Complementarity to the microRNA (miRNA) seed region has long been recognized as the primary determinant in target recognition by the Argonaute-miRNA complex. Recently, we reported that pairing to miRNA 3′-supplementary region (nucleotides 13–16) can increase target affinity by more than an order of magnitude beyond seed-pairing alone. Here, we present biochemical evidence that supplementary interactions can drive robust differential targeting between equivalently seed-matched target RNAs in vitro. When mixed together, Ago2–miRNA complexes initially bind seed-matched targets equally but then redistribute between targets based on the strength of supplementary interactions. Thus, while initial target recognition was driven by seed-pairing, the distribution of Ago2–miRNA complexes between targets was determined by retention of Ago2 on target RNAs via supplementary interactions. Mathematical modeling and biochemical data predict that targets with strong supplementary interactions could be more strongly repressed than seed-only matched targets, even when vastly outnumbered by seed-only targets. The combined results raise the possibility that supplementary interactions could play a role in specifying specific miRNA targets for enhanced repression.
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