Figure 2.

Illustration of the affinity-specificity trade-off in hybridization reactions. Under conditions in which the oligonucleotide binds very tightly (e.g., longer oligonucleotide) to the target RNA (bottom left), off-target RNAs will also be bound. Higher selectivity can be achieved under conditions in which on-target binding is only marginally favorable (e.g., shorter oligonucleotide, bottom right), but at the cost of yield. Optimal conditions (yellow shaded region) balance affinity and specificity. In this example, reaction conditions were simulated under fixed conditions ([RNA_target] = [RNA_off-target] = 10 pm, [capture oligonucleotide] = 100 nm). In this simulation, the capture oligonucleotide prefers the target RNA (green) over the off-target RNA (red) by a constant energetic difference (3 kcal/mol). This example depicts the trade-off when designing the chemical composition of capture oligonucleotides, but similar considerations apply when choosing conditions for hybridization.