Triple Helical Structures Involving Inosine:  There Is a Penalty for Promiscuity

• Published in: Biochemistry (Easton) 1996-10, Vol.35 (41), p.13338-13344
• Main Authors: Mills, Martin, Völker, Jens, Klump, Horst H
• Format: Article
• Language: English
• Subjects: Base Composition; Binding Sites; Circular Dichroism; Cytosine - chemistry; Hydrogen Bonding; Hydrogen-Ion Concentration; Inosine - chemistry; Molecular Structure; Nucleic Acid Conformation; Oligodeoxyribonucleotides - chemistry; Osmolar Concentration; Thermodynamics; Thymine - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi960193w

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.