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Free Solution Assay Signal Modulation in Variable-Stem-Length Hairpin Aptamers.
Michael N Kammer1, Amanda K Kussrow1, Ian R Olmsted1
1Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, United States.
ACS Omega
|June 2, 2020
Summary
Aptamer probes designed with varying stem lengths showed altered signals in free solution assays (FSAs). Longer stems decreased binding affinity, demonstrating rational design potential for label-free aptamer beacons using FSAs.
Area of Science:
- Biophysical Chemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Free solution assays (FSAs) measure molecular binding events.
- Aptamers are short DNA or RNA molecules that bind to specific targets.
- Understanding aptamer conformational changes is key for biosensor development.
Purpose of the Study:
- To investigate how varying aptamer stem lengths affect FSA signals.
- To demonstrate the rational design of aptamer probes for FSA readout.
- To correlate aptamer structure with binding affinity and conformational changes.
Main Methods:
- Synthesized a series of hairpin aptamers with varying stem lengths (2-10 base pairings) targeting tenofovir.
- Performed interferometric measurements using free solution assays (FSAs).
- Calculated thermodynamic parameters, including Gibbs free energy (ΔG) and dissociation constants (KD).
Main Results:
- Aptamers with longer stem regions exhibited decreased FSA signals.
- Increased stem length correlated with reduced binding affinity (higher KD).
- Thermodynamic analysis showed a decrease in ΔG with increasing stem length.
Conclusions:
- Aptamer conformational changes upon binding directly influence FSA signals.
- Rational design of aptamer stem length can modulate binding affinity and FSA response.
- FSAs offer a label-free readout for aptamer beacon design and characterization.
