Structural Implications of Homopyrimidine Base Pairs in the Parallel‐Stranded d(YGA) Motif

Abstract

DNA can adopt many other structures beyond the canonical B-form double helix. These alternative DNA structures have become increasingly significant as new biological roles are found for them. Additionally, there has been a growing interest in using non-canonical base pairs to provide structural diversity for designing DNA architectures for nanotechnology applications. We recently described the crystal structure of d(ACTCGGATGAT), which forms a tetraplex through parallel-stranded homo-base pairs and nucleobase intercalation. The homoduplex region contains a d(YGA⋅YGA) motif observed in crystal and solution structures. Here, we examine the structural implications of the homopyrimidine base pair within this motif. We determined crystal structures of two variants that differ from the original structure in the homopyrimidine base pairs and number of d(YGA) motifs. Our results show that the intercalation-locked tetraplex motif is predictable in these different sequence contexts and that substituting C⋅C base pairs for T⋅T base pairs introduces asymmetry to the homoduplex. These results have important implications for utilizing d(YGA) motifs in DNA crystal design and could provide a basis for understanding how local structures could be associated with repeat expansions.

Stability of the pH-Dependent Parallel-Stranded d(CGA) Motif

“…The CD spectrum for (CGA)6 at pH 5.5 showed a prominent positive band at 265 nm and a negative band at 245 nm, consistent with parallel-stranded homoduplex formation (Figure 2A) (26,27). A clear apparently two-state UV absorbance melting transition was observed for (CGA)6 at pH 5.5, with a concentration-dependent melting temperature (Tm) indicating a reaction molecularity greater than one ( Figure 2B), consistent with the parallel bimolecular complexes seen in crystal and solution structures containing the d(CGA) motif (25)(26)(27)(28)(29). At pH 7.0, the measured Tm is independent of concentration, indicating a unimolecular structure, and the CD spectrum is characteristic of anti-parallel strands (Figure 2A, B).…”

“…CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made repeats (n £ 4) in solution (27) or within a complex crystal structure (29,31). Additional structural studies on sequences containing (CGA)n triplets (n > 4) will be useful in providing in-depth insight into the behavior of long stretches of tandem d(CGA) repeats to further confirm the specific interactions responsible for the difference in thermodynamic stability at pH 5.5 and 7.0.…”

“…Though the d(CGA) repeat motif is capable of adopting multiple structural forms, only its parallelstranded form has been extensively structurally characterized (23,25,(28)(29)(30)(31). Originally called P-DNA, the parallel motif is stabilized by hemi-protonated C-CH + , G-G minor groove edge, and A-A Hoogsteen-face homo-base pairs ( Figure 1B) (26,31).…”

“…A crystal structure containing both d(CGA) and internal d(TGA) triplets revealed that interstrand GA stacking is identical in the d(TGA) triplet and the d(CGA) motif, but there is increased structural asymmetry and duplex bending associated with the C-CH + base pair (29). Additionally, the same cross-strand GA dinucleotide step seen in d(CGA) and d(TGA) was observed in parallel 5′-d(GGA) sequences both in solution and in the non-canonical motif of a continuously base-paired 3D DNA lattice (33,35).…”

Parallel‐stranded DNA and RNA duplexes – structural features and potential applications

“…G‐rich sequences have the capacity to adopt four‐stranded helical structures with parallel strand orientation, in the presence of monovalent cations. These structures are formed from at least two stacks of four guanosines, held together by Hoogsteen hydrogen bonds . The quadruplexes that rely on parallel‐stranded base pairing motifs can be useful tools for a wide range of targets, such as thrombin, cancer cells or pathogenic molecules .…”