Does Cobalt Hexamine induce DNA-DNA Attraction? Investigations to Understand Nucleic Acid Compaction in the Cell

Abstract

Nucleic acid compaction is a fundamental process in all living systems, exemplified by the tight packaging of DNA into chromatin in eukaryotic nuclei, as well as nucleic acid compaction in the viral capsid. The strong negative charge on the DNA backbone must be mitigated by positively charged species, including polyvalent ions and proteins, to achieve the necessary close packing. To dissect the electrostatic interactions driving DNA compaction, the xHEED (X-linking of Helices to measure Electrostatic Effects at Distance) method, previously developed in the Russell Lab, was used to measure distant-dependent interhelical encounter frequencies, and the atomic forces that mitigate these mechanisms as previous poisson-boltzmann models did a poor job of it. Rate constants were measured that reflect the equilibrium constant for transient DNA-DNA encounters under varying ionic conditions. Using the xHEED method, DNA-DNA crosslinking interactions were compared to DNA-Aldrithiol, a neutral molecule, to understand the effect of positive ions mitigating negative charge repulsion from the DNA Backbones. Previous work indicated that the maximal DNA-Aldrithiol neutral rate constant was 1.3 x 104 M-1s-1. This work indicated that crosslinking at moderate millimolar concentrations of Co(NH₃)₆³⁺ not only fully screen interhelical repulsion but also induce net attractions at higher concentrations, eventually reaching a plateau at 3.5 x 104 M-1s-1, faster than the neutral reaction, where further increases in Co(NH₃)₆³⁺ concentration have no additional effect. However, new work conducted in the lab indicates that the neutral rate constant at lower concentrations of Co(NH₃)₆³⁺ is lower than the maximal rate constant, indicating that there is still net attraction induced by Co(NH₃)₆³⁺ at lower concentrations, removing support for the net-neutral screening regime. Future side-by-side comparisons between cross-linked DNA and aldrithiol-mediated reactions at the same concentration of Co(NH₃)₆³⁺ are needed to fully clarify the impact of Co(NH₃)₆³⁺ on electrostatic interactions and the potential existence of an attractive regime. Measuring DNA-DNA crosslinking and comparing it to DNA-Aldrithiol at lower concentrations of Co(NH₃)₆³⁺ will clarify if Co(NH₃)₆³⁺ can induce attraction at lower concentrations of trivalent ions. This will refine understanding of in vivo DNA Compaction and provide further understanding on the role of multivalent compounds that naturally exist in the nucleus, such as spermine and spermidine.Biochemistr