Thiol-modified oligonucleotides

Thiol-modified oligonucleotides have a number of uses, including reaction with labels that contain an α,β-unsaturated ketone, maleimide or other Michael acceptor. They can also be used for reaction with cysteines in proteins to made disulfide bonds and for binding to gold nanoparticles. For further information on uses of thiol-modified oligos see 5′-Thiol oliognucleotides and alkaline phosphatase probes.

Thiol-modified oligos can be synthesised by incorporating the thiol modification during solid-phase phosphoramidite oligonucleotide synthesis, at either the 5′- end or the 3′-end of the oligo (Figure 1).

Structures of oligonucleotides containing a 5'-thiol (thiohexyl; C6) modification (left), and a 3'-thiol (C3) modification (right)

Figure 1 | Structures of oligonucleotides containing a 5′-thiol (thiohexyl; C6) modification (left), and a 3′-thiol (C3) modification (right)

Synthesis of thiol-modified oligonucleotides

Protection of the thiol

The thiol group must be protected during phosphoramidite oligonucleotide synthesis: thiols are strong nucleophiles and interfere with phosphoramidite chemistry, and unprotected thiols spontaneously form disulphides in neutral aqueous solution. Two methods are commonly used for thiol protection during phosphoramidite synthesis: disulphide protection and trityl protection.

Disulphide protection

One method of synthesizing thiol-modified oligonucleotides is to protect the thiol as a disulphide. A commercially available phosphoramidite (Figure 2) is used to incorporate the disulphide into the oligonucleotide during solid-phase synthesis.

Structures of a disulphide phosphoramidite (left), used in the synthesis of 5'-thiol oligonucleotides, and a disulphide resin (right), used in the synthesis of 3'-thiol-modified oligonucleotides

Figure 2 | Structures of a disulphide phosphoramidite (left), used in the synthesis of 5′-thiol oligonucleotides, and a disulphide resin (right), used in the synthesis of 3′-thiol-modified oligonucleotides

After oligonucleotide synthesis and deprotection, the disulfide protecting group can be removed by reaction with a reducing agent such as dithiothreitol (DTT), to yield the free thiol (Figure 3). Gel filtration is then carried out to remove excess DTT.

Scheme showing the removal of a the disulfide protecting group to yield a 5'-thiol oligonucleotide after solid-phase oligonucleotide synthesis

Figure 3 | Scheme showing the removal of a the disulfide protecting group to yield a 5′-thiol oligonucleotide after solid-phase oligonucleotide synthesis

Disulphide-protected oligos should be deprotected just before use, to prevent the deprotected oligos from forming disulphides in solution:
  • treat the disulphide-protected oligo with a 100 mM solution of DTT in an aqueous buffer at pH 5.0, for 30 minutes at room temperature
  • then pass the solution through a gel filtration (desalting) column (e.g. a NAP-10 column)
Trityl protection

Another method of synthesizing 5′-thiol oligos is to protect the thiol with a trityl group. A commercially available phosphoramidite (Figure 4) is used to incorporate the trityl-protected thiol into the oligonucleotide at the end of solid-phase synthesis.

Structure of a trityl-protected thiol phosphoramidite, used in the synthesis of 5'-thiol oligonucleotides

Figure 4 | Structure of a trityl-protected thiol phosphoramidite, used in the synthesis of 5′-thiol oligonucleotides

Following oligo synthesis and deprotection, the trityl group is removed by reaction by reaction with silver nitrate. Excess silver nitrate is then removed by treatment with DTT (which forms an insoluble complex with silver). Finally, excess DTT is removed by gel filtration.

Scheme showing the removal of a the trityl protecting group to yield a thiol-modified oligonucleotide after solid-phase oligonucleotide synthesis

Figure 5 | Scheme showing the removal of a the trityl protecting group to yield a thiol-modified oligonucleotide after solid-phase oligonucleotide synthesis

One problem with the trityl protection method is that the DNA sticks to the DTT-silver precipitate, which tends to result in low oligonucleotide yields. As treatment with DTT is necessary (to remove excess silver), the disulphide protection method (which also requires the use of DTT, but not silver) is a cleaner method for the synthesis of thiol-modified oligonucleotides, and typically gives better yields.

Order of deprotection: thiol deprotection after oligo deprotection

If the sulfur of the thiol oligonucleotide is deprotected before the ammonia deprotection step, the thiol will react with the acrylonitrile liberated by deprotection of the cyanoethyl phosphotriesters (Figure 6). This potentially serious side-reaction is avoided by leaving the trityl protecting group on sulfur during the ammonia treatment and removing acrylonitrile from the oligonucleotide by evaporation or gel-filtration before the free thiol is liberated.

Mechanism of formation of a thiol cyanoethyl adduct during oligonucleotide deprotection

Figure 6 | Mechanism of formation of a thiol cyanoethyl adduct during oligonucleotide deprotection