Alkynyl phosphonate DNA: a versatile "click"able backbone for DNA-based biological applications

Area of Science:

• Oligonucleotide Chemistry
• Chemical Biology
• Biotechnology

Background:

• DNA-based applications face challenges in cell delivery, nonspecific effects, toxicity, and enzyme stability.
• Existing oligonucleotide analogues lack a versatile methodology to systematically address these issues.

Purpose of the Study:

• To introduce a novel, clickable chemistry for rapid functionalization of DNA analogues.
• To synthesize and evaluate the biological properties of triazolylphosphonate (TP) modified oligonucleotides (ODNs).

Main Methods:

• Development of an alkynyl phosphoramidite synthon compatible with solid-phase synthesis.
• Postsynthetic functionalization using "Click" chemistry to create TP internucleotide linkages.
• Automated solid-phase synthesis of mixed backbone ODNs with TP, phosphate, or thiophosphate linkages, including 2'-OMe and LNA modifications.

Main Results:

• TP linkages exhibit high resistance to 5'- and 3'-exonucleases.
• TP modification causes slight destabilization upon hybridization with RNA.
• Fluorescently labeled TP-ODNs demonstrate efficient cellular uptake.
• Subcellular distribution of TP-ODNs is cell-type dependent, with notable nuclear uptake in HeLa cells.

Conclusions:

• Triazolylphosphonate ODNs represent a versatile new class of oligonucleotide analogues.
• This clickable chemistry approach provides a powerful tool for designing novel biological research reagents.
• TP-modified ODNs offer enhanced stability and tunable cellular uptake for various applications.