Identification of cell membrane protein stress-induced phosphoprotein 1 as a potential ovarian cancer biomarker using aptamers selected by cell systematic evolution of ligands by exponential enrichment.

In this paper, we describe the elucidation of the target of an aptamer against ovarian cancer previously obtained by cell-SELEX (SELEX = systematic evolution of ligands by exponential enrichment). The target's identity, stress-induced phosphoprotein 1 (STIP1), was determined by mass spectrometry and validated by flow cytometry, using siRNA silencing and protein blotting. Initial oncologic studies show that the aptamer inhibits cell invasion, indicating that STIP1, which is currently under investigation as a potential biomarker for ovarian cancer, plays a critical role in this process. These results serve as an excellent example of how protein target identification of aptamers obtained by cell-SELEX can serve as a means to identify promising biomarker candidates and can promote the development of aptamers as a new drug class to block important oncological processes.

In vitro selection with artificial expanded genetic information systems.

Artificially expanded genetic information systems (AEGISs) are unnatural forms of DNA that increase the number of independently replicating nucleotide building blocks. To do this, AEGIS pairs are joined by different arrangements of hydrogen bond donor and acceptor groups, all while retaining their Watson-Crick geometries. We report here a unique case where AEGIS DNA has been used to execute a systematic evolution of ligands by exponential enrichment (SELEX) experiment. This AEGIS-SELEX was designed to create AEGIS oligonucleotides that bind to a line of breast cancer cells. AEGIS-SELEX delivered an AEGIS aptamer (ZAP-2012) built from six different kinds of nucleotides (the standard G, A, C, and T, and the AEGIS nonstandard P and Z nucleotides, the last having a nitro functionality not found in standard DNA). ZAP-2012 has a dissociation constant of 30 nM against these cells. The affinity is diminished or lost when Z or P (or both) is replaced by standard nucleotides and compares well with affinities of standard GACT aptamers selected against cell lines using standard SELEX. The success of AEGIS-SELEX relies on various innovations, including (i) the ability to synthesize GACTZP libraries, (ii) polymerases that PCR amplify GACTZP DNA with little loss of the AEGIS nonstandard nucleotides, and (iii) technologies to deep sequence GACTZP DNA survivors. These results take the next step toward expanding the power and utility of SELEX and offer an AEGIS-SELEX that could possibly generate receptors, ligands, and catalysts having sequence diversities nearer to that displayed by proteins.