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, Ln this paper we present a series of oligonucleotides with a 5'pur(9)-pyr(9)-pyr(9) 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 tripler, the inosine-containing third strand is incorporated in parallel orientation to the purine strand of the duplex. The influence of inosine on the tripler-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) degrees C, and replacing a single protonated cytosine drops the T-m by 32.1 (+/-1.0) degrees 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) degrees C; on introducing two inosines in place of cytosines, the T-m drops by 44.5 (+/-1.0) degrees 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) degrees 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.