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Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

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KF polymerase-based fluorescence aptasensor for the label-free adenosine detection.

Dongli Liao¹, Huping Jiao, Bin Wang

¹State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, PR China.

|December 21, 2011

Summary

This study presents a novel, label-free method for detecting adenosine using Klenow fragment polymerase and DNA aptamers. The assay offers a simple and inexpensive way to quantify adenosine levels through fluorescence enhancement.

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Area of Science:

Biochemistry
Molecular Biology
Analytical Chemistry

Background:

Klenow fragment polymerase (KF polymerase) possesses both 5' to 3' DNA polymerase and 3' to 5' exonuclease activity.
Single-stranded DNA can be degraded by the exonuclease activity of KF polymerase.
DNA aptamers can be designed to bind specific molecules, such as adenosine.

Purpose of the Study:

To develop a simple, inexpensive, and label-free method for the selective detection and quantification of adenosine.
To utilize the enzymatic activity of KF polymerase and the binding properties of DNA aptamers for adenosine detection.

Main Methods:

A DNA aptamer specific for adenosine was split into two single-stranded DNA fragments (aptamer-A1 + aptamer-A2).
In the absence of adenosine, KF polymerase's exonuclease activity degraded the single-stranded aptamer fragments, resulting in low background fluorescence with SYBR Green dye.
In the presence of adenosine, the aptamer fragments bound to adenosine, hybridized to form a duplex DNA, which initiated DNA polymerization and led to significant fluorescence enhancement with SYBR Green dye.

Main Results:

The developed method demonstrated selective detection of adenosine.
The assay showed a significant increase in fluorescence in the presence of adenosine due to DNA polymerization.
The fluorescence enhancement was proportional to the adenosine concentration, enabling quantification.

Conclusions:

The developed method provides a sensitive and selective approach for adenosine detection.
This label-free assay is a cost-effective alternative for adenosine quantification.
The combination of KF polymerase and DNA aptamers offers a versatile platform for detecting small molecules.