Structural variability of DNA-containing Mg-pyrophosphate microparticles: optimized conditions to produce particles with desired size and morphology

Abstract

Our previous studies demonstrated the formation of structurally diverse DNA-containing microparticles (DNA MPs) in PCR with Mg-pyrophosphate (MgPPi) as the structure-forming component. These DNA MPs were referred to major structural types: microdisks (2D MPs) with nanometer thickness and 3D MPs with sophisticated morphology and constructed from intersecting disks and their segments. Little is known about factors that influence both the morphology and size of DNA MPs, and the present study was aimed at fulfilling this gap. We showed that the addition of Mn cations to PCR mixtures caused the profound changes in MPs morphology, depending on DNA polymerase used (KlenTaq or Taq). Asymmetric PCR with 20-fold decrease in the concentration of one of two primers facilitated the predominant formation of microdisks with unusual structure. The addition of 1 mM Na-pyrophosphate to PCR mixtures with synthesized DNA and subsequent thermal cycling (10-15 cycles) were optimal to produce microdisks or nanometer 3D particles. Using electron microscopy, we studied also the structure of inorganic micro- and nanoparticles from MgPPi, formed during multiple heating and cooling cycles of a mixture of Mg and Na-pyrophosphate in various regimes. Also, we found the conditions to yield planar (Mg·Mn)PPi nanocrystals (diameter ~100 nm and thickness ~10 nm) which efficiently adsorbed exogenous DNA. These inorganic nanoparticles are promising for DNA delivery in transfection studies. Mechanisms to be involved in structural modifications of MPs and perspectives of their practical application are discussed.

Sustained Release of Minor-Groove-Binding Antibiotic Netropsin from Calcium-Coated Groove-Rich DNA Particles

“…Using the circular DNA template, Ф29 DNA polymerase polymerized the elongated strand of DNA. Pyrophosphate (PPi), which is produced during the polymerization process, and Mg 2+ , a cofactor of Ф29 DNA polymerase, were reacted to form magnesium pyrophosphate (Mg 2 PPi), inducing DNAp synthesis with a spherical particle shape (Figure S1) [49,50].…”