Oligonucleotide tagging for copper-free click conjugation.

Area of Science:

• Chemical Biology
• Oligonucleotide Synthesis
• Bioconjugation Chemistry

Background:

• Copper-free click chemistry offers mild and selective oligonucleotide conjugation.
• Existing methods for site-specific probe introduction are limited, especially for automated synthesis.
• Reported cyclooctynes are often large and hydrophobic, posing challenges for oligonucleotide modification.

Purpose of the Study:

• To develop a robust method for site-specific incorporation of bicyclo[6.1.0]nonyne (BCN) into synthetic oligonucleotides.
• To present phosphoramidite building blocks compatible with automated solid-phase synthesis.
• To demonstrate the utility of BCN-modified oligonucleotides in various conjugation applications.

Main Methods:

• Development of BCN and azide phosphoramidite building blocks.
• Standard solid-phase phosphoramidite chemistry for oligonucleotide synthesis.
• Demonstration of conjugation via biotinylation, fluorescent labeling, dimerization, and polymer attachment.

Main Results:

• Successful incorporation of BCN into synthetic oligonucleotides using standard automated protocols.
• Availability of a range of phosphoramidite building blocks for BCN and azide incorporation.
• High-yielding and straightforward conjugation of BCN-modified oligonucleotides for diverse applications.

Conclusions:

• The developed phosphoramidite chemistry enables facile site-specific introduction of BCN into oligonucleotides.
• This approach overcomes limitations of existing methods, offering a versatile tool for oligonucleotide modification.
• The BCN-modified oligonucleotides are readily conjugated, facilitating advanced applications in molecular biology and diagnostics.