Genetically Encoding Ultra-Stable Virus-like Particles Encapsulating Functional DNA Nanostructures in Living Bacteria

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 9 . Moreover, by exposing these NCs to hostile biological conditions, such as human blood serum, we exhibit that the VLPs serves as an excellent protective shell. To the best of our knowledge, these engineered NCs pose key properties that are yet unattainable by current fabrication methods. Wild-type (WT) MS2 bacteriophage is a 27 nm particle consisting of a single of the protein and 180 copies of the coat proteins (organized as 90 homodimers) arranged into an icosahedral shell 22 . 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 genome 23,24 . Recently, it has been shown that MS2 self-assembly can be initiated in vitro in the presence of a 20 nt TR-DNA sequence analogous to the MS2 TR-RNA 25 .

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 9 . Moreover, by exposing these NCs to hostile biological conditions, such as human blood serum, we exhibit that the VLPs serves as an excellent protective shell. To the best of our knowledge, these engineered NCs pose key properties that are yet unattainable by current fabrication methods.

Main
Based on straightforward Watson-Crick base pairing, DNA nanotechnology offers exceptional control over the shape, size, geometry, and site-specific functionalization of DNA nanostructures [10][11][12][13][14][15] . DNA nanostructures can be further designed as dynamic molecular devices, also known as "DNA nanomachines" [16][17][18] . However, in addition to rapid degradation under real-world conditions, DNA nanotechnology suffers from certain limitations. For instance, the size of a sequence-specific ssDNA that can be chemically synthesized is limited to 200 nt 9, 19 . Furthermore, for certain applications such as gene therapy and drug delivery, it is necessary to retain the DNA structures' functionality, a feature that is commonly overlooked when developing DNA encapsulation and delivery technologies 20,21 .
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) (organized as 90 homodimers) arranged into an icosahedral shell 22 . 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 genome 23,24 . Recently, it has been shown that MS2 self-assembly can be initiated First, we aimed to encapsulate a DNA nanostructure in the form of a " light-up " aptamer (see methods) which can be used as a sensor. MS2 CP dimers were purified from E. coli and incubated in the presence of the ssDNA 32,33 . The ssDNA included the TR-DNA and Malachite Green "light-up" DNA aptamer 33  The success of producing active ssDNA-VLPs NCs has motivated us to produce more intricate NCs: MS2 VLPs encapsulating functional pH-dependent DNA nanotweezers 34 . The nanotweezers are responsive to a specific environmental trigger (see methods) and it was previously reported that WT MS2 maintains its stability under these conditions 35  Hence, complex DNA nanomachines such as DNA nanotweezers can be encapsulated inside MS2 VLPs, resulting in novel tweezers-VLPs NCs.
Next, the tweezers-VLPs NCs functionality was confirmed. This was done by measuring the fluorescence signal under both neutral and acidic conditions (pH 7.3 and pH 5.3, respectively, see methods) following a DNase I treatment. Under neutral pH conditions, the tweezers-VLPs NCs produced a significant fluorescence signal, whereas a low fluorescence signal was observed in the control group (unencapsulated DNA nanotweezers) (Figure 3b and c, Figures S11-S13). These results suggest that the VLPs shell was able to both maintain the nanotweezers structure-activity and to protect it from degradation. Under acidic conditions, both the tweezers-VLPs NCs and the control group showed reduced fluorescence signal. Hence, the nanotweezers maintained autonomous functionality inside the VLPs and the tweezers-VLPs NCs acted as a pH sensor.
Our next goal was to mass-produce ssDNA-VLPs NCs in vivo. The capability to synthesize any ssDNA sequence and encapsulate it inside a protective shell, such as the MS2 VLPs, holds great potential in nanotechnology for various applications.
However, a synthetic genetic system dictating the encapsulation of any ssDNA within VLPs in living cells has not yet been shown. We recently engineered a system that utilizes the human immunodeficiency virus reverse transcriptase (HIVRT) and murine leukemia reverse transcriptase (MLRT) to produce ssDNA in vivo 36,37 . This technology can specifically convert engineered non-coding RNAs (r_oligo) into a ssDNA of choice. We hypothesize that this ssDNA production technology can be re-engineered to initiate ssDNA-VLPs NCs assembly in vivo We aimed to surpass the 200-nt ssDNA production limit and the engineered r_oligo was designed at different lengths ranging from 200 to 1500-nt. The purified samples were analyzed using TEM and Cryo-TEM, where MS2 VLPs of ~30 nm diameter (similar to WT MS2) were observed only in the presence of all the system's parts (RTs, CPs, r_oligo) ( Figure 4b). The deletion of the TR-DNA sequence from the r_oligo, the absence of the CP, or the absences of the ssDNA prevented the ssDNA-VLPs NCs' assembly ( Figure S14). To evaluate the length of the encapsulated ssDNA, the ssDNA was purified directly from the ssDNA-VLPs NCs and visualized using gel electrophoresis (Figure 4c). This result suggests that the ssDNA-VLPs NCs produced in vivo can produce ssDNA, which significantly exceeds the 200-nt limit. Furthermore, the ssDNA production yield from the ssDNA-VLPs NCs was evaluated to be 27 mg ssDNA/L of bacteria and 2.5*10^11 VLPs/mL (see methods). These yields are more than 1000-fold higher than the purification yields of unencapsulated ssDNA for 1 L of bacteria 36 .
The purified ssDNA was further characterized by Sanger sequencing; as expected, sequencing results correspond to the engineered desired ssDNA sequence ( Figure 4d). The ssDNA encapsulated inside the MS2 VLPs is not limited in its sequence as long as the TR-DNA activator sequence remains in the 3' end. Linear DNA is highly prone to degradation under cellular conditions and, therefore, needs to be designed in structures or to be chemically modified in order to reduce its degradation rate. However, with our technology the VLPs' shell protects the DNA under cellular conditions and in the presence of DNase (see methods). As a result, in the described in vivo technology, the ssDNA does not require additional structural design or chemical modifications to increase its stability in vivo. Thus, the newly developed technology was able to genetically encode, produce and encapsulate long ssDNA inside MS2 VLPs in vivo.
We examined the ability of the in vivo technology to encapsulate functional DNA structures inside the MS2 VLPs. For this purpose, as a reporter element, we used the MGA "light-up" aptamer, which was successfully used in the in vitro technology.
We have incorporated the MGA sequence into the r_oligo gene together with the TR-DNA activator sequence ( Figure 5). By measuring the MGA fluorescence signal we were able to determine whether the encapsulated ssDNA maintained its structural folding properties. Aptamer-VLPs NCs were purified from cells; then, Malachite Green dye was added to the samples. As expected, a strong fluorescence signal was observed in the aptamer-VLPs NCs, whereas in the sample containing VLPs encapsulating random ssDNA sequences no fluorescence signal was observed (Figure 5a). This confirms that the precise DNA As a means to study the importance of the TR-DNA sequence for the VLPs selfassembly process, we designed four different r_oligo with 2-5-nt deletions in their TR-DNA sequence ( Figure S17). Fluorescence measurements were performed on living bacteria expressing the different variants. The high fluorescence signal was detected in the WT TR-DNA variant and a decrease in the fluorescence signal was observed in correlation with the number of nucleotides that were deleted from the TR-DNA sequence (Figure 5b). We suggest that the deletion of these nucleotides may prevent the formation of the TR-DNA bulge that contains a free adenine nucleotide, which was previously reported to be a key structural feature in the TR-RNA recognition and the self-assembly process of WT MS2 bacteriophages 23,24 .
A major goal of DNA nanotechnology is to engineer molecular tools that can operate in harsh biological environments, enabling drug delivery or diagnostic functionality. However, it is well known that free ssDNA is highly prone to degradation under various biological environments and its functionality is severely limited in cell culture medium with 10% fetal bovine serum (FBS) 6 . Moreover, exogenously delivered nucleic acids can trigger an innate immune response through the activation of toll-like receptors (TLRs) 6,38 . The novel ssDNA-VLPs NCs can serve as a new nanotechnology tool that can be applied under these natural environments and real-world conditions. As a proof of principle, free aptamer and aptamer-VLPs NCs were incubated with 100% human blood serum, which is a nuclease rich environment 7 . Aliquots from the samples were collected at different time points and fluorescence measurements were conducted (Figure 5c). These results suggest that aptamer-VLPs NCs were able to maintain the fluorescence signal with more than 60% preservation ratio following 1hr incubation with 100% human blood serum. In contrast, the free aptamer fluorescence signal rapidly decreased with less than 50% preservation ratio following 20 minutes incubation. Therefore, the aptamer-VLPs NCs were significantly more stable than the free MGA, showing a protective quality of the VLPs under real-world conditions. These results demonstrate the potential of this technology to mass-produce NCs that can serve both as functional particles under harsh biological environments and as delivery platforms of operational DNA nanostructures.

Conclusion
Inspired by the bacteriophage's reproduction mechanism and its ability to protect its nucleic acid content in endonucleases rich environments, we developed new technologies for the production of novel and functional ssDNA-VLPs NCs. The ssDNA-VLPs NCs present diverse ssDNA sizes, sequences, structures, and functionalities. Moreover, the size of ssDNA sequences that can be produced using this technology is greater than what can be chemically synthesized today (more than 7-fold). The high DNA purification yields hold great potential for the development of a cheap and smart infrastructure for the production of DNA and RNA nanotechnology. Furthermore, this technology offers the ability to generate highly durable NCs encapsulating functional DNA nanomachines, which can also serve as a targeted drug delivery platform 30,31,39,40 .
In conclusion, the reported technologies can serve as one of the missing links between in vitro experiments and real-world applications of DNA nanotechnology.

Acknowledgments
We thank Dafna Greitzer for her help with graphical design. We also thank Dr.

Naama Koifman from The Technion Center for Electron Microscopy of Soft Matter
for preforming the Cryo-TEM images. In addition, we thank the Gazit and Elbaz lab members for fruitful discussions.  DNA activator sequence (red) leading to the VLPs' self-assembly and the encapsulation of the ssDNA within the VLPs. Following their self-assembly, the ssDNA-VLPs NCs are purified from the expressing bacteria.   20 Representative sequencing analysis of the in vivo produced ssDNA. The redmarked sequence represents the TR-DNA sequence.