title: Direct visualization of electrophoretic mobility shift assays using nanoparticle-aptamer conjugates.
abstract: Here, we demonstrate that aptamers tethered to gold nanoparticles enable direct visualization of protein-oligonucleotide interactions during gel electrophoresis. This technique is used to confirm that an aptamer previously identified as binding to C-reactive protein (CRP) only binds to the monomeric form of CRP. While native, pentameric CRP (pCRP) is used in clinical assays to predict cardiovascular disease (CVD) risk, it is the monomeric isoform that is more strongly associated with pro-inflammatory and pro-atherogenic effects. To visualize this selectivity, the CRP-aptamer was conjugated to streptavidin-coated gold nanoparticles and the mobility of the free oligonucleotide-nanoparticle conjugate (ON-NP) and the protein/ON-NP complex bands were visualized and recorded during electrophoresis using a simple digital camera. At a concentration of 6 μg/mL, monomeric CRP showed a significant decrease in the observed ON-NP mobility, whereas no change in mobility was observed with pCRP up to 18 μg/mL. Advantages of this nanoparticle-based electrophoretic mobility shift assay (NP-EMSA) over the traditional EMSA include real-time detection of protein-oligonucleotide interactions, the avoidance of harmful radioisotopes, and elimination of the need for expensive gel imagers. The availability of both the NP-EMSA technique and an mCRP-specific probe will allow for improved clinical diagnostic to more accurately predict future CVD risk.
authors.affiliation: Department of Chemistry, University of Colorado Denver, Denver, CO 80217-3364, USA.
authors.forename: Min S
authors.initials: MS
authors.lastname: Wang
authors.forename: Scott M
authors.initials: SM
authors.lastname: Reed
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism; D015415:Biomarkers; D002097:C-Reactive Protein / Q000032:analysis / Q000378:metabolism; D002318:Cardiovascular Diseases; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D006046:Gold / Q000737:chemistry; D053768:Metal Nanoparticles / Q000737:chemistry*; D008956:Models, Chemical
chemical_list: D052157:Aptamers, Nucleotide; D015415:Biomarkers; D006046:Gold; D002097:C-Reactive Protein