Fluorescent modifications in aptamer switches-positional, structural, and neighboring pair effects on sensor...

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Aptamers are nucleic acid ligands used in biosensors for precision medicine.
• Fluorescent switches are created by conjugating aptamers with reporter molecules.
• Reporter molecule conjugation can negatively impact aptamer function and biosensor performance.

Purpose of the Study:

• To evaluate the impact of fluorophore-quencher modifications on aptamer switches.
• To investigate the interplay between reporter identity, environment, and labeling positions.
• To understand how these modifications affect aptamer binding properties, kinetics, specificity, and signal resolution.

Main Methods:

• Utilized a combinatorial array of modifications on aptamer switch sequences.
• Probed the interplay between reporter identity, local environment, and labeling positions.
• Analyzed changes in apparent binding affinities and kinetic response rates.

Main Results:

• Tuned aptamer switch binding properties over a 2-order-of-magnitude range (~230 to 2.3 μM).
• Adjusted apparent kinetic response rates from ~1000 s to sub-second resolution.
• Demonstrated that reporter environment and spatial positioning are critical for performance.

Conclusions:

• Reporter modifications significantly influence aptamer switch performance, including binding affinity, kinetics, specificity, and signal resolution.
• Strategic design of fluorescent modifications and their placement is essential for optimizing biosensor development.
• This study provides insights for designing next-generation biosensors with tailored performance for precision medicine.