Self-Assembly of Rolling Circle Amplification DNA into Nano-particles using Mg2+ and ppi
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Rolling circle amplification (RCA) is an effective isothermal amplification technique that can generate long singlestrandedDNA (ssDNA) with a high yield and sequence programmability. In this study, we studied the production of the DNAnanoparticles by self-assembly RCA with ionic condensation. The padlock DNA was converted into a dumbbell-shaped circularDNA using the mediate RNA and SplintR ligase, followed by amplification with phi29 DNA polymerase at 30°C. The resultingssDNA, containing repetitive sequences, was condensed into well-defined DNA nanoparticles through the addition of magnesium(Mg2+ ) and pyrophosphate (ppi) ions. Morphologies and surface electrostatic potentials were observed using optical microscopy,atomic force microscopy (AFM), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and surfacepotential mapping via Kelvin probe force microscopy (KPFM) and electrostatic force microscopy (EFM). Molecular dynamics (MD)simulations were used to investigate a hierarchical self-assembly pathway involving initialMg2+and ppi cluster formation, DNA–Mg2+coordination, and subsequent DNA incorporation. These findings provide mechanistic insight into RCA-derived DNAnanoparticle formation and highlight the critical role of ionic interactions in directing nanoscale self-assembly. This paper providesinsights into the padlock DNA design and scalable production of functional DNA based nanomaterials for applications in drug delivery,biosensing, and nanotechnology.
Abstract
Rolling circle amplification (RCA) is an effective isothermal amplification technique that can generate long singlestrandedDNA (ssDNA) with a high yield and sequence programmability. In this study, we studied the production of the DNAnanoparticles by self-assembly RCA with ionic condensation. The padlock DNA was converted into a dumbbell-shaped circularDNA using the mediate RNA and SplintR ligase, followed by amplification with phi29 DNA polymerase at 30°C. The resultingssDNA, containing repetitive sequences, was condensed into well-defined DNA nanoparticles through the addition of magnesium(Mg2+ ) and pyrophosphate (ppi) ions. Morphologies and surface electrostatic potentials were observed using optical microscopy,atomic force microscopy (AFM), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and surfacepotential mapping via Kelvin probe force microscopy (KPFM) and electrostatic force microscopy (EFM). Molecular dynamics (MD)simulations were used to investigate a hierarchical self-assembly pathway involving initialMg2+and ppi cluster formation, DNA–Mg2+coordination, and subsequent DNA incorporation. These findings provide mechanistic insight into RCA-derived DNAnanoparticle formation and highlight the critical role of ionic interactions in directing nanoscale self-assembly. This paper providesinsights into the padlock DNA design and scalable production of functional DNA based nanomaterials for applications in drug delivery,biosensing, and nanotechnology.