Triple helical structures involving inosine: There is a penalty for promiscuity

Inosine has the ability to act as a 'wild-card' binding nonspecifically to both A-T and G-C base pairs. This has obvious implications for the design of oligonucleotide site-directed probes. In this paper we present a series of oligonucleotides with a 5'pur₉-pyr₉-pyr₉ motif which are designed to fold up sequentially into intramolecular triple helices. One or more inosines are incorporated into the Hoogsteen strands in place of T's and/or C's. Once folded into the triplex, the inosine-containing third strand is incorporated in parallel orientation to the purine strand of the duplex. The influence of inosine on the triplex-duplex equilibrium, characterized by the melting temperature (T(m)), and on the phase boundaries, as a function of pH and/or ionic strength, has been assessed by means of UV and CD spectroscopy. There are two distinguishable influences of third-strand inosines which affect binding, namely, backbone distortion due to bulkiness (I for T and I for C⁺) and/or loss of intramolecular ion pairs between protonated cytosines and the backbone phosphates (I for C⁺). A single thymine replacement drops the T(m) by 25.0 (±2.1) °C, and replacing a single protonated cytosine drops the T(m) by 32.1 (± 1.0) °C at pH 6.0. On introducing two inosines in place of thymines, the T(m) at pH 6.0 of the triple helix to hairpin transition is lowered by 35.5 (±1.4) °C; on introducing two inosines in place of cytosines, the T(m) drops by 44.5 (±1.0) °C, and on replacing a cytosine and a neighboring thymine with inosines, the T(m) of the same transition is lowered by 29.2 (±1.6) °C. Replacing more than two thymines or cytosines, respectively, eliminates the binding of the Hoogsteen strand at room temperature altogether. Under no circumstances does inosine replacement stabilize the triple helix: it is a poor substitute and its role as a wild- card is limited.