title: Viral nanoparticle-encapsidated enzyme and restructured DNA for cell delivery and gene expression.
abstract: Packaging specific exogenous active proteins and DNAs together within a single viral-nanocontainer is challenging. The bacteriophage T4 capsid (100 × 70 nm) is well suited for this purpose, because it can hold a single long DNA or multiple short pieces of DNA up to 170 kb packed together with more than 1,000 protein molecules. Any linear DNA can be packaged in vitro into purified procapsids. The capsid-targeting sequence (CTS) directs virtually any protein into the procapsid. Procapsids are assembled with specific CTS-directed exogenous proteins that are encapsidated before the DNA. The capsid also can display on its surface high-affinity eukaryotic cell-binding peptides or proteins that are in fusion with small outer capsid and head outer capsid surface-decoration proteins that can be added in vivo or in vitro. In this study, we demonstrate that the site-specific recombinase cyclic recombination (Cre) targeted into the procapsid is enzymatically active within the procapsid and recircularizes linear plasmid DNA containing two terminal loxP recognition sites when packaged in vitro. mCherry expression driven by a cytomegalovirus promoter in the capsid containing Cre-circularized DNA is enhanced over linear DNA, as shown in recipient eukaryotic cells. The efficient and specific packaging into capsids and the unpackaging of both DNA and protein with release of the enzymatically altered protein-DNA complexes from the nanoparticles into cells have potential in numerous downstream drug and gene therapeutic applications.
authors.affiliation: Center for Bio/Molecular Science and Engineering, US Naval Research Laboratory, Washington, DC 20375; jinny.liu@nrl.navy.mil lblack@umaryland.edu.
authors.forename: Jinny L
authors.initials: JL
authors.lastname: Liu
authors.affiliation: Department of Biochemistry, School of Medicine, University of Maryland at Baltimore, Baltimore, MD 21201;
authors.forename: Aparna Banerjee
authors.initials: AB
authors.lastname: Dixit
authors.affiliation: Engility Corporation, Alexandria, VA 22314; and.
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Robertson
authors.affiliation: Science and Engineering Apprenticeship Program, US Naval Research Laboratory, Washington, DC 20375.
authors.forename: Eric
authors.initials: E
authors.lastname: Qiao
authors.affiliation: Department of Biochemistry, School of Medicine, University of Maryland at Baltimore, Baltimore, MD 21201; jinny.liu@nrl.navy.mil lblack@umaryland.edu.
authors.forename: Lindsay W
authors.initials: LW
authors.lastname: Black
mesh_terms: D001287:Attachment Sites, Microbiological; D017122:Bacteriophage T4 / Q000737:chemistry*; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry*; D016923:Cell Death; D045744:Cell Line, Tumor; D002470:Cell Survival; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D042003:DNA Packaging; D004270:DNA, Circular / Q000378:metabolism; D005434:Flow Cytometry; D005453:Fluorescence; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D008969:Molecular Sequence Data; D053758:Nanoparticles / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D013194:Staining and Labeling; D014170:Transformation, Genetic
chemical_list: D004270:DNA, Circular; D004247:DNA; C045073:Cre recombinase; D019426:Integrases
keywords: DNA packaging; Hoc; Soc; capsid decoration proteins; terminase