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.

Figure 1

NP-EMSA and 3′Cy5-EMSA. Photographs of (A) NP-EMSA and (D) 3′Cy5-EMSA gel after electrophoresis. Western blots of the (B) NP-EMSA gel and (E) 3′Cy5-EMSA gel. Superimposed images of (C) NP-EMSA and (F) 3′Cy5-EMSA gel with their respective Western blots showing the overlapping regions of aptamer and mCRP. Lanes: (1) ON-NP or 3′Cy5-RNA, (2–6) ON-NP or 3′Cy5-RNA incubated with 0.12, 0.24, 0.60, 1.2 and 1.8 mg/mL mCRP, (7) 1.8 mg/mL mCRP, (8–12) ON-NP or 3′Cy5-RNA incubated with 0.12, 0.24, 0.6, 1.2 and 1.8 mg/mL pCRP, (13) 1.8 mg/mL pCRP, and (14) ON-NP or 3′Cy5-RNA incubated with 1.8 mg/mL BSA.

Figure 2

Migration of ON-NP/CRP complex. ON-NP/CRP complex were separated by electrophoresis at 45V for 30 min. The migration distances of ON-NP/mCRP (black) and ON-NP/pCRP (white) complex were measured from the center of the well to middle of the band at the end of the 30 min electrophoresis. Data was reported as the mean distance ± SD. n = 3; *, p<0.05; **, p<0.01. (B) Migration rates of ON-NP with increasing concentrations of mCRP and pCRP.

Scheme 1

Preparation of ON-NP. Citrate-capped gold nanoparticles were incubated with (i) streptavidin followed by (ii) biotinylated aptamer to generate ON-NP. The nanoparticles were centrifuged between each step to remove unbound molecules in the supernatant. Binding of (iii) mCRP to the ON-NP was visually detected by EMSA.