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

Anatoly Zinchenko,Eisuke Inagaki,Shizuaki Murata

doi:10.1021/acsomega.8b02865

Anatoly Zinchenko, Eisuke Inagaki + Show 1 more

Open Access

https://doi.org/10.1021/acsomega.8b02865

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Journal: ACS omega	Publication Date: Jan 7, 2019
Citations: 2	License type: cc-by

Affiliation: Nagoya University

Abstract

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.

Highlights

Encapsulation of nucleic acids is an important research area from both fundamental and applied points of view
CaCO3 beads are dissolved by ethylendiaminetetraacetate dihydrate (EDTA) to release the DNA into the interior of a capsule (Figure 2D)
Conformational behavior of single-sub-megabase long DNA molecules can be monitored by fluorescence microscopy (FM)

Summary

■ INTRODUCTION

Encapsulation of nucleic acids is an important research area from both fundamental and applied points of view. In vitro systems containing submegabase-sized DNA macromolecules inside a micrometersized confinement are highly relevant to the state of DNA in living cells, in which several meters long DNA is confined inside a tiny nucleus of several micrometers size. Such systems can be utilized for a better understanding of DNA structure and behavior in vivo.[15] A number of “artificial cell” models containing DNA in a microconfinement were elaborated on the basis of water-in-oil[16−18] or water-in-water microdroplets,[19] liposomes,[20] and giant vesicles[21−24] (Figure 1) to Figure 1. By comparing encapsulation of long bacteriophage DNA and short salmon sperm DNA (ca. 300 bp), we make clear a difference in the partitioning of long and short DNAs inside polyelectrolyte microcapsules

■ RESULTS AND DISCUSSION

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES