In addition to the well-known G-tetrad serving as the building block of G-quadruplexes (G4), other types of homogeneous or heterogeneous base-tetrads are also possible. In DSSR, all these base tetrads are generally termed multiplets where three or more bases associate in a co-planar fashion via H-bonding interactions.

In the context of G4 structures, U-tetrads are the most common. Fig. 1A shows an example of U-tetrads in PDB entry 4rne reported in the paper titled Structural Variations and Solvent Structure of r(UGGGGU) Quadruplexes Stabilized by Sr²⁺ Ions.. In the structure (Fig. 1B), two terminal U-tetrads cap the six-layered G4 structure in the middle. The four U’s in the U-tetrad are paired in parallel orientation (i.e., U+U), just as the G+G pairs in the G-tetrad of G4 structures (Fig. 1C). On the other hand, there is only one H-bond (O4…N3) in the U+U pair of the U-tetrad, in contrast to the two H-bonds in the G+G pair of the G-tetrad (Fig. 1C). In the PDB entry 4rne, DSSR also detects two octads where the middle G-tetrad is surrounded by four U’s in anti-parallel orientation (G’s filled in green vs. U’s empty, see Fig. 1C for an example).

Similarly orientated C-tetrad (C+C pair, Fig. 1D) or A-tetrad (A+A pair, Fig. 1E) are also possible. PDB entry “6a85”, associated with the paper High-resolution DNA quadruplex structure containing all the A-, G-, C-, T-tetrads., reported a high-resolution crystal structure of sequence 5'-AGAGAGATGGGTGCGTT-3' which contains all the homogeneous A-, G-, C-, T-tetrads, and the heterogeneous A:T:A:T tetrads. As of this writing (Feb. 19, 2019), the status for the PDB entry “6a85” is still “HPUB” (‘processing complete, entry on hold until publication’) even though the paper was published several months ago. Using mutate_bases in 3DNA, I generated a C-tetrad and an A-tetrad as shown in Fig. 1D and 1E. As the U-tetrad (Fig. 1A), the C- and A-tetrads also have only one H-bond in their M+N type pairs. The G-tetrad, with two H-bonds in its connecting pairs, is more stable than the other homogeneous base tetrads, leading to wide-spread G4 structures.

In the homogeneous base tetrads shown in Fig.1A-E, pairs are of the parallel M+N type and the bases are associated via their Watson-Crick and major-groove (Hoogsteen) edges. Two canonical (WC or G—U wobble) pairs can also associate via their minor-groove edges, as seen in PDB entries 2hk4 and 2lsx. Fig. 1F gives an example with two G—U wobble pairs (of anti-parallel M—N type, filled U in blue vs. empty G) in PDB entry 2lsx reported in the paper titled A minimal i-motif stabilized by minor groove G:T:G:T tetrads..

Figure 1. Non-G base tetrads automatically identified or modeled by 3DNA-DSSR. The images were created using DSSR and PyMOL.