Typical Applications of Thiol-Modified Oligonucleotides

Conjugated addition reaction of thiol to Michael receptor is a rapid and selective chemical reaction which is sometimes the preferred method for attaching labels in oligonucleotides. In the synthesis process of alkaline phosphatase-linked oligonucleotides, maleimide is added to the appropriate amino group of the alkaline phosphatase, then the maleimide functionalized enzyme is linked to the thiol group on the oligonucleotide to achieve the purpose. Finally, the product is purified by gel filtration and anion exchange chromatography. Thiolated oligo works as probes in assays involving fluorescence, colorimetry, or chemiluminescence, such as in situ hybridization, which is used to detect specific RNA sequences, especially the highly expressed mRNA in tumor cells.

Fig.1 Thiol-modified oligonucleotide conjugation to maleimide ligand. (CD BioGlyco)

Thiol-based Oligonucleotide Modification Service at CD BioGlyco

To support your diverse applications, CD BioGlyco offers 3' end modification, 5' end modification, and internal modification strategies on oligonucleotides. Our dedicated experts help you choose the right solution.

• 5'-thiol linker: The thiol group is usually added to the 5' end in the form of a disulfide to protect thiol from being destroyed in other chemical reactions. Reducing agents like dithiothreitol (DTT) should be used to reduce the protective group and obtain the active thiolated oligo before its application. The activated thiol terminal can be linked to maleimide, iodoacetamide, or thiol-reactive dye and used as a probe in hybridization experiments or PRC. The oligonucleotide can also be attached to a solid surface to prepare a DNA microarray. CD BioGlyco offers the 5'-thiol disulfide modifier with a six-carbon arm (5'-thiol S-S C6 linker), 5'-dithiol linker (5'-DTPA), and 5'-S-S dithiol serinol linker (5'-DTSPA).

Fig.2 Chemical structures of 5'-thiol S-S C6 linker, 5'-DTPA, AND 5'-DTSPA. (CD BioGlyco)

• 3'-thiol linker: The modification strategy of 3'-thiol linker is similar to that of 5'-thiol linker. Thiol group is added in the form of a disulfide bond, requiring reduction of the protective group before the application. CD BioGlyco provides customers with 3'-thiol S-S C3 linker and 3'-thiol S-S C6 linker, with spacers in different lengths (C3, C6) to reduce spatial interaction of oligonucleotides with labels to varying degrees. We also have a choice of 3'-DTPA or 3'-DTSPA.

Fig.3 Chemical structures of 3'-thiol S-S C3 linker, 3'-thiol S-S C6 linker, 3'-DTPA, and 3'-DTSPA. (CD BioGlyco)

• Internal thiol linker: When the thiol S-S C6 linker is incorporated into the interior, the two oligonucleotides are separated by a disulfide-bonded C12 spacer. The reduction of the disulfide group results in 3'-modified and 5'-modified oligonucleotides. Functionalized oligonucleotides can also be synthesized by thiol-attached thymine deoxynucleotides (thiol-C6-dT), which can be coupled to solid surfaces for application. To support your special needs, we also have an internal dithiol linker and internal dithiol dT.

Fig.4 Chemical structures of internal thiol S-S C6 linker, thiol-C6 dT, internal dithiol linker, and internal dithiol dT. (CD BioGlyco)

Applications

• Preparing functionalized oligonucleotide probes.
• Performing in situ hybridization experiments.
• Preparing DNA microarrays.
• Analyzing gene expression in tumor cells.

Highlights

• Superb technology in oligonucleotide modification.
• Oligonucleotide products with high purity.
• Custom modification sites.
• Intimate pre-sales and after-sales service.

CD BioGlyco offers almost all kinds of Oligonucleotide Modification Service. If you do not find the linker modification you need, please feel free to contact us for a custom solution to support your special needs. Look forward to working with you.