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4. Seal the vial containing partially protected oligo(2'-0-alkylribonucleotide) in ammonia solution, and keep at 60 °C for 8-10 h (generally overnight) to remove the heterocyclic protection.

5. When cool, evaporate the solution to dryness at room temperature using a Speedvac.

6. Dissolve the residue in about 250 pi of 0.1 M triethylammonium acetate buffer and purify the 5'-0-dimethoxytrityl-oligo(2'-0-alkylribonucleotide) by reversed-phase HPLC on a p-Bondapak C18 or equivalent column using a gradient of acetonitrile in 0.1 M aqueous triethylammonium acetate, pH 7.0. The oligonucleotide product will elute at about 35-40% acetonitrile.

7. Lyophilize the product fraction on a Speedvac.

8. Dissolve the residue in 500 pi of 80% acetic acid and keep at room temperature for 30 min to cleave the 5'-0-dimethoxytrityl group.

9. Add 500 pi of H20 and transfer the whole solution to a 25 ml pear-shaped glass flask.

10. Add 5 ml of diethyl ether and mix well by shaking. Leave on the bench until the phases separate. The oligonucleotide remains in the lower (aqueous) phase. Discard the upper phase.

11. Repeat step 10 twice more.

12. Lyophilize the ether-extracted oligonucleotide solution (i.e. the aqueous phase from the final extraction) on a rotary evaporator.

13. Resuspend the oligonucleotide in 1 ml of H20. The oligonucleotide is now ready for use and its concentration can be determined by measuring 4260 in a spectrophotometer (y4260 = 1.0 is equivalent to an oligonucleotide concentration of 25pg/ml).

3. Antisense affinity selection of RNAs or RNP complexes

3.1 Strategy

Specific RNAs or RNP complexes can be isolated directly from crude cellular extracts using an affinity selection approach with biotinylated antisense probes. For this application it is important to use antisense probes which form duplexes with the target RNA sequence characterized by high melting temperatures (Tm). It is also important that the resulting duplexes are not substrates for RNase H, which is commonly present in crude nuclear and cellular extracts.

The complex formed between the RNP particle and biotinylated antisense probe is purified on a solid support to which is bound either avidin, streptavidin, or anti-biotin antibodies. A wide range of such products are commercially available and suitable for use. Reproducibly reliable results have been obtained with streptavidin-agarose and so protocols based on this technology are described below (Section 3.3).

3.2 Probes for affinity selection

3.2.1 Advantages of substituted oligoribonucleotide probes

The use of either 2'-0-methyl or 2'-0-allyl substituted oligoribonucleotide probes is particularly recommended since they are nuclease resistant and form extremely stable, RNase H-resistant hybrids with complementary RNAs (8). The RNA:2'-0-alkylRNA heteroduplexes actually have significantly higher Tm values than the corresponding RNA:RNA hybrids of identical nucleotide sequence. These probes can be synthesized as described in Section 2. Lower backgrounds of non-specific probe binding are often obtained by using 2' -O-methylinosine in place of 2'-0-methylguanosine.

3.2.2 Biotinylation of probes

The most efficient method of incorporating biotin into the oligonucleotides is during synthesis by using a phosphoramidite substrate consisting of a modified base coupled to biotin via a flexible spacer arm (4, 5). An alternative, though less efficient, method is to incorporate an amino linker in the oligonucleotide and then to couple biotin to this linker after synthesis using a biotin ester such as sulphosuccinimidyl 6-(biotinamido)hexanoate [NHS-LC-biotin] (10). Suitable reagents for post-labelling probes can be purchased from Pierce. The best results have been obtained using probes incorporating two to four tandem biotin residues at either the 3' or 5' terminus of the oligonucleotide. However, since the position of biotin residues can, in certain cases, significantly affect the efficiency of affinity selection of targeted complexes, presumably due to steric hindrance from proteins in the RNP particle (11), it is better to incorporate two biotin residues at each end of the probe.

Protocol 3. Solid-phase synthesis of biotinylated probes Equipment and reagents

• Equipment and reagents listed in Protocol 2

• Biotin-coupled phosphoramidite, e.g. 5'-0-dimethoxytrityl-/\/4-methyl, NA-[N-methyl, /V-(/V-{4-tert.-butylbenzoyl}bio-

Method

1. Follow step 1 of Protocol 2 and in addition prepare a 0.1 M solution of the biotin-containing phosphoramidite in anhydrous acetonitrile. Place the biotin reagent in a spare monomer position on your synthesizer.

tinyl )-8-amino-3,6-dioxaoctyl ] 2 '-deoxy-cytidine-3'-0-(2-cyanoethyl A/,/V-diisopro-pylphosphoramidite) (MWG-Biotech) or biotin-dT (Glen Research)

2. Use a 3'-terminal dT controlled pore glass support to start your synthesis, first couple on two biotin-containing monomers then synthesize your desired sequence as in step 3 of Protocol 2. Now add on two more biotin-containing monomers before performing the 'trityl on auto' end procedure. If the oligonucleotide being made needs to be efficiently 5' phosphorylated (for example, for 5' labelling with 32P-phosphate) it is recommended that an additional 5' nucleotide (such as dT) is added to the probe after the biotinylated residues. In this case add a dT monomer before performing the 'trityl on auto' end procedure.

3. Follow steps 4-10 of Protocol 2 to obtain a tetrabiotinylated 2'-0-alkyl RNA probe. Note however that biotinylated oligonucleotides will elute at a slightly higher acetonitrile concentration than unbiotinylated oligo nucleotides of otherwise identical sequence.

An alternative, but less convenient, procedure is postsynthesis biotinylation. However, this requires the incorporation of amino-modifier phosphoramidites instead of the biotin-containing monomer. Suitable reagents are the amino modifier-dT available from Glen Research or the amino modifier II from Cruachem. The oligonucleotide thus synthesized and purified containing primary amino groups is readily biotinylated using a suitable activated ester of biotin (10).

3.2.3 Nucleotide sequence and design of probes

There is no simple rule for knowing which region of an RNA will be the best target for an antisense oligonucleotide. In general, there is a good correlation between regions susceptible to nuclease cleavage or chemical modification and efficient probe binding (12-14). All RNase H sensitive sites are good candidates for targeting biotinylated probes. Note, however, that 2'-0-alkyl RNA probes can also bind stably to target sequences which are not, or are only poorly, cleaved by RNase H. This is probably the result of both the higher stability of RNA:2'-0-alkyl RNA hybrids as opposed to RNA:DNA hybrids and the inaccessibility of the bulky RNase H enzyme to certain sites of hybrid formation. Sequences known to be binding sites for proteins are not likely to be suitable targets for antisense probes. In contrast, RNA sequences predicted to form stems can sometimes be successfully targeted, since, as mentioned in Section 2.1, antisense probes may be able to displace intramolecular secondary structures due to the higher stability of the 2'-0-alkyl RNA:RNA hybrids compared to RNA:RNA duplexes (13).

Since the 2' -O-alkyl oligoribonucleotides form very stable hybrids with RNA, it is usually not essential to use long probes to obtain efficient affinity selection. Very good results can often be achieved with probes in the size range 11-16 nt, depending on the base composition of the target sequence. The stability of short hybrids can be additionally increased by including the modified base 2-aminoadenine in the oligonucleotide to base pair with uracil in the target sequence (14). Three hydrogen bonds are formed between 2-aminoadenine and uracil instead of the usual two hydrogen bonds formed by adenine. As discussed above, in the interest of minimizing background it can often be worth replacing 2'-Oalkylguanosine residues with 2'-0-alkylinosine. However, in cases where only a short region of the RNA target is available for interaction with the antisense probe, guanosine should be retained.

3.3 Biotin-streptavidin affinity selection of RNP complexes 3.3.1 Preblocking streptavidin-agarose beads

Affinity chromatography of RNP complexes (or RNAs) bound to biotinylated antisense oligonucleotides requires highly specific, biotin-dependent binding to the streptavidin-agarose beads. Therefore, to reduce non-specific binding, the beads should be preblocked shortly before use. This is described in Protocol 4.

Protocol 4. Preparation of preblocked streptavidin-agarose beads

Equipment and reagents

• Rotating wheel stirrer

• Streptavidin-agarose beads (normally supplied as a slurry in 10mM sodium phosphate, pH 7.2, containing 0.15 M NaCI and 0.02% sodium azide); Sigma streptavidin-agarose is particularly recommended

Method

1. Centrifuge the beads in a microcentrifuge at 4000 r.p.m. (1400^) for 20 sec. Note that centrifuging the agarose beads too hard or too long can damage them and so should be avoided. This also applies to all subsequent washing steps.

2. Carefully remove and discard the supernatant and note the packed bead volume.

3. Gently resuspend the beads in an equal volume of preblock buffer.

4. Stir the beads for 1 5 min at 4 °C on the rotating wheel stirrer.

5. Collect the beads by centrifugation in a microcentrifuge at 4000 r.p.m. (1400gf) for 20 sec.

6. Resuspend the beads in an equal volume of wash buffer and then centrifuge them again as in step 5. Repeat this washing step twice more.

7. Remove the supernatant wash buffer from the beads after the final wash step. Keep the preblocked beads on ice until ready for use. They can be kept like this for at least several hours without loss of activity.

• Preblock buffer (20 mM Hepes-KOH, pH 7.9, 0.3 M KCl, 0.01 % Nonidet P-40, 50 Mg/ml glycogen, 0.5mg/ml BSA, 50 (jg/ml yeast tRNA)

• Wash buffer (20 mM Hepes-KOH, pH 7.9, 50 mM KCl, 0.1% Nonidet P-40)

3.3.2 Affinity selection procedure

Protocol 5 describes the affinity selection of RNP complexes from a cell or nuclear extract using a biotinylated oligo(2' -O-alkylribonucleotide) targeted to the RNA component of the complex. The following points should be noted:

(a) The optimal concentration of antisense probe required to select a particular RNP complex depends upon the abundance of the RNP in the cell extract and the accessibility of the targeted complementary region of the RNA. Ideally the optimum concentration of the probe required should be determined in a pilot titration experiment. However, in practice approximately 0.2-0.5 pmol of biotinylated antisense probe per microgram of total protein in the extract is generally sufficient for efficient selection of even abundant RNP complexes. Note that if high concentrations of oligonucleotide are used, it may be necessary to increase the quantity of streptavidin-agarose beads in the selection. This can also be checked in the pilot titration experiment; if there is a decrease in selection efficiency when the probe concentration is increased, then it is very likely that the amount of streptavidin-agarose beads used is too low.

(b) The optimum time for incubation of the probe with the cell extract may also vary for different targeted RNPs. As a general guide, a 30-60 min incubation at 30 °C is usually sufficient.

(c) Incubation at lower temperatures (i.e. below 20 °C) may reduce selection efficiency.

(d) It is advisable to incubate the probe with the cell extract in the presence of ATP and creatine phosphate since this can help prevent protein aggregation and precipitation. The mechanism responsible for this preventative effect of ATP is not clear. The cell extract normally contains sufficient endogenous creatine kinase, so supplementation with purified enzyme is not required.

Protocol 5. Affinity selection of RNA complexes using biotinylated oligonucleotides

Equipment and reagents

• Rotating wheel stirrer

• 50mM creatine phosphate

(see Protocol 4, prepared fresh and stored on ice)

• Biotinylated antisense oligonucleotide probe (see Protocols 2 and 3)

• Cell or nuclear extract (normally at 10 mg protein/ml), for preparation of extracts see Chapters 3 and 6, and Volume II, Chapter 3.

• WB300 (20 mM Hepes-KOH, pH 7.9, 0.3 M KCI, 0.1% Nonidet P-40)

• Preblocked streptavidin-agarose beads protocol 5. Continued Method

Note: If a pilot titration experiment is to be performed to determine the concentration of probe required, set up a series of 100 pi reactions according to step 1, but vary the amount of antisense oligonucleotide, typically over a range from 50-500 pmol. Make up the volume of each reaction to 100 |jl with sterile H20. Proceed as in the remaining steps of the Protocol. In the subsequent experiment, use the concentration of probe found to give the maximum recovery of RNP complexes.

1. In a 1.5 ml microcentrifuge tube, mix:

• cell or nuclear extract volume containing 0.5 mg protein

• biotinylated oligonucleotide probe volume containing 200 pmol

2. Incubate the mixture for 45-60 min at 30 °C.

3. Centrifuge in a microcentrifuge at 12000 r.p.m. (12700 g) for 3 min to remove any aggregated material which may have formed during the incubation. This step can significantly reduce the non-specific background.

4. Transfer the supernatant to a fresh microcentrifuge tube and discard the pellet.

5. Gently centrifuge down the preblocked streptavidin-agarose beads at 4000 r.p.m. ( 1400 g) for 20 sec and discard all of the supernatant.

6. Add an equal volume of preblocked streptavidin-agarose beads (i.e. packed bead volume) to the supernatant from step 4 and stir the suspension at 4 °C for 45-60 min using the rotating wheel stirrer. It may be necessary to add a larger quantity of streptavidin-agarose beads if the oligonucleotide probe is used at high concentration (see the text).

7. Recover the beads by centrifugation at 4000 r.p.m. (1400 g) for 20 sec in a microcentrifuge and remove the supernatant. The supernatant can either be discarded or retained for RNA analysis (see Protocol 6) to determine the efficiency of selection of the targeted RNP.

8. Gently resuspend the streptavidin-agarose beads in 2-3 vol. of WB300.

9. Stir the suspension for 5 min at 4 °C and recover the beads as in step 7.

10. Repeat steps 8 and 9 twice. Further washing generally does not improve the ratio of the selected RNP complex to the non-specific background.

11. Elute the affinity-selected RNP complex from the beads (see Protocol 6).

• 50 mM creatine phosphate

to 100 pi final volume

4: RNP complexes 3.3.3 Recovery of affinity-selected RNA

The method of elution of affinity-selected RNA from streptavidin-agarose beads will be dictated by the type of analysis which is to be carried out subsequently. Protocol 6 describes recovery of RNA components from streptavidin-agarose using proteinase K/phenol. The analysis of these RNAs is described in Section 7, whereas the analysis of proteins from affinity-selected complexes is covered in Section 5.

Protocol 6. Recovery of affinity-selected RNA from streptavidin-agarose beads

Reagents

• Streptavidin-agarose with bound affinity- »PK buffer (0.1 M NaCI, 10 mM Tris-HCI, selected RNP complexes (or RNAs) pH 7.6, 1 mM EDTA, 0.5% SDS) prepared as in Protocol 5, step 10 »Phenol¡chloroform (1:1 v/v)

• Proteinase K (20 mg/ml) «TE buffer (10 mM Tris-HCI, pH 7.5, 1 mM

Method

1. Centrifuge the washed streptavidin-agarose beads (from Protocol 5, step 10) in a microcentrifuge for 20 sec at 4000 r.p.m. (1400 g). Remove the supernatant and resuspend the beads in 0.3 ml of PK buffer.

2. Add 35 pi of proteinase K and 20 pi of glycogen stock solutions.

3. Incubate the mixture at 65 °C for 45 min.

4. Pellet the beads by centrifuging the mixture in a microcentrifuge for 1 min at 4000r.p.m. (1400g).

5. Transfer the supernatant to fresh tube, taking care not to transfer any streptavidin-agarose beads, and extract it by vortexing it with an equal volume of phenokchloroform.

6. Centrifuge the mixture for 3 min at 10 000 r.p.m. (8800 g). Collect the upper (aqueous) phase and mix with 3 vol. of absolute ethanol to precipitate the RNA. There is no need to add additional salt at this stage. Leave either overnight at -20 °C or 1 h in a dry ice/ethanol bath.

7. Recover the RNA precipitate by centrifuging for 10 min in a microcentrifuge at 12 000 r.p.m. (12 700g).

8. Wash the precipitate twice with 0.5 ml of 70% ethanol, dry briefly, and resuspend the washed RNA in 25 pi of TE buffer for storage. Alternatively, redissolve the RNA directly in the appropriate gel loading buffer for immediate analysis as in Section 7.

An example of the affinity selection of an snRNA from a HeLa cell nuclear extract is shown in Figure 2 (lane 3).

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