Thermal stabilisation of the short DNA duplexes by acridine-4-carboxamide derivatives

The short oligodeoxynucleotide (ODN) probes are suitable for good discrimination of point mutations, however, they suffer from low melting temperatures. In this work, the strategy of using acridine-4-carboxamide intercalators to improve thermal stabilization is investigated. The study of large series of acridines revealed that optimal stabilization is achieved upon decoration of acridine by secondary carboxamide carrying sterically not demanding basic function bound through two-carbon linker. Presence of secondary carboxamides plays a key role in stabilization of the duplex. Two highly active intercalators were attached to short probes (13 or 18 bases; designed as a part of HFE gene) by click chemistry into positions 7 and/or 13 and proved to increase the melting temperate (T_m) of the duplex by almost 8°C for the best combination. The acridines interact with both single- and double-stranded DNAs with substantially preferred interaction for the latter. The study of interaction suggested higher affinity of the acridines toward the GC- than AT-rich sequences. Good discrimination of two types of point mutations was shown.