Selection of DNA aptamers for ovarian cancer biomarker HE4 using CE-SELEX and high-throughput sequencing

Abstract

The development of novel affinity probes for cancer biomarkers may enable powerful improvements in analytical methods for detecting and treating cancer. In this report, we describe our use of capillary electrophoresis (CE) as the separation mechanism in the process of selecting DNA aptamers with affinity for the ovarian cancer biomarker HE4. Rather than the conventional use of cloning and sequencing as the last step in the aptamer selection process, we used high-throughput sequencing on an Illumina platform. This data-rich approach, combined with a bioinformatics pipeline based on freely available computational tools, enabled the entirety of the selection process—and not only its endpoint—to be characterized. Affinity probe CE and fluorescence anisotropy assays demonstrate the binding affinity of a set of aptamer candidates identified through this bioinformatics approach.Graphical AbstractA population of candidate aptamers is sequenced on an Illumina platform, enabling the process by which aptamers are selected over multiple SELEX rounds to be characterized. Bioinformatics tools are used to identify enrichment of selected aptamers and groupings into clusters based on sequence and structural similarity. A subset of sequenced aptamers may be intelligently chosen for in vitro testing.

Microfluidics in the selection of affinity reagents for the detection of cancer: paving a way towards future diagnostics

“…Furthermore, potential aptamer candidates can be reduced from 2000 to only 20 nucleotide sequences (with strong affinity), thus reducing the expensive and tedious sequencing and cloning process. 76 When compared to high-affinity regents (Kd ≦ 10 nM), these selected aptamers exhibited lower affinities. For instance, capillary electrophoresis (CE) SELEX, 67 sol-gel SELEX, 68 magnetic bead-based SELEX, 69,70 agarose-droplet SELEX 71 and cell-SELEX [72][73][74] have been performed with microfluidic systems.…”

In Vitro Selection and Characterization of DNA Aptamers to a Small Molecule Target

“…One recent breakthrough technology is high-throughput sequencing, which has led to advancements even in small molecule selection. Recent examples of high-throughput sequencing applied to aptamer selection include selections for tyramine (Valenzano et al, 2016), thrombin (Riley et al, 2015), and ovarian cancer biomarkers CA125 and HE4 (Eaton et al, 2015;Scoville, Uhm, Shallcross, & Whelan, 2017). Recent examples of high-throughput sequencing applied to aptamer selection include selections for tyramine (Valenzano et al, 2016), thrombin (Riley et al, 2015), and ovarian cancer biomarkers CA125 and HE4 (Eaton et al, 2015;Scoville, Uhm, Shallcross, & Whelan, 2017).…”

“…High-throughput sequencing has permitted a deeper understanding of the selection efficiency between rounds and identification of rare sequences that may be lost through conventional sequencing methods (Hoinka et al, 2015;Valenzano et al, 2016). Recent examples of high-throughput sequencing applied to aptamer selection include selections for tyramine (Valenzano et al, 2016), thrombin (Riley et al, 2015), and ovarian cancer biomarkers CA125 and HE4 (Eaton et al, 2015;Scoville, Uhm, Shallcross, & Whelan, 2017).…”

Applications of High-Throughput Sequencing for In Vitro Selection and Characterization of Aptamers

“…In contrast, all the candidates with a negative meta-Z-score did not show any binding. However, when MPBind was used by another lab to analyze another SELEX against the ovarian cancer marker HE4, the correlation was weaker between the score and the affinity of the sequences [46]. Indeed, the best binders showed a negative Z-score.…”