Encapsulation of Long Genomic DNA into a Confinement of a Polyelectrolyte Microcapsule: A Single-Molecule Insight

Encapsulation of nucleic acids is an important technology in gene delivery, construction of "artificial cells", genome protection, and other fields. However, although there have been a number of protocols reported for encapsulation of short or oligomeric DNAs, encapsulation of genome-sized DNA containing hundreds of kilobase pairs is challenging because the length of such DNA is much longer compared to the size of a typical microcapsule. Here, we report a protocol for encapsulation of a ca. 60 μm contour length DNA into several micrometer-sized polyelectrolyte capsules. The encapsulation was carried out by (1) compaction of T4 DNA with multivalent cations, (2) entrapment of DNA condensates into micrometer-sized CaCO₃ beads, (3) assembly of polyelectrolyte multilayers on a bead surface, and (4) dissolution of beads resulting in DNA unfolding and release. Fluorescence microscopy was used to monitor the process of long DNA encapsulation at the level of single-DNA molecules. The differences between long and short DNA encapsulation processes and morphologies of products are discussed.