New Approaches for Separation of DNAs with Different Structures by Selective Sedimentation Using Nanoparticles and Metal Ions
Malla, Shubha Rajya Laxmi
Circular DNAs have been widely used for gene therapy, CRISPR therapy, RNA interference therapy and DNA vaccines. Currently, there is no inexpensive, scalable way to separate small circular DNAs from large linear chromosomal DNAs. In this study we have investigated new approaches for the separation and purification of nucleic acids with different structures. Our particular focus was on improving separation of circular DNAs from linear DNAs. The analysis of selective binding of nucleic acids to nanoclays (halloysite, kaolinite, and montmorillonite) identified experimental conditions that allowed one form of DNA to be preferentially bound to the clays. For example, circular plasmid DNA showed better binding to halloysite (HNT) nanotubes as compared to linear DNA. The importance of metal ions on DNA-clay interactions was examined in detail. Kaolinite demonstrated weak binding to all forms of DNA, much lower than that seen with HNT nanotubes. Montmorillonite showed strikingly higher binding affinity for linear DNA than circular plasmid DNA in the absence of metal ions. Divalent metals were shown to induce DNA sedimentation when the pH was raised above 11.0. This was not the case for monovalent metals. Several experiments indicated that DNA is partially denatured at pH 12.0 and forms single-stranded DNAs that aggregate and cause sedimentation of DNA. Although metals form insoluble hydroxides at high pH, the results demonstrated that there was no correlation between the efficiency of DNA sedimentation and the amount of formation of metal hydroxides in solution. The results of many experiments performed for this project strongly supported a model involving charge neutralization and cation bridging by the metals to aggregate and sediment DNA at high pH. Heating DNAs to 95° C and cooling in the presence of divalent metal ions produced strong sedimentation with divalent metals but not monovalent metals. Other experiments revealed that Ca2+ ions were the most effective among divalent metals tested at DNA sedimentation. Chemical denaturants such as formamide and DMSO also demonstrated an ability to induce DNA sedimentation. Several experiments indicated that there are differences in the process of denaturation and renaturation between linear and circular DNAs because of differences in their structures. Linear DNAs denature completely into separated single strands when heated at 95° C, whereas circular DNAs denatured but could not separate. The strands remained interlocked and could quickly snap back to re-form double-stranded DNA by base-pairing upon cooling. Separated complementary linear ssDNAs formed aggregated complexes upon cooling in the presence of divalent metals, whereas circular plasmids could not. Because of the differences in the denaturation and renaturation of linear and circular DNA, we were able to selectively remove linear DNAs from circular DNAs using metals under appropriate conditions. The new methods developed in this project using nanoparticles and metals to purify circular DNA can be a beneficial tool in gene therapy and other molecular biology and biomedical techniques to improve understanding and treatment of cancer and genetic diseases.
nanoparticles, nanoclays, DNA sedimentation, divalent metals, high pH
Malla, S. R. L. (2021). New approaches for separation of DNAs with different structures by selective sedimentation using nanoparticles and metal ions (Unpublished dissertation). Texas State University, San Marcos, Texas.