Method Modifies Oligonucleotides Without Special Phosphoramidites

Researchers in Denmark have developed an easy and inexpensive method for linking other molecules to DNA sequences with desired functions—a breakthrough that will ease production of modern DNA- and RNA-based drugs like the COVID-19 vaccine.

The production of short DNA and RNA sequences, called oligonucleotides, is essential to diagnostics, DNA sequencing, gene synthesis for synthetic biology, and gene therapies such as CRISPR-Cas9 methods. The technology of oligonucleotide production has been advanced since it was developed in 1981, but the underlying chemistry remains largely unchanged—and dependent on unstable phosphoramidites.  

Kurt Gothelf and his research group at Aarhus University previously showed a more efficient way to produce the building blocks for oligonucleotides by developing a flow-based method to make phosphoramidites immediately before they are to be used, avoiding degradation.

But now there is a great need for oligonucleotides that have been modified with special functionalities. For example, oligonucleotides modified with certain sugars have been shown to be taken up by the liver. Based on this, several oligonucleotide drugs are used to regulate proteins that are, in fact, produced in the liver and thus can cure diseases. It is also important to be able to attach dyes to oligonucleotides as this is often used in diagnostics and research.

Usually, these functionalities are inserted into oligonucleotides by phosphoramidites produced with a specific chemical handle or the desired functional group. There are commercial catalogs with several hundred varieties of these modified phosphoramidites. However, such modified phosphoramidites are, in most cases, very expensive to obtain and unstable in liquids at room temperature.

In a recently published paper in Nucleic Acids Research, Gothelf and Angel Santorelli present an easy and inexpensive method of introducing modifications to oligonucleotides without the need for special phosphoramidites. They use chemical compounds called sulfonyl azides, which can synthesize oligonucleotides with many different functionalities.

“Compared to phosphoramidite-based modifiers, the use of sulfonyl azides offers several potential advantages,” according to the paper. “They are easy to synthesize, air and moisture stable, and their incorporation is not limited to terminal positions. In addition, the formation of sulfonylphosphoramidates does not compromise the sequence of the oligonucleotide, nor do they introduce gaps and nicks in the backbone. In this work, we demonstrate a versatile and straightforward protocol to modify oligonucleotides with a variety of different functional groups of interest using sulfonyl azides applied in solid phase oligonucleotide synthesis (SPOS).”

The method is compatible with the previously mentioned automated and flow-based method for oligonucleotide synthesis, where degradation of the phosphoramidites is avoided.