A two-step stimulus-response cell-SELEX method to generate a DNA aptamer to recognize inflamed human aortic endothelial cells as a potential in vivo molecular probe for atherosclerosis plaque detection

Abstract

Aptamers are single-stranded oligonucleotides that are capable of binding wide classes of targets with high affinity and specificity. Their unique three-dimensional structures present numerous possibilities for recognizing virtually any class of target molecules, making them a promising alternative to antibodies used as molecular probes in biomedical analysis and clinical diagnosis. In recent years, cell-systematic evolution of ligands by exponential enrichment (SELEX) has been used extensively to select aptamers for various cell targets. However, aptamers that have evolved from cell-SELEX to distinguish the "stimulus-response cell" have not previously been reported. Moreover, a number of cumbersome and time-consuming steps involved in conventional cell-SELEX reduce the efficiency and efficacy of the aptamer selection. Here, we report a "two-step" methodology of cell-SELEX that successfully selected DNA aptamers specifically against "inflamed" endothelial cells. This has been termed as stimulus-response cell-SELEX (SRC-SELEX). The SRC-SELEX enables the selection of aptamers to distinguish the cells activated by stimulus of healthy cells or cells isolated from diseased tissue. We report a promising aptamer, N55, selected by SRC-SELEX, which can bind specifically to inflamed endothelial cells both in cell culture and atherosclerotic plaque tissue. This aptamer probe was demonstrated as a potential molecular probe for magnetic resonance imaging to target inflamed endothelial cells and atherosclerotic plaque detection.

Post-synthesis DNA modifications using a trans-cyclooctene click handle

“…N37 is a known aptamer selectively targeting the thrombospondin-1 (TSP-1) protein on the surface of inflamed endothelial cells with high potential of atherosclerosis. 46, 47 Using the same PCR amplification conditions described above, N37 was used as new template for amplifications. Tetrazine-FITC (fluorescein isothiocyanate reacting with an amino handle of tetrazine, Tz-FITC, Figure 4B, see SI for details) was synthesized for post-PCR fluorescent labelling of TCO-TTP modified DNA.…”

“…Tetrazine-FITC (fluorescein isothiocyanate reacting with an amino handle of tetrazine, Tz-FITC, Figure 4B, see SI for details) was synthesized for post-PCR fluorescent labelling of TCO-TTP modified DNA. We studied the incorporation of fluorescein into a known aptamer 46 N37 and the applicability of using such a fluorophore-labelled DNA for binding applications. TCO-TTP modified N37 (TCO37) (Lane 3, Figure 4A) was obtained and analyzed with 15% PAGE after KOD-XL catalyzed PCR.…”

“…In natural N37 aptamer studies, the binding affinity with TSP-1 was determined with 5’-FAM labelled primer, which has a single fluorophore attached in each aptamer sequence. 46 However, with the ability to incorporate a fluorophore using the tetrazine- trans -alkene chemistry, it was possible to introduce a fluorophore in multiple positions of the TCO-modified aptamer N37 (TCO37). Such labelled aptamers could have higher fluorescent intensity without losing binding affinity.…”

Applications of Aptamers in Targeted Imaging: State of the Art

“…A detectable change in MRI signal is observed with 25 nM thrombin in human serum, which was not observed with control proteins. In a recent study, one aptamer targeting activated endothelial cells was used for MRI detection of atherosclerosis plaque since the initiation of atherosclerosis is upon the dysfunction of endothelial cells [62]. Although only cellular MRI was conducted to validate its specificity, this aptamer can be used for detecting early stage atherosclerotic plaques.…”

“…In addition, the conventional SELEX involves a number of time-consuming steps which reduce the efficiency and efficacy of aptamer selection. Optimization such as stimulus-response cell SELEX (utilizing asymmetric PCR) was adopted which enables aptamer selection to distinguish activated cells or “diseased” cells and greatly reduces time and cost [62]. …”