Assembly of split aptamers by dynamic pH-responsive covalent ligation

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Split aptamers offer modularity for biosensor construction.
• Developing efficient and specific ligation strategies for split aptamers is crucial.
• Existing ligation methods may suffer from background noise or lack dynamic control.

Purpose of the Study:

• To investigate the utility of reversible pH-responsive N-methoxyoxazolidine formation for ligating split aptamer fragments.
• To evaluate the performance of this ligation strategy using different models of the cocaine-binding aptamer (CBA).
• To assess the dynamic nature and substrate-dependent assembly of the ligated aptamers.

Main Methods:

• Synthesis and characterization of N-methoxyoxazolidine modified aptamer fragments.
• Assembly of twice-split and thrice-split cocaine-binding aptamer (CBA) models.
• pH-controlled ligation experiments to form functional aptamers.
• Assessing aptamer assembly dynamics and substrate-dependent binding.

Main Results:

• Successful ligation of split aptamer fragments was achieved using pH-responsive N-methoxyoxazolidine chemistry.
• Both twice-split and thrice-split CBA models demonstrated efficient assembly.
• Aptamer assembly was found to be dynamic and directly proportional to the substrate (cocaine) concentration.
• The ligation process occurred without significant interfering background ligation.

Conclusions:

• Reversible pH-responsive N-methoxyoxazolidine formation is a viable strategy for dynamic split aptamer ligation.
• This method provides a controllable and specific approach for assembling aptamer-based biosensors.
• The observed substrate-proportional assembly highlights the potential for quantitative aptamer sensing applications.