We study the energy contributions for the formation and targeting of unusual DNA structures. A combination of spectroscopic and calorimetric techniques was used to investigate: (1) the molecular forces controlling the stability of DNA triplexes and complexes containing triplex-duplex motifs; (2) the stability of a variety of DNA structures with single 2-aminopurine substitutions; (3) the energetic contributions for the association of triplex, hairpins and other unusual structures, with their complementary strands; and (4) the interaction of DNA with poly(ethylene glycol)-b-poly-L-lysine (PEG-PLL) for cellular delivery purposes.

In summary: (a) Triplex stability is dependent on their molecularity, and follows the order: monomolecular > bimolecular > trimolecular. DNA complexes with joined triplex-duplex motifs have a similar trend, i.e., the intramolecular complex is the more stable one. (b) Both loop length and loop sequence affect the stability of an intramolecular triplex. Intramolecular triplexes with 5 bases in the loop are the most stable; however, smaller or larger loops yielded triplexes with lower stability. The helical stem of triplexes can be further stabilized by replacing the less base-stacked pyrimidine loop with a well-stacked ^5'−GCAA sequence. (c) 2-aminopurine incorporations are an efficient method to investigate the unfolding of DNA structures because its fluorescence emission is sensitive to the local environment that it is experiencing. (d) Each single-strand used in the targeting experiments is able to invade and disrupt the corresponding intramolecular DNA complex. The favorable free energy terms of these targeting reactions are enthalpy driven, resulting from a compensation of exothermic contributions, due to formation of additional base-pair stacks or base-triplet stacks in the product, and the immobilization or removal of water and endothermic contributions from disruption of base-base stacking interactions of the reactant single-strands. (e) The binding affinity of PEG-PLL with DNA molecules depends on the length of the poly-lysine chain and DNA.

These results will help to improve (a) the rational design of gene-targeting reagents; (b) strategies to target specific secondary structures, such as the ones found in mRNA; and (c) identification of appropriate polycations for the proper cellular delivery.