Quantitative Characterization of Stringency in SELEX

High stringency (e.g., low target concentration) is key for obtaining high-affinity aptamers in SELEX; however, excessive stringency greatly increases the probability of SELEX failure. The control of stringency in SELEX remains a technical art based solely on intuition. A major reason for this is the lack of a measure of stringency. Here we introduce the Binder-to-Nonbinder Ratio at the output of partitioning (BNR), a parameter that characterizes stringency quantitatively: increasing stringency leads to decreasing BNR. BNR is determined experimentally by simply measuring by qPCR the quantities of oligonucleotides after partitioning in the presence and absence of target. The theory suggests, and our SELEX experiments with two targets confirm, that BNR must be kept statistically significantly greater than zero to avoid SELEX failure due to excessive stringency. Using BNR will help experimenters to rationalize the choice of conditions which the stringency depends on, e.g., target concentration and time of partitioning.