The impact of base stacking on the conformations and electrostatics of single-stranded DNA

Abstract

Single-stranded DNA (ssDNA) is notable for its interactions with ssDNA binding proteins (SSBs) during fundamentally important biological processes including DNA repair and replication. Previous work has begun to characterize the conformational and electrostatic properties of ssDNA in association with SSBs. However, the conformational distributions of free ssDNA have been difficult to determine. To capture the vast array of ssDNA conformations in solution, we pair small angle X-ray scattering with novel ensemble fitting methods, obtaining key parameters such as the size, shape and stacking character of strands with different sequences. Complementary ion counting measurements using inductively coupled plasma atomic emission spectroscopy are employed to determine the composition of the ion atmosphere at physiological ionic strength. Applying this combined approach to poly dA and poly dT, we find that the global properties of these sequences are very similar, despite having vastly different propensities for single-stranded helical stacking. These results suggest that a relatively simple mechanism for the binding of ssDNA to non-specific SSBs may be at play, which explains the disparity in binding affinities observed for these systems.

Mechanism of sequence‐based separation of single‐stranded DNA in capillary zone electrophoresis

“…Strand T had fewer excess ions than the other five strands, which had similar values. This is consistent with results reported for dT 30 and dA 30 that were obtained from ICP‐AES, which showed that there were more excess ions associated with dA 30 [26]. In our work, strand T has the fewest excess ions and should therefore have the largest effective charge and, ignoring differences in effective mass due to intrinsic differences in base composition as well as differences in numbers of excess ions traveling with the strand, the highest µ EP .…”

“…In Tris buffer, where sequence‐based separation was not obtained in CZE [11], there were no significant differences among the R g values of the 15mers. In 0.14 M K‐PO 4 buffer, where separation was achieved, R g did show differences: R g was significantly smaller for T than for the other strands, followed by A and G, and then by the strands containing both A and G. This result is consistent with a previous report in the literature [26], in which SAXS data were analyzed using ensemble fitting methods to compare the global properties of single‐stranded 30mers (dT 30 and dA 30 ) at high salt concentrations. They found that the mean R g from transient conformation distributions was almost identical, but the transient conformation distributions showed different populations for certain conformations [26].…”

“…In 0.14 M K‐PO 4 buffer, where separation was achieved, R g did show differences: R g was significantly smaller for T than for the other strands, followed by A and G, and then by the strands containing both A and G. This result is consistent with a previous report in the literature [26], in which SAXS data were analyzed using ensemble fitting methods to compare the global properties of single‐stranded 30mers (dT 30 and dA 30 ) at high salt concentrations. They found that the mean R g from transient conformation distributions was almost identical, but the transient conformation distributions showed different populations for certain conformations [26]. Since R g represents an average, it is not an adequate representation of the conformation distribution of DNA strands, especially considering that ssDNA is very flexible.…”

“…The dA 30 distribution had more compact backbones than dT 30 , which had more elongated structures. They also showed that dA 30 has a higher degree of base stacking, which could explain the difference in lengths of the structures, and therefore the higher mobility of polyA [26]. The high ionic strength of the buffer used in the present work might further increase the difference in base stacking strength between purines and pyrimidines and increase the differences in mobility.…”

“…It is well recognized that purines (A, G) exhibit stronger base stacking than pyrimidines (T, C) [26], and that increasing the purine content increases the compactness of average solution structures of ssDNA [29‐31]. As noted above, a SAXS study of the R g of dT 30 and dA 30 showed that the transient conformation distributions were different for the two strands [26]. The dA 30 distribution had more compact backbones than dT 30 , which had more elongated structures.…”