Reducing Measurement Deviation by Metastable DNA Probes for Aptamer Thermodynamic Characterization

Area of Science:

• Biophysical Chemistry
• Molecular Biology
• Thermodynamics

Background:

• DNA reaction equilibrium calculations are valuable for thermodynamic characterization.
• Significant measurement deviations in equilibrium concentration limit their application.
• Accurate measurement of aptamer binding energy is crucial for developing aptasensors and therapeutics.

Purpose of the Study:

• To introduce a novel strategy, metastable DNA reference calorimetry (MDRC), for standardized aptamer binding energy measurement.
• To demonstrate the advantages of metastable DNA hybridization in reducing quantification deviation.
• To develop MDRC-based algorithms for calculating thermodynamic parameters of aptamers with varying binding sites.

Main Methods:

• Utilized metastable DNA hybridization to minimize equilibrium concentration deviation.
• Developed and applied MDRC algorithms for aptamers with single and multiple binding sites.
• Incorporated a correlative model to account for cross-effects between binding sites in multi-site aptamers.

Main Results:

• MDRC successfully reduced quantification deviation, enabling accurate thermodynamic characterization.
• The correlative model revealed that for ATP aptamers, the first binding event can negatively impact the affinity for the second ATP molecule.
• Demonstrated the universality of MDRC by calculating thermodynamic parameters for protein-specific and cell-specific aptamers.

Conclusions:

• MDRC offers a universal and standardized approach for precise aptamer binding energy measurement.
• The method is applicable to diverse aptamer systems, including those with multiple binding sites and complex biological targets.
• MDRC significantly advances the thermodynamic characterization of DNA-aptamer interactions.