A template-based approach was used to design unprecedented architectural motifs into a knownDNA framework. The structure formed by the sequence d(GCGGTTGGAT) in 0.1 M Na+ solution hasbeen determined using molecular dynamics simulations constrained by distance and dihedral restraintsderived from NMR experiments. The molecular topology has been previously observed for the sequenced(GCGGTGGAT) (Webba da Silva, M. (2003) Biochemistry 42, 14356-65). Insertion of a single thymineinto the double chain reversal formed by the segment GGTGG results in the unprecedented experimentaldemonstration of a T:(G:G:G:G):T hexad. The bi-stranded hexad results from the pairing alignment oftwo G(T-G) triads. Each triad results from recognition of the sheared edge of a guanine by the Watson-Crick edge of a thymine of the segment GGTTGG. The alignment is stabilized by base-stacking of thethymine to the sugar pucker of the preceding thymine. The latter is involved in formation of the T:A:A:Ttetrad alignment by forming a hydrogen bond with the free amino proton of a Watson-Crick alignedA:A mispair. We have thus established that residues in double chain reversal loops linking juxtaposedtetrads of a quadruplex stem may facilitate formation of yet unknown hydrogen bond alignments. Byemploying a systematic approach analysis of sequence motifs appearing in double chain reversals, bridgingtetrad layers should allow for the prediction of topologies and architectural motifs appearing in biologicallyrelevant genomic regions.