Folding of single-stranded DNA quadruplexes containing an autonomously stable mini-hairpin loop

The single-stranded DNA quadruplex motif TG₃-L ₁-G₃-L₂-G₃-L₃-G ₃T (where L₁, L₂ and L₃ are the three loop sequences) was used as a template for probing the effects of the loop sequences on stability and folding topology. An autonomously stable mini-hairpin sequence (ACGTAGT) was inserted into the central loop (L ₂) of different sequences with intrinsic propensities to form either parallel or anti-parallel structures. Single nucleotides (T) at positions L ₁ and L₃ strongly favour the formation of a parallel structure with the L₂ hairpin insert affecting stability in the same way as a T₇ loop. However, in the context of an anti-parallel quadruplex with T₃ loops in positions L₁ and L ₃, the mini-hairpin in the central loop forms a stable structure which enhances the T_m of the quadruplex by ∼10 °C when compared with the T₇ insert. The CD and UV melting data show that base pairing interactions within the ACGTAGT hairpin loop sequence, when accommodated as a diagonal loop in an anti-parallel structure, can enhance stability and lead to novel quadruplex structures, adding complexity to the folding landscape and expanding the potential repertoire of sequences that are able to regulate gene expression in vivo.