A double chain reversal loop and two diagonal loops define the architecture of a unimolecular DNA quadruplex containing a pair of stacked G(syn)center dot G(syn)center dot G(anti)center dot G(anti) tetrads flanked by a G center dot(T-T) triad and a T center dot T center dot T triple

The architecture of G(.)G(.)G(.)G tetrad-aligned DNA quadruplexes in monova lent cation solution is dependent on the directionality of the four strands , which in turn are defined by loop connectivities and the guanine syn/anti distribution along individual strands and within individual G G G G tetrad s. The smallest unimolecular G-quadruplex belongs to the d(G(2)N(n)G(2)N(2) G(2)N(n)G(2)) family, which has the potential to form two stacked G-tetrads linked by N-n loop connectivities. Previous studies have focused on the th rombin-binding DNA aptamer d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), where N-n was T- 2 for the first and third connecting loops and TGT for the middle connectin g loop. This DNA aptamer in K+ cation solution forms a unimolecular G-quadr uplex stabilized by two stacked G(syn)(.)G(anti)(.)G(syn)(.)G(anti) tetrads , adjacent strands which are antiparallel to each other and edge-wise conne cting T,, TGT and T,loops. We now report on the NMR-based solution structur e of the d(G(2)T(4)G(2)CAG(2)GT(4)G(2)T) sequence, which differs from the t hrombin-binding DNA aptamer sequence in having longer first (T-4) and third (GT(4)) loops and a shorter (CA) middle loop. This d(G(2)T(4)G(2)CAG(2)GT( 4)G(2)T) sequence in Na+ cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)(.)G(syn)(.)G(anti)(.)G(anti) tetrads, adja cent strands which have one parallel and one antiparallel neighbors and dis tinct non-edge-wise loop connectivities. Specifically, the longer first (T- 4) and third (GT(4)) loops are of the diagonal type while the shorter middl e loop is of the double chain reversal type. In addition, the pair of stack ed G(.)G(.)G(.)G tetrads are flanked on one side by a G (T-T) triad and on the other side by a (TTT)-T-.-T-. triple. The distinct differences in stran d directionalities, loop connectivities and syn/anti distribution within G( .)G(.)G(.)G tetrads between the thrombin-binding DNA aptamer d(G(2)T(2)G(2) TGTG(2)T(2)G(2)) quadruplex reported previously, and the d(G(2)T(4)G(2)CAG( 2)GT(2)G(2)T) quadruplex reported here, reinforces the polymorphic nature o f higher-order DNA architectures. Further, these two small unimolecular G-q uadruplexes, which are distinct from each other and from parallel-stranded G-quadruplexes, provide novel targets for ligand recognition. Our results d emonstrate that the double chain reversal loop connectivity identified prev iously by our laboratory within the Tetrahymena telomere d(T(2)G(4))(4) qua druplex, is a robust folding topology, since it has now also been observed within the d(G(2)T(4)G(2)CAG(2)GT(2)G(2)T) quadruplex. The identification o f a G(.)(T-T) triad and a (TTT)-T-.-T-. triple, expands on the available re cognition alignments for base triads and triples. (C) 2001 Academic Press.