RESEARCH PAPER
Segmented hybridization probes: modulating target affinity and base pairing selectivity

Short hybridization probes bind their targets with greater base pairing fidelity, but with lower affinity than longer

probes. Furthermore, their target sequence is shorter, and thus more likely not to be unique in a given genome.

Long hybridization probes provide increased affinity, and their sequences are more unique, but their duplexes

tolerate mismatches more readily, without a significant depression in melting point. It was reasoned that segmenting

longer hybridization probes by introducing flexible, abasic linkers might lead to oligonucleotides that

retain some of the sequence selectivity of short probes without losing too much of the target affinity of their unsegmented

counterparts. A model study led to 1,3-propandiol-phosphates as linker residues. These spacer residues

were introduced at different positions of hybridization probes 8-20 residues in length and their hybridization

properties were studied in UV-melting curves with RNA or DNA target strands. Increases in base pairing selectivity

(ΔΔTm of up to -7.4°C for a single mismatch) and decreased target affinities (ΔΔTm between -15 and -25°C)

were found for the segmented probes when compared to their unsegmented counterparts, and so was a decreased

selectivity for insertions at the site of the linker. Also, the increases in selectivity are not uniform in their

magnitude and depend on sequence context and position. A favorable case appears to be a hybridization probe

that contains two spacers, with one octamer as core segment, flanked by a heptamer and a pentamer as terminal

segments.