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An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

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Direct visualization of electrophoretic mobility shift assays using nanoparticle-aptamer conjugates.

Min S Wang¹, Scott M Reed

¹Department of Chemistry, University of Colorado Denver, Denver, CO 80217-3364, USA.

Electrophoresis

|December 16, 2011

Summary

Gold nanoparticles with aptamers visualize protein interactions during gel electrophoresis. This confirms aptamers bind only monomeric C-reactive protein (CRP), not pentameric CRP, aiding cardiovascular disease risk prediction.

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Area of Science:

Biochemistry
Nanotechnology
Molecular Diagnostics

Background:

C-reactive protein (CRP) exists in monomeric (mCRP) and pentameric (pCRP) forms.
Pentameric CRP (pCRP) is a clinical biomarker for cardiovascular disease (CVD) risk.
Monomeric CRP (mCRP) is more closely linked to pro-inflammatory and pro-atherogenic effects, suggesting its diagnostic potential.

Purpose of the Study:

To develop a method for direct visualization of protein-oligonucleotide interactions.
To confirm the binding specificity of a C-reactive protein (CRP) aptamer to the monomeric form.
To establish a novel diagnostic tool for assessing CVD risk.

Main Methods:

Aptamers targeting CRP were conjugated to gold nanoparticles (AuNPs).
The resulting oligonucleotide-nanoparticle conjugates (ON-NP) were used in a nanoparticle-based electrophoretic mobility shift assay (NP-EMSA).
Interactions between ON-NP and CRP isoforms were visualized during gel electrophoresis using a digital camera.

Main Results:

The CRP-aptamer ON-NP conjugate showed decreased mobility in the presence of monomeric CRP (mCRP).
No significant change in mobility was observed with pentameric CRP (pCRP) even at higher concentrations.
The NP-EMSA allowed for real-time detection without radioisotopes or specialized imagers.

Conclusions:

Gold nanoparticle-aptamer conjugates enable direct, real-time visualization of protein-oligonucleotide binding.
The developed NP-EMSA confirms aptamer specificity for monomeric CRP.
This technique offers a sensitive and accessible method for improved CVD risk diagnostics.