Abstract
DNA nanotechnology is leading the field of in vitro molecular-scale device engineering, accumulating to a dazzling array of applications. However, while DNA nanostructures' function is robust under in vitro settings, their implementation in real-world conditions requires overcoming their rapid degradation and subsequent loss of function. Viruses are sophisticated supramolecular assemblies, able to protect their nucleic acid content in inhospitable biological environments. Inspired by this natural ability, we engineered in vitro and in vivo technologies, enabling the encapsulation and protection of functional DNA nanostructures inside MS2 bacteriophage virus-like particles (VLPs). We demonstrate the ssDNA-VLPs nanocomposites' (NCs) abilities to encapsulate single-stranded-DNA (ssDNA) in a variety of sizes (200-1500 nucleotides (nt)), sequences, and structures while retaining their functionality. Moreover, by exposing these NCs to hostile biological conditions, such as human blood serum, we exhibit that the VLPs serve as an excellent protective shell. These engineered NCs pose critical properties that are yet unattainable by current fabrication methods.
Highlights
Encoding Ultra-Stable Virus-like Particles Encapsulating Functional DNA Nanostructures in Living Bacteria Shai Zilberzwige-Tal1,4, Dan Mark Alon1,4, Danielle Gazit1, Shahar Zachariah1, Amit Hollander1, Ehud Gazit1,2,3 and Johann Elbaz1*
Wild-type (WT) MS2 bacteriophage is a 27 nm particle consisting of a single copy of the maturation protein and 180 copies of the coat proteins (CP) arranged into an icosahedral shell22
The bacteriophage's assembly is driven by a specific 20 nt RNA sequence forming a stem-loop structure called “translational repression RNA” (TR-RNA), which is encoded in the MS2 genome23,24
Summary
Encoding Ultra-Stable Virus-like Particles Encapsulating Functional DNA Nanostructures in Living Bacteria Shai Zilberzwige-Tal, Dan Mark Alon, Danielle Gazit, Shahar Zachariah, Amit Hollander, Ehud Gazit and Johann Elbaz1*. Viruses are incredibly sophisticated supramolecular assemblies, able to protect their nucleic acid content in the relatively inhospitable biological environment8 Inspired by this natural ability, we engineered both in vitro and in vivo technologies, enabling the encapsulation and protection of functional DNA nanostructures inside MS2 bacteriophage virus-like particles (VLPs). We demonstrate the ssDNA-VLPs nanocomposites (NCs) abilities to encapsulate single-stranded-DNA (ssDNA) of an unprecedented variety of sizes (200–1500 nucleotides (nt)), sequences, and structures while retaining their functionality. Utilizing viruses for shielding functional DNA structures under complex conditions has not been demonstrated yet We report both in vitro and in vivo technologies for the production of functional ssDNA-VLPs NCs that meet the requirements of ssDNA protection and functionality under real-world conditions, whereas the MS2 VLPs are the shield and the functional DNA is the cargo. In the in vivo technology, we genetically encoded the integration of ssDNA production with the expression of MS2 CP in vivo, resulting in the self-assembly of novel functional ssDNA-VLPs NCs within living bacteria (Figure 1b)
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