I recently came across the paper by Zurkowski et al. (2025), titled "Detecting polynucleotide motifs: Pentads, hexads, and beyond.". The authors introduce LinkTetrado, a software tool that is described as "the first fully automated method for detecting polyadic motifs in the three-dimensional structures of nucleic acids." I am somewhat surprised by this claim, as I believe it overlooks the 2015 DSSR paper, which includes a dedicated section on "Higher-order coplanar base associations (multiplets)" as shown below:

DSSR defines multiplets as three or more bases associated in a coplanar geometry via a network of hydrogen-bonding interactions. Multiplets are identified through inter-connected base pairs, filtered by pair-wise stacking interactions and vertical separations to ensure overall coplanarity (Supplementary Figures S1, S3, S4 and S7). The abundant A-minor motifs (33) (types I and II, Supplementary Figures S3, S4 and S7) are base triplets, the smallest multiplet. The G-tetrad motif, where four guanines are associated via four pairs in a square planar geometry, is another special case of a multiplet.

In fact, DSSR multiplets are all-encompassing, including pentads, hexads, heptads, octads, etc.

The DSSR User Manual has extensive discussions (see Section 3.2.4 "Multiplets (higher-order coplanar base associations)") and several examples of multiplets, including:

• Figure 8: The GUA triplet auto-identified by DSSR in PDB entry 1msy.
• Figure 12: Base pentad (AUAAG) auto-identified by DSSR in PDB entry 1jj2. The five nts (A306,U325,A331,A340,G345) are all within the 23S rRNA.

DSSR can successfully identify the multiplets reported in the Zurkowski et al. paper, although there may be minor differences due to variations in cutoffs and definitions. For instance, using PDB ID 6w9p (shown in Fig. 7F of the Zurkowski et al. paper), DSSR can perform the following:

x3dna-dssr -i=6w9p.pdb -o=6w9p.out
x3dna-dssr -i=dssr-multiplets.pdb --select-model=4 -o=G4T3.pdb

The relevant portions of DSSR output (6w9p.out) are shown below:

List of 4 multiplets
   1 nts=4 GGGG A.DG4,A.DG10,A.DG16,A.DG22
   2 nts=4 GGGG A.DG5,A.DG11,A.DG17,A.DG23
   3 nts=4 GGGG A.DG7,A.DG13,A.DG19,A.DG25
   4 nts=7 GTGTGTG A.DG6,A.DT9,A.DG12,A.DT15,A.DG18,A.DT21,A.DG24

...
2 dssr-multiplets.pdb -- an ensemble of multiplets

DSSR can further render the extracted G4T3.pdb into the following image using PyMOL:

DSSR has far more to offer than meets the eye. See the DSSR User Manual and the practical guide to DSSR-PyMOL integration for more details.

References

Lu,X.-J. et al. (2015) DSSR: an integrated software tool for dissecting the spatial structure of RNA. Nucleic Acids Res, gkv716.

Zurkowski,M. et al. (2025) Detecting polynucleotide motifs: Pentads, hexads, and beyond. PLoS Comput Biol, 21, e1013633.