High-yield method to fabricate and functionalize DNA nanoparticles from the products of rolling circle amplification

DNA condensation is a facile method to construct DNA nanostructure with a high biostability and low cost, which is mainly used in DNA separation and gene transfection. The recent emerging condensed DNA nanostructures from the rolling circle amplification (RCA), i.e., the complexes between RCA products and magnesium pyrophosphate (RCA-MgPPi), have quickly become attractive biomedical materials with broad application potential because they combine the advantages of the designable and high-throughput isothermal amplification technique and the high stability of DNA condensation structures. However, we find that only approximately 10% of RCA products can be condensed after an RCA reaction, which limits the practical application of the RCA-MgPPi nanostructures. Therefore, in this paper, we investigate how to control the condensation efficiency of RCA-synthesized DNAs in depth. The very long RCA products, which show high charge densities, can be efficiently condensed by an excessive amount of Mg²⁺ to form RCA-MgPPi nanostructures at a yield approaching 100%. Additionally, the new condensation approach is general and is not limited to the RCA products, which can be applied to other polymeric DNAs. These RCA- MgPPi nanoparticles exhibit a high biostability and low toxicity, in addition, which can be efficiently functionalized with foreign components to create hierarchical properties. Finally, as a proof of concept, based on RCA-MgPPi nanostructures, a ratiometric fluorescence sensor system has been constructed and demonstrated to be an efficient lysosomal pH tracker.