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

Abstract

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 !7.4EC for a single mismatch) and decreased target affinities (ΔTm between !15 and !25EC) 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.

Low temperature Hydrogen Reduction of High Surface Area Anatase and Anatase/β‐TiO2 for High‐Charging‐Rate Batteries

“…The structure presented here shows how an easy‐to‐append cap can enhance affinity and base pairing fidelity of DNA probes for RNA targets such as microRNAs 9. 23 The prolinol‐glycine linker positions the oxolinic acid residue well for stacking on the terminal base pair, leading to a network of contacts to both nucleobases and (deoxy)riboses. The same linker might be useful for testing other quinolones, including fluoroquinolone antibiotics, known to inhibit the resealing of duplexes that is part of the mechanism of action of bacterial gy‐ rases 24.…”

“…For the ogOA cap, melting point increases of up to 13.1 °C were measured for duplexes with target strands 9. Its ability to reinforce probes for miRNAs was demonstrated,9, 23 and a synthesis of a phosphoramidite building block for incorporating the ogOA cap has been developed 22. The mode of binding remained enigmatic, though, as additional substituents or side chains were not tolerated in several positions 9.…”