March 28, 1998
Thiopropyl Sepharose 6B has been commonly used to enrich thiolated RNA(1). More recently, attempts have been made to exploit its reactivity with thiols to generate affinity matrices. Thiopropyl Sepharose 6B (Figure 1) contains activated disulfide groups which can react with thiol-containing compounds including thiophosphates and aliphatic thiols at the same time releasing the activating group, thiopyridone (Figure 2). The covalently-linked thiol containing compounds may later be eluted from the column by washing it with DTT or BME, which reduces the covalent adduct with thiopropyl sepharose (Figure 3). 100 mM BME is adequate for elution of thiophosphates. 100 mM DTT is generally required to elute aliphatic thiols. (2)
Some of the chemistry and uses of Thiopropyl Sepharose 6B are outlined in the technical bulletin from Pharmacia(3). The procedures outlined relate mainly to purification of proteins on Thiopropyl Sepharose 6B. However, they can also serve as a starting point for development of techniques for RNA work.
Reaction of thiols with thiopropyl sepharose 6B releases the activating group, thiopyridone. Since thiopyridone is a chromophore, the reaction can be monitored photometrically. The extinction coefficient of this chromophore at its maximum, 343 nm, is 8.08x103 M-1cm-1. (The number quoted in the Pharmacia technical bulletin, 8.08x108 M-1cm-1, is in error by 5 orders of magnitude. The correct value is available in a Pierce product bulletin, Instructions for ImmunoPure Biotin-HPDP, Pierce Product Number 21341). The stated concentration of 2-pyridyl disulfide active groups on the gel is 18-31 Ámol/mL drained gel. The stated coupling capacity for proteins is 10-20 mg protein/ml drained gel.
The Pharmacia bulletin suggests using urea or guanidine to denature proteins prior to reaction with the thiopropyl sepharose 6B. In our experience, however, urea has been found to interfere with the coupling of thiophosphates to the matrix and therefore should only be added after reaction of the gel and adduct.
Wash Buffer + 3M Urea
100 mM BME in 0.5x Wash Buffer
9 M Urea
Thiopropyl Sepharose 6B, 15g/pkg Pharmacia Product Number 17-0420-01
Preparation of the Gel
The gel can be swollen by adding 10 mL H2O to 1 mL dry gel powder in a plastic centrifuge tube. After agitating the gel for 15 minutes at room temperature, the gel should be adequately hydrated, and buffer salts dissolved. The mixture is now centrifuged to pellet the gel. Supernatant is pipetted off until the gel represents approximately half of the total volume of the mixture. When shaken, the mixture produces a slurry that is ~50% gel by volume. The technical bulletin recommends storage of the gel at neutral pH in a bacteriostat such as 20% ethanol. Early versions of the bulletin recommended storing the gel in 0.02% sodium azide. More recent versions caution against this practice because of the reactivity of azide with activated disulfides, the 2-pyridyl disulfide moieties. Merthiolate and phenyl mercury reagents should not be used for storage of the gel. In its activated disulfide (oxidized) form, the gel is stable for many weeks. Exposure to reducing agents should be avoided since this very easily produces the sulfhydryl (reduced) form. If the reduced form of the gel is desired, it should be prepared from the oxidized form shortly before use by reducing the oxidized form with BME, then rinsing for many volumes.
200 mL of slurry is transferred to a BioRad mini-column. The gel is washed with 60 column volumes (6 mL) 25 mM HEPES pH 7.4 + 1 mM EDTA. The column is capped on the bottom. At this point it is ready for the sample to be loaded.
Binding and Elution of the RNA Sample
Thiophosphorylated RNA in 1 mL 25 mM HEPES pH 7.4 + 1 mM EDTA is added to the column. The column is rotated for 1 hour. (This is probably excessive. Experiments have suggested that 15 minutes should be sufficient in most cases.) 0.5 mL 9 M (0.5 mL) urea is added to the column, enough to give a final urea concentration of 3 M urea. The column is rotated for an additional 15 minutes, then rinsed with the following buffer systems:
The thiophosphorylated RNA is eluted in the BME fraction. An example of such a column following radiolabelled thiophosphorylated RNA is shown (Figure 4).
In this experiment, 62% of the counts eluted with BME and 8.5% remained on the column after BME elution. The RNA used in this study was 288 bases long. Shorter length RNA moledules generally exhibit less nonspecific binding to the column. Non-specific binding is also decreased by more stringent denaturing washes.
In the Pharmacia technical bulletin, suitable buffer systems that are suggested for binding of proteins to the gel generally include a buffer of pH 7-8 such as Tris HCl, phosphate or acetate, 0.1-0.5 M NaCl. Often, 1 mM EDTA is included to chelate contaminating heavy metals. Since the gel has a partial positive charge at neutral pH, the high ionic strength of the binding buffer may serve to neutralize repulsive electrostatic interactions between the gel and basic proteins. Since nucleic acids are negatively charged, low ionic strength may facilitate reaction with the gel. Subsequent washing of the gel should have higher ionic strength to reduce nonspecific binding of the RNA due to electrostatics.
Elution may be achieved with BME or DTT, or with such reagents as reduced glutathione, or cysteine.
(2) C. Lauhon, and J.W. Szostak, unpublished results. (3) Thiopropyl Sepharose 6B Instructions, Edition AA, Catalog number 71-7105-00, Pharmacia Biotech, Uppsala Sweden, (1995).
(3) Thiopropyl Sepharose 6B Instructions, Edition AA, Catalog number 71-7105-00, Pharmacia Biotech, Uppsala Sweden, (1995).