The first question to ask is how confident are you that the lyophilized oligo material is present as oligo monomer as opposed to oligo--S-S-oligo dimers? If you have a small amount of the disulfide form, you don't need to worry too much. However, usually, you simply don't know, in which case I usually do the following.

1. Reconstitute the oligo in a small volume of pH 8.0 buffer (e.g. phosphate). Calculate the concentration of oligo and add excess DTT. As DTT is a very powerful reductant you don't need a great excess, maybe 3-fold (this assumes the worst case scenario, that all your oligo is disulfide linked). Incubate 15 min room temp.

2. Desalt the sample using commercially available disposable columns (e.g. NAP5). It is important to know where the oligo elutes compared with the DTT and if you have not used the column before I'd try out oligo and DTT separately, just to get used to the elution positions and peak widths. If you don't want to do this, just collect SMALL fractions to be safe, and pool those that have oligo, but not DTT. You can track DTT using DTNB reagent to make sure you don't pool any DTT-containing fractions. DTT will elute later than oligo, but there will likely be some small overlap of the trailing edge of the oligo peak and the front edge of the DTT peak. Those oligo fractions should be discarded.

From a desalting perspective, any column buffer will work of course, but from a conjugation/storage perspective you need to choose carefully. If you intend to use all the oligo IMMEDIATELY you can desalt into a suitable buffer (MOPS or phosphate) with a pH of around 7. I would not go any higher than pH 7.5 otherwise the maleimides may start to hydrolyze in your subsequent conjugation reaction.

However, if you need to store some of the oligo, I'd desalt into a weak acidic buffer. e.g. 10mM acetate, pH 5. Snap freeze the surplus oligo and store at -70 if you can, or -20. The reason for using a slightly acidic buffer is to protonate the thiolate anion to give -SH, which is unreactive. Only -S minus reacts to give oligo dimers.

3. For conjugation, mix your reduced oligo and maleimide particles and leave the reaction 1h. You cannot leave the reaction for too long, so you can do overnight if you have no previous knowledge of the rate of the reaction. It should be pretty fact but will depend on the exact conditions used. Obviously the oligo needs to be in excess of the maleimide groups, at least if you want to label them all. (If you desalted into weak acidic buffer, you will need to adjust the pH of the conjugation reaction. The fact the acidic buffer is weak facilitates any adjustment that needs to be made.

Note:

Since you have magnetic beads it is easy to wash away excess oligo, and you could in principle add a great excess to drive the reaction and then clean up easily.

As for concern over oligos reacting with themselves rather than maleimides, you really do not need to worry. The concern is whether you are starting with reduced material, not losses by oxidation during conjugation, which will be both minimal and irrelevant (by the fact you use excess oligo).

I hope this helps.

Typo, I meant of course: ..It should be pretty fast, ....

Hi Nick Gee.

That’s a synthetic oligo with 2 thiol groups inserted at the 3’ tail. The company confirmed that the lyophilyzed oligos are in the oxydized form. So I need to dissolve them in a buffer containing DTT.

After desalting from DTT, I should freeze the reduced Thiol-oligis quickly.

Now, I am looking for a protocol of conjugation between Thiol-oligo and Maleimide-bead.

I assume the probability of the reaction between Thiol-Maleimide is more than thiol-thiol of 2 oligos.

Okay thanks. Perhaps you have a particular reason for wanting two thiols, but your final conjugate is likely to have unconjugated thiols on the surface, which may require an extra step in the protocol to block them.

If the two thiols are present in the SAME molecule and are favorably disposed to form a cyclic structure, then intuitively the risk of re-oxidation is slightly higher than with molecules with single thiols. The strategy is essentially the same but I'd now lean towards desalting into a weak acidic buffer and freezing the pooled material in small aliquots prior to conjugation. Additionally, I would add 0.1-1mM EDTA to the column buffer and to your subsequent conjugation reaction, the rationale being that trace metal ions can catalyze re-oxidation of thiols. In fact, I would add EDTA regardless of the detail of the thiolated oligo on further reflection. However, check with bead supplier that EDTA is not contraindicated.

Another thought is that you might have a linear disulfide structure and only one thiol attached to the oligo AFTER reduction? Anyway, it changes little in the approach except that the blocking step is not needed if you have one thiol per reduced oligo, since only unconjugated oligos can have free thiols, and they will be removed in the washing steps anyway.

As for the protocol, there is very little to say really, the generic advice that follows applies to almost any thiol maleimide reaction. You will have to determine the specifics empirically. Suspend your magnetic beads in conjugation buffer. There are many suitable buffers and conditions, I'd choose from 50mM Hepes, phosphate or MOPS at around pH 7 (up to pH 7.5 is okay), Include EDTA, but it can be omitted if it is incompatible with the bead type. Then add the desalted oligo. The best molar ratio (for your application) of oligo to bead is something you will have to work out, unless you want maximum incorporation, in which case just add a considerable excess of oligo. Since your oligo is in a weakly acidic buffer, your conjugation buffer needs to be able to overpower any acid introduced with the oligo. I cannot give any specific advice as I don't know what volume of oligo you will add or your preferred conjugation buffer.

The extra thiol (if there is one) potentially could cause crosslinking of beads or other issues later on, and so I would use a short conjugation time (1-3h, to be optimized) and introduce a capping step post conjugation. The most commonly used blockers are iodoacetamide or NEM, I'd go with the latter as it is more reactive and easier to store. Add the blocker to the conjugation reaction in 2-3 fold molar excess over the thiols. Hopefully you have a linear disulfide and these steps are redundant.

Finally, wash the beads with whatever buffer is needed for your application.

Good luck with it!