Detecting short RNA strands with high fidelity at any of the bases of their sequence, including the termini, can be challenging, since fraying, wobbling, and refolding all compete with canonical base pairing. We performed a search for 5'-substituents of oligodeoxynucleotides that increase base pairing fidelity at the terminus of duplexes with RNA target strands. From a total of over 70 caps, differing in stacking moiety and linker, a phosphodiester-linked sequence of the residues of L-prolinol, glycine, and oxolinic acid, dubbed ogOA, was identified as a 5'-cap that stabilizes any of the four canonical base pairs, with ΔT(m) values of up to +13.1 °C for an octamer. At the same time, the cap increases discrimination against any of the 12 possible terminal mismatches, including mismatches that are more stable than their perfectly matched counterparts in the control duplex, such as A:A. A probe with the cap also showed increased selectivity in the detection of two closely related microRNAs, let7c and let7a, with a ΔT(m) value of 9.2 °C. Melting curves also yielded thermodynamic data that shed light on the uniformity of molecular recognition in the sequence space of DNA:DNA and DNA:RNA duplexes. Hybridization probes with fidelity-enhancing caps should find applications in the individual and parallel detection of biologically active RNA species.
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.
Protocols for the synthesis of oligodeoxynucleotides with a short peptidyl substituent linked to the 5'-O-terminus through a phosphodiester bond are presented. The example given is a peptidyl cap consisting of the residues of L-prolinol, glycine, and the acyl residue of oxolinic acid. DNA probes with this cap, also known as ogOA cap, give melting point increases for duplexes with RNA targets and improve mismatch discrimination at the terminus. The cap is either introduced in one step, using a newly developed phosphoramidite reagent, or assembled on the DNA chain. The step-wise assembly of the peptidyl chain is advantageous for combinatorial studies aimed at the optimization of a cap structure. The block coupling method, introducing the preassembled cap in one step, is attractive for routine use of a cap already optimized for a given application. Cap-bearing probes can increase fidelity of hybridization in a genomic context. They can be synthesized by automated DNA synthesis.
To cap or… …not to cap? In their Full Paper on page 11813 ff., S. Egetenmeyer and C. Richert describe a covalently attached cap for the 5'-terminus of oligonucleotides that increases base pairing fidelity by binding tightly to the helix when a canonical base pair is formed, but providing little stabilization in the case of a mismatch. The resulting enhancement in affinity and selectivity aids the detection of microRNAs.
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