Figure 1

Rational probe design results and high predicted affinity. (a) A target sequence, with HindIII restriction site (bold) resulting in a toehold, and the complementary LNA-probe. LNA nucleotides are red, DNA nucleotides black. (b) Schematic representation of the strand invasion. First, the probe binds the toehold on the target. Fraying of the DNA ends allows for strand invasion, and finally a stable hybrid is formed. In addition, secondary structures can form between the displaced target strand, and un-incorporated nucleotides from the probe, resulting in alternative structures. Two alternative structures with 12 bases incorporated are shown on the left. (c) Calculations of the free energy change of hybridization, as the number of hybridized nucleotides of the probe increases. The red line depicts the hybrid, in which the target strand hybridizes linearly with the probe. Black dots indicate alternative LNA-DNA hybrids between the displaced strand and the probe. (d) Alternative structures can stabilize intermediate structures. In equilibrium the full hybrid accounts for 87% of the hybridization products, calculated by the Boltzmann probability distribution.