient, and can easily be scaled up or down.

It has previously been reported that small latex beads used as a solid support for DNA had hybridization kinetics comparable to free solution reaction kinetics [1], but that handling was not easy due to the need for centrifugation or filtration steps. The introduction of monosized superparamagnetic beads [2] as a solid support avoids the need for both centrifugation and filtration and reduces handling requirements to the application of a magnetic field. In this way the solid phase magnetic beads carrying the reactants are pelleted within a few seconds without affecting other substances that might be present. Hence the starting material may be quite crude. The usefulness of such systems, which combine the fast reaction kinetics of beads with the simple and rapid magnetic isolation, has already been reported for several difFrom the Apothekernes Laboratorium A.S., Harbitzall~en 3, Oslo, Norway. Address correspondence to: Dr. Erik Hornes, Apothekernes Laboratorium A.S., Harbitzall~en 3, N-0275 Oslo 2, Norway. Received February 1, 1990; revised June 6, 1990; accepted July 10, 1990.

ferent applications in cell biology and immunology [3, 4]. Biotinylated DNA fragments have successfully been attached to streptavidin coated Dynabeads (Dynal, Oslo, Norway) and applied in magnetic solid phase DNA sequencing [5] and magnetic purification of DNA-binding proteins [61. In the present report we show that biotinylated oligonucleotide probes attached to Dynabeads M-280 Streptavidin function well in hybridization reactions and have extremely fast hybridization kinetics. Furthermore, we show that these hybridization properties combined with the rapid magnetic separation technique provide an excellent method for purifying messenger RNA from eukaryotic cells. The number of handling steps and overall purification time is reduced to a minimum, resulting in a greatly reduced risk of physical and enzymatic degradation of the mRNA, while retaining as good or better efficiency compared with conventional preparations.

Materials and Methods Magnetic Beads Dynabeads M-280 Streptavidin (Dynal A.S., Box 158, N-0212 Oslo) were used as solid phase. These are m o n o d i s p e r s e s u p e r p a r a m a g n e t i c polymer particles with a diameter of 2.8 ~m covalently coated with streptavidin.

Biotin Binding Capacity One hundred microliters of 6 x SSPE [7] containing 1 nmol ~4C-Biotin (Amersham) was added to 0.5 mg Dynabeads (prewashed in 6 x SSPE) and placed on a roller mixer (Coulter) at room temperature for 15 minutes. After two separate washes in 6 x SSPE the fraction of bound ~4CBiotin was measured by scintillation counting.

Oligonucleotides 5' Aminomodified oligonucleotides were made using AminolinklI on an Applied Biosystems 381A D N A synthesizer. The immunoglobulin light kappa chain probe used was: 5'-TCACTGGATGGTGGGAAGATGGATACAGTTGGTGCA-3'.

Biotinylation of Probes Biotin XNHS ester (Clontec) was used as recommended by the supplier. Excess labeling reagent and buffer were removed in a Sephadex G50 spin column. The 5' Biotin oligo (dT)z5 was end-la-

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146 GATA 7(6): 145-150, 1990

E. Hornes and L. Korsnes

beled using Klenow polymerase, a-32p-dTTP and oligo(dA)25 as template. Excess label was removed using a Sephadex G50 spin column.

pending on the subsequent use of the purified mRNA, SDS may be omitted in the last washing step and in the elution buffer.

Preparation of Oligo(dT) Dynabeads (T-Beads)

Extraction of Total RNA

Two hundred micrograms of Biotinylated oligo(dT)z5 (24 nmol) in 2.5 ml 6 × SSPE was mixed with 50 mg prewashed Dynabeads M-280 Streptar vidin and incubated on a roller mixer for 15 minutes at room temperature. After two washes in 6 x SSPE the beads were stored at 4°C in 6 × TE buffer pH 7.4 [7] containing 0.1% SDS.

Oligonucleotide Hybridization Assay In the standard assay to measure hybridization capacity of different batches of T-beads, 0.I mg beads were pipetted into an eppendorf tube and washed once with 6× SSPE, 0.1% SDS. A magnet rack (MPC-E, Dynal A.S., Oslo) was used to pellet beads between each step. After removal of washing buffer, 50 txl hybridization solution, consisting of 6× SSPE, and 0.1% SDS, containing 50 pmol of oligo(dA)~5 with a trace amount ( 1 - 2 × 105 cpm) of a-32p-dATP-labeled oligo(dA)25, was added. After gentle mixing the reactants were hybridized by incubation for 2 minutes at room temperature. The hybridized beads were washed twice with 2 × SSPE, 0.1% SDS at room temperature and the percentage of oligo(dA)z5 hybridized to the oligo(dT)25 Dynabeads was measured in a scintillation counter.

Labeling of Poly(A) mRNA Tracer One microgram of poly(A) RNA was mixed with 2.5 pmol oligo(dT)z5 in 10 I~! 5 x Klenow buffer [7], 1 U RNAasin (Promega), 10 mM DDT. After 2 minutes at room temperature 10 ~Ci c ~ - 3 2 P dATP, 1 U Klenow polymerase (Amersham) and H20 up to 50 I~l were added and incubation continued for 60 minutes at 15°C. Excess a-32p-dATP was removed using a Sephadex spin column.

Buffers for Poly(A) mRNA Hybridization to Dynabeads M-280 Streptavidin Coated With Oligo(dT)e5 The buffers and solutions recommended by Ausubel et al. [8] were used. Binding buffer: 0.5 M LiC1, 10 mM Tris-HC1, pH 7.5, 1 mM EDTA, 0.1% SDS. Middle wash buffer: 0.15 M LiCI, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% SDS. Elution buffer: 2 mM EDTA, 0.1% SDS. De-

Extraction of total RNA from cell cultures was carried out using sarcocyl-, LiCI-, urea-method according to the protocol of Auffray and Rougeon [91. Results

DNA Coupling and Hybridization Capacity The Dynabeads M-280 Streptavidin used in the present experiments were found to bind 390 pmol 14C-Biotin per milligram of beads. The amount of attached 5' Biotinylated oligo(dT)z5 was determined using a labeled oligo(dT)25 tracer in the coupling reaction and found to be 250 pmol Biotin oligo(dT)25 per milligram of beads. To determine the maximum hybridization capacity of these magnetic oligo(dT)z5 beads a standard assay was assigned using a (dA)25 oligonucleotide as described in materials and methods. The batch of T-beads made and used in the present study were found to have a hybridization capacity of 193 pmol oligo(dA)zJmg. In a control experiment to measure nonspecific binding, beads attached with a noncomplementary probe (a 42mer Tn917 probe) bound less than 10 attomol of oligo(dA)25/mg beads.

Hybrid&ation Kinetics With Oligonucleotides Before carrying out the magnetic mRNA isolation experiments it was necessary to study the hybridization kinetics of the system. Hybridization experiments were set up using a less than twofold excess of T-bead hybridization capacity for the complementary oligo(dA)25 target nucleotide. Figure 1 shows the hybridization kinetics and that saturation was complete within 1 minute.

Hybridization Efficiency With Oligonucleotides To test how efficiently target nucleic acids were separated from a mixture, two different experiments were set up. In the first experiment increasing amounts of target oligonucleotides (oligo(dA)zs) were added

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147 Rapid Purification of poly(A) mRNA

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to a fixed a m o u n t o f T-beads (100 ~g) with a k n o w n m a x i m u m h y b r i d i z a t i o n c a p a c i t y of 19 pmol. The results shown in Figure 2A demonstrate that the T-beads were able to bind at least 99% of the target oligonucleotides even when the molar ratio of target to bead capacity approached 1:1. In the second experiment, five different concentrations of oligo(dA)25 from 1 pmol down to 100 a t t o m o l were added to 100 ~xg aliquots of beads. The total amount of nucleic acids present in each mixture was adjusted up to 10 pmol with a nonsense probe. In a negative control experiment without target oligonucleotides the noncomplementary probe was labeled in order to detect nons p e c i f i c b i n d i n g . A f t e r h y b r i d i z a t i o n for 2 m i n u t e s f o l l o w e d by t w o w a s h i n g s t e p s the amount of oligo(dA)25 hybridized was measured. The results shown in Figure 2B illustrate that even with a target oligonucleotide amount as low as 100 attomol (0.001% of total nucleic acids present) the magnetic oligo(dT)25 beads r e m o v e more than 95%.

Magnetic Isolation of Poly(A) mRNA To determine the maximum poly(A) m R N A binding capacity of the T-beads a 32p-labeled control RNA of known concentration was used. This was a 1,200 nucleotide kanamycin transcript having 30 A's in the 3' end (Promega). The strategy for determining the binding capacity was as described for the oligonucleotide binding assay, but using the buffers r e c o m m e n d e d by Ausubel et al. [8]. The binding capacity of this particular poly(A)30

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pmol target in solution Figure 2. (A) Hybridization efficiency of Dynabeads with ~ 2 5 probes to oligonucleotides. One hundred micrograms of oligo(dT)25 beads was hybridized with variable amounts of target oligo in 50 I~1 hybridization solutions: 1.0, 2.5, 5.0, 7.5, 10.0, 15.0, and 20.0 pmol target oligo(dA)25respectively with 2 minutes hybridization time. (B) Hybridization efficiency of Dynabeads with oligo(dT)25probes to oligonucleotides. A similar experiment as in Figure 2A but with 1.0, 0.1, 0.01, 0.001, and 0.0001 pmol target oligo(dA)2sin solution. In addition, the total amount of oligo present was adjusted to l0 pmol with a nonsense probe.

mRNA was found to be 33 pmol (13 p.g) mRNA per mg T-beads.

Kinetics and Efficiency of poly(A) mRNA Hybridization to T-Beads Using Isolated Total RNA Total RNA was extracted from 1 × 108 cells of the hybridoma cell line ABI [I0] using the method of Auffray et al. [9]. The kinetics of mRNA hybridization to the oligo(dT)25 beads were determined using 10 ~g total RNA together with trace amount (150 ng) o f riP-labeled mouse pancreas poly(A) mRNA (Clontec), and an approximately fivefold calculated capacity excess of T-beads in 150 Ixl hybridization buffer. Figure 3 shows the hybridization kinetics with poly(A) RNA, and that close to 90% of the poly(A) mRNA in the sample was hybridized to the beads in less than 2 minutes, with 78% occurring in the first 30 seconds.

© 1990 Elsevier Science PublishingCo., Inc., 655 Avenue of the Americas, New York, NY 10010

148 E. Hornes and L. Korsnes

GATA 7(6): 145-150, 1990

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Direct Magnetic Isolation of Poly(A) mRNA From Cell Cytoplasm Cells from a culture of the hybridoma cell line AB1 (1 x 106) were washed once and resuspended in 50 Ixl PBS (Dulbecco, 041-04190) and Triton X-100 was added to a final concentration of 0.5%. After lysis for 1 minute the cell debris, including nuclei, was pelleted by a 10 second spin in an Eppendorf centrifuge. The supernatant was added to T-beads in 100 ixl binding buffer [9] of double concentration and allowed to hybridize for 2 minutes. This was followed by magnetic separation of the beads. The hybridized mRNA was detached from the beads in 2 mM EDTA at 65°C and beads removed using the magnet. Figure 4A shows a Northern blot of poly(A) mRNA after detachment from the T-beads and probing with an immunoglobulin kappa light chain probe. In this experiment aliquots of 1 X 10 6 hybridoma cells were added to varying amounts of T-beads, 200 Ixg, 100 I~g, 50 Ixg, 20 Ixg and 200 Ixg of Dynabeads M-280 Streptavidin without probe. The results from the blotting experiment shown in Figures 4A and 4B indicate that the yield of mRNA increases with increased amounts of beads, while the remaining mRNA in the supernatant is correspondingly reduced. This is also clearly demonstrated in Figure 5, which shows a densitometric scan of the x-ray film (the smear in 4B is because the samples are direct total lysate). The results presented in Figures 4 and 5 show that approximately 120 Ixg of T-beads is sufficient to isolate more than 90% of cytoplasmic poly(A) mRNA from 106 cells. Streptavidin beads without probe gave no detectable mRNA binding. To show the size distribution of the mRNA iso-

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B Figure 4. (A) Magnetic isolation of poly(A) mRNA from cell cytop['~m. One hundred microliters of cytoplasm from 1 x 106 cells lysed with 0.5% Triton X-100 was added to varying amounts of oligo(dT) beads in 100 I~1 2 x concentrated binding buffer. Northern blot analysis of mRNA isolated with 200 Izg Dynabeads M-280 Streptavidin with no oligo(dT)25 attached (lane 1), 20 lzg, 50 txg, 100 txg, and 200 I~g oligo(dT)25 beads (lanes 2-5), probed with immunoglobulin kappa light chain probe, C k to demonstrate quality and quantity. (B) Poly(A) mRNA remaining in the supernatant after magnetic isolation. Ten percent of the supernatant after magnetic isolation of mRNA from the experiment shown in Figure 4A was loaded on a gel for Northern blot analysis. Supernatant from control beads (lane 1) and 20-200 Izg beads (lanes 2-5).

lated from cytoplasm, a Northern blot was probed with poly dT25 (Figure 6A). The mRNA appears as a smear from approximately 9 kb to about 0.3 kb. In order to test its functionality, the isolated

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149 Rapid Purification of poly(A) mRNA

GATA 7(6): 145-150, 1990

ography (Figure 6B) and demonstrated discrete bands up to 100 kb. N

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mRNA (SDS being omitted from the washing and elution steps) was directly translated in a rabbit reticulocyte lysate. The synthesized proteins were examined by electrophoresis and autoradi-

Figure 6. (A) Size range of isolated mRNA. Northern gels wl-]ih--m~NA from 5 × 105 cells were blotted to Hybond N (Amersham) and probed with kappa light chain probe (lane 1). The same gel was reprobed with oligo (dT)25 (lane 2). (B) In vitro translation of isolated poly(A) mRNA in rabbit reticulocyte lysate. Isolated mRNA (SDS was omitted in both the washing and elution buffers) from 1 x 106 cells were translated in a rabbit reticulocyte lysate (Promega) and one-third loaded on a 15% polyacrylamide gel. Translation is shown of mRNA isolated without SDS in binding buffer (lane 1) and with 0.2% SDS (lane 2). Lane 3 contains size markers ranging from 14,000-200,000 (Amersham).

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In this report we have demonstrated that 5'-biotin labeled oligonucleotides can be affinity bound to streptavidin-coated monodisperse superparamagnetic polymer particles (Dynabeads). The beads used in our experiments were documented by the manufacturer to have a surface area of 4.3 m2/g which, if the probes were randomly distributed, would give an average distance between neighboring oligonucleotides of 54 A However, as streptavidin is a tetramer and may bind four biotin molecules, more than one probe can be attached to each streptavidin molecule. The binding capacity of the T-beads was found to be 193 pmol/mg for oligo(dT)25 and 33 pmol/mg for mRNA. The reduced capacity for mRNA is probably the result of steric hindrance, but as this binding is achieved in a single step with 90% efficiency, it is as good or better than previously reported oligo(dT) cellulose chromatography, which in addition depends on two cycles of sample loadings. One of the basic results of this study is that hybridization kinetics of DNA probes attached to Dynabeads is extremely fast. Complete hybridization was observed within 60 seconds both with oligonucleotides (Figure 1) and with poly(A) m R N A (Figure 3). This observation agrees well with the result of Wolf et al. [I], who found that DNA probes attached to latex particles have hybridization kinetics similar to nucleic

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150 E. Homes and L. Korsnes

GATA 7(6): 145-150, 1990

acids in free solution. One of the great advantages of magnetism as a separation procedure is that it functions well even in solutions of crude material. The magnetic m R N A purification system makes it possible to detach mRNA from the beads in very small volumes, as low as 5-20 ~1 of H20. This is especially important if small numbers of cells have been used as starting material. The need for concentration and buffer change by e t h a n o l p r e c i p i t a t i o n is t h e r e b y omitted, and the mRNA can be used directly for Northern blot analysis as shown in Figure 4A, and for in vitro translation experiments (Figure 5B). Figures 6A and 6B demonstrate the isolation of high molecular weight mRNA with this magnetic purification system, indicating little degradation. The results in this report demonstrate that magnetic purification of poly(A) mRNA is a rapid and efficient method for the isolation of high quality poly(A) mRNA from total RNA and culture cells. Future work would be to adapt this technology to isolation of mRNA directly from tissue. The magnetic bead technology can easily be scaled up, or more interesting, scaled down, providing a method for efficient isolation of RNA from small samples of cell material. We thank Prof. John Ugelstad and his group at SINTEF, Trondheim, for having developed and modified the M-280

monosized superparamagnetic polymer particles for biological applications, and Dr. Kjell Nustad, The Norwegian Cancer Hospital, for developing the streptavidin beads. We are also grateful to Gro Lindberg for all cell cultivation and handling, and to Kari Solberg for word-processing the manuscript. This work was supported by grant NNPT.24741 from the Norwegian Technical Res. Council (NTNF).

References 1. Wolf SF, Haines L, Fisch J, Kremsky JN, Dougherty JP, and Jacobs K: Nucl Acids Res 15:2911-2926, 1987 2. Ugelstad J, Mfutakamba HR, MCrk PC, Ellingsen T, Berge A, Schmid R, Holm L, JCrgedal A, Hansen FK, and Nustad K: J Polymer Sci 72:225-240, 1985 3. Funderud S, Nustad K, Lea T, Vartdal F, Gauderuack G, Stenstad P, and Ugelstad J: IRL Press, Oxford 14:55-65, 1987 4. Lea T, Vartdal F, Nustad K, Funderud S, Berge A, Ellingsen T, Schmid R, Stenstad P, and Ugelstad J: J Mol Recog 1:9-17, 1988 5. Hultman T, Staahl S, Hornes E, and Uhlen M: Nucl Acids Res 17:4937-4946, 1989 6. Gabrielsen OS, Homes E, Korsnes L, Ruet A, and Oyen TB: Nucl Acid Res 17:6253-6267, 1989 7. Maniatis T, Fritsch EF, and Sambrook J: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press, 1982 8. Ausubel FM, Brent R, Kingston RE, Moore DD, and Smith JA: Current Protocols in Molecular Biology. Harvard Medical School. New York, Greene, 1987 9. Auffray C, and Rougeon F: Eur J Biochem 107:303-314, 1980 10. Melsom H, Funderud S, Lie SO, and Godal T: Scand J Haemotol 33:27-34, 1984

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