Hi‐Fi SELEX: A high‐fidelity digital‐PCR based therapeutic aptamer discovery platform

Abstract

Current technologies for aptamer discovery typically leverage the systematic evolution of ligands by exponential enrichment (SELEX) concept by recursively panning semi-combinatorial ssDNA or RNA libraries against a molecular target. The expectation is that this iterative selection process will be sufficiently stringent to identify a candidate pool of specific high-affinity aptamers. However, failure of this process to yield promising aptamers is common, due in part to (i) limitations in library designs, (ii) retention of non-specific aptamers during screening rounds, (iii) excessive accumulation of amplification artifacts, and (iv) the use of screening criteria (binding affinity) that does not reflect therapeutic activity. We report a new selection platform, High-Fidelity (Hi-Fi) SELEX, that introduces fixed-region blocking elements to safeguard the functional diversity of the library. The chemistry of the target-display surface and the composition of the equilibration solvent are engineered to strongly inhibit non-specific retention of aptamers. Partition efficiencies approaching 10(6) are thereby realized. Retained members are amplified in Hi-Fi SELEX by digital PCR in a manner that ensures both elimination of amplification artifacts and stoichiometric conversion of amplicons into the single-stranded library required for the next selection round. Improvements to aptamer selections are first demonstrated using human α-thrombin as the target. Three clinical targets (human factors IXa, X, and D) are then subjected to Hi-Fi SELEX. For each, rapid enrichment of ssDNA aptamers offering an order-nM mean equilibrium dissociation constant (Kd) is achieved within three selection rounds, as quantified by a new label-free qPCR assay reported here. Therapeutic candidates against factor D are identified.

Aptamers as targeted therapeutics: current potential and challenges

“…With the increase of PCR cycles and iterative selection rounds, excessive accumulation of amplification artifacts hampers the enrichment of high-affinity aptamers, and may even cause selection failure 54 . Recently, emulsion PCR (ePCR) or droplet digital PCR (ddPCR) have been incorporated into the selection protocol to reduce the propagation of byproducts and avoid PCR bias 45, 56–58 . These modified techniques preserve library diversity and prevent the loss of highly structural aptamer sequences that are difficult to amplify in a conventional PCR system.…”

Nucleic acid aptamer-based methods for diagnosis of infections

“…First, it has been reported that natural nucleic acids do not possess the full spectrum of chemical functional groups and conformational space needed to yield high-quality aptamers for many proteomic targets. To solve this problem, a number of methods (e.g., Hi-Fi SELEX (Ouellet et al, 2015), array-based strategies (Cho et al, 2015) and multiparameter particle display strategies ) have been developed, which have led to greater selection efficiencies (Zhang et al, 2016). In this light, chemical methods for DNA synthesis have enabled the incorporation of special functional groups onto oligonucleotides and have led to a significant advancement in the number of diverse aptamers that are available (Kimoto et al, 2013).…”

Selection and characterization of a DNA aptamer inhibiting coagulation factor XIa

“…These include interference from non-degenerate sequences flanking the variable regions in the aptamer library; non-specific retention of sequences not binding the desired target; and accumulation of amplification artifacts and artifactual sequences arising from library regeneration 20 . We observed many examples of such artifacts using high throughput sequencing after Round 4, some of which persisted to Round 10, as well as the selection of aptamers with barely detectable affinity for FXIa (e.g.…”

“…SELEX selects for ssDNA or ssRNA molecules able to adopt stable three-dimensional structures and bind molecular targets from a pool of ~10 14 unique strands 20 . Although aptamers against numerous coagulation factors have been developed, to our knowledge none have targeted FXIa 21–27 .…”