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  • In this paper we describe a


    In this paper, we describe a facile and universal method for in situ crosslinking of DNA micelles, using spherically directed reduction of metal ions. As shown in Scheme 1A, a single-stranded DNA (ssDNA) consisting of a ligand domain, a template domain, and a lipid domain was designed as a monomer to form the DNA micelle. Some reported specific DNA sequences were incorporated here as a template domain to selectively adsorb different kinds of metal ions (poly C for Ag,12, 13, 14 poly A for Au,15, 16 and poly T for Cu). In the presence of a reducing agent, metal ions enriched around the template domain will be reduced to zero-valent metal under very mild conditions at room temperature and finally crosslinked into a hollow (Cu) or solid (Ag and Au) metal core, resulting from the dense packing of DNA ligands in the micelle. The size of the metal core can be tuned by simply changing the length of the template domain, whereas the ligand remains on the surface of the metal core as a DNA corona because only the template domain will induce the enrichment of metal ions. The detailed structural profile of DNA micelle was further determined for the first time after a series of calculations and simulations, and the results matched very well with the subsequent experiments. The metal-crosslinked DNA micelle, or MDM, is an interesting DNA-micelle/DNA-nanoparticle conjugate that combines the facile preparation and dense ligand distribution of a DNA micelle with the physical properties of the widely used spherical nucleic acids (SNAs) reported by Mirkin and coworkers. Compared with the typical SNA preparation approach, which involves synthesizing gold nanoparticles (AuNP) in boiling solution and modification of DNA ligand using the well-established Au-S chemistry, our spherically directed strategy is simply a one-pot reaction at room temperature and much more universal for different kinds of metals. In addition, the extremely low CMC of DNA Biocytin enables highly efficient micelle formation in solution and, together with the templated-reduction procedure, permits much higher utilization efficiency of DNA strands than the regular DNA-particle conjugation method. Because of the metal crosslink, the internalization efficiency of MDM is much greater than that of DNA micelles. On the basis of all the above properties, we further prepared a metal-crosslinked DNA micelle flare (MDMF) for intracellular ATP imaging by adapting an aptamer-toehold-based strategy. Benefitting from the excellent biocompatibility of MDMs, MDMFs showed negligible cytotoxicity and effective self-delivery as an intracellular sensing platform, demonstrating the huge potential of MDMs in biological applications. Apart from lipid-based ssDNA micelles, by using double-stranded DNA (dsDNA) with two cholesterol modifications to form a micelle, we prove that these dsDNA micelles can also be used to form MDMs by designing a specific dsDNA template domain, which is an extension of our strategy and strong evidence of the flexibility of our method.
    Discussion In summary, using a spherically directed synthesis of metal-crosslinked DNA micelles, as well as a series of simulation algorithms, we succeeded in acquiring a detailed structural profile of a DNA micelle. The programmability of DNA strands and the selectivity of the DNA template permitted the effective crosslinking of DNA micelles by three kinds of metal ions, including copper, silver, and gold, generating a hollow or solid core structure. This strategy gives a facile approach for crosslinking DNA micelles, as well as a universal and time-saving method for preparing DNA-metal nanoparticle conjugates. The size of the metal core can be controlled by designing the length or sequence of the template domain, which makes it a flexible parameter in programmable synthesis. Compared with the typical SNA preparation approach, which consists of synthesizing AuNPs in boiling solution and modification of DNA ligand via Au-S chemistry, our spherically directed strategy is simply a one-pot reaction at room temperature and is much more universal for different kinds of metals. Unlike DNA micelles, which fuse with the cell membrane, MDMs were found to have good cellular internalization. Furthermore, by integrating an aptamer-toehold-mediated ATP detection strategy, MDMFs were prepared for effective intracellular imaging, which demonstrated the feasibility of MDMs in biological applications. In addition, this is the first report of micelle formation by cholesterol-modified dsDNA instead of the usual single-stranded cholesterol-DNA. These results not only extend the preparation methods of metal-crosslinked DNA micelles but also inspire numerous potential DNA micelle applications.