Tandem Oligonucleotide Synthesis Enabled by a Reducible Linker

Area of Science:

• Biotechnology
• Synthetic Chemistry
• Molecular Biology

Background:

• Tandem oligonucleotide synthesis (TOS) enables the assembly of multiple oligonucleotide fragments within a single sequence using cleavable linkers.
• Disulfide-containing building blocks are typically used for introducing thiol functionality but can be repurposed for TOS.

Purpose of the Study:

• To explore the utility of a commercially available disulfide building block for tandem oligonucleotide synthesis (TOS).
• To demonstrate the compatibility of this TOS approach with standard oligonucleotide production and subsequent bio-conjugation techniques.

Main Methods:

• Incorporation of a disulfide-containing building block into oligonucleotide sequences.
• Cleavage of the disulfide bond via reduction to release oligonucleotide fragments with terminal thiol modifications.
• Assembly of a model crosslinked small interfering RNA (siRNA) construct.

Main Results:

• The disulfide linkage proved orthogonal to standard DNA/RNA synthesis workflows.
• Efficient cleavage of the disulfide bond upon reduction yielded thiol-modified oligonucleotide fragments.
• Successful assembly of a crosslinked siRNA construct and demonstrated compatibility with bio-conjugation.

Conclusions:

• The revisited TOS method using disulfide linkers is efficient and compatible with existing workflows.
• This approach facilitates the synthesis of complex oligonucleotide structures, including crosslinked siRNA.
• The developed methodology holds potential for diverse applications in biotechnology and nucleic acid-based therapeutics.