Hi-Fi SELEX: A High-Fidelity Digital-PCR Based  Therapeutic Aptamer Discovery Platform.
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.
Eric
E
Ouellet
Jonathan H
JH
Foley
Edward M
EM
Conway
Charles
C
Haynes
D052157:Aptamers, Nucleotide / Q000302:isolation & purification* / Q000494:pharmacology*; D011416:Complement Factor D / Q000378:metabolism; D055808:Drug Discovery / Q000379:methods*; D015949:Factor IXa / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D006801:Humans; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D052156:SELEX Aptamer Technique / Q000379:methods*; D013917:Thrombin / Q000378:metabolism
D052157:Aptamers, Nucleotide; D005170:Factor X; D015949:Factor IXa; D011416:Complement Factor D; D013917:Thrombin