Single-Round Patterned DNA Library Microarray Aptamer Lead Identification.

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Aptamers are crucial for biosensors, but traditional selection methods (SELEX) can be time-consuming and challenging for surface immobilization.
• Solution-based aptamer selection may not always translate to effective binding when the aptamer is immobilized on a surface.

Purpose of the Study:

• To develop a single-round method for identifying aptamers using custom DNA microarrays.
• To create a DNA library computationally designed for enhanced structural complexity and binding probability.
• To investigate potential differences in aptamer binding mechanisms between surface-immobilized and solution-based strategies.

Main Methods:

• Computationally designed DNA microarrays with patterned libraries.
• High-throughput screening of DNA libraries for target binding via fluorescence intensity.
• Surface plasmon resonance (SPR) for specificity confirmation.
• Imino proton NMR/2D NOESY for structural analysis (G-quartet formation).

Main Results:

• A DNA aptamer with high fluorescence intensity upon thrombin addition was identified.
• The identified aptamer demonstrated specific binding to thrombin confirmed by SPR.
• Structural analysis indicated a lack of G-quartet formation in the microarray-derived aptamer.

Conclusions:

• A computational, microarray-based approach can efficiently identify aptamers in a single round, offering an alternative to SELEX.
• The absence of G-quartets in microarray-derived aptamers may suggest distinct binding mechanisms compared to solution-based methods.
• This method is beneficial for developing biosensors, overcoming challenges with immobilizing solution-selected aptamers.