A novel method to produce armored double-stranded DNA by encapsulation of MS2 viral capsids

Abstract

With the rapid development of molecular diagnostic techniques, there is a growing need for quality controls and standards with favorable properties to monitor the entire detection process. In this study, we describe a novel method to produce armored hepatitis B virus (HBV) and human papillomavirus (HPV) DNA for use in nucleic acid tests, which was confirmed to be stable, homogeneous, noninfectious, nuclease resistant, and safe for shipping. We demonstrated that MS2 bacteriophage could successfully package double-stranded DNA of 1.3-, 3-, 3.5-, and 6.5-kb length into viral capsids with high reassembly efficiency. This is the first application of RNA bacteriophage MS2 as a platform to encapsulate double-stranded DNA, forming virus-like particles (VLPs) which were indistinguishable from native MS2 capsids in size and morphology. Moreover, by analyzing the interaction mechanism of pac site and the MS2 coat protein (CP), we found that in addition to the recognized initiation signal TR-RNA, TR-DNA can also trigger spontaneous reassembly of CP dimers, providing a more convenient and feasible method of assembly. In conclusion, this straightforward and reliable manufacturing approach makes armored DNA an ideal control and standard for use in clinical laboratory tests and diagnostics, possessing prospects for broad application, especially providing a new platform for the production of quality controls for DNA viruses.

Design of virus-based nanomaterials for medicine, biotechnology, and energy

“…More recently, a return to the Armored RNA roots was taken with the demonstration of encapsulation of dsDNA in MS2, which resulted in extra stability over lambda as was found for fd phage but without being limited to ssDNA. 421 By conjugating sulfhydryl-modified DNA sequences of interest to an amine-modified stem-loop DNA structure, assembly of dissociated MS2 CPs could then be triggered around the stem-loops to form Armored DNA. Using this strategy, the formation of MS2 capsids packaging HBV and HPV DNA sequences with lengths ranging from 1.3 to 6.5 kb was accomplished, which is astonishing given that the genome of MS2 is only 3.5 kb.…”

External quality assessment for the molecular detection of MERS-CoV in China

“…JX869059.2) were synthesized separately: the first was 394 bp, consisting of part of the upE gene, and the other was 1629 bp, which encompassed part of the ORF 1a, ORF 1b, N, and RdRp genes. The chimeric sequences were amplified (primers listed in Table 1), gel-purified, and separately subcloned into Kpn I/Pvu I and Kpn I/Pac I sites of the pACYC-MS2 vector [14,15] to form the recombinant plasmids, pACYC-MS2-upE and pACYC-MS2-ORF 1ab/N, respectively. These two kinds of MS2 virus-like particles (VLPs) packaging specific sequences of MERS-CoV were expressed and purified according to previously published protocols of our laboratory [14][15][16].…”

“…The chimeric sequences were amplified (primers listed in Table 1), gel-purified, and separately subcloned into Kpn I/Pvu I and Kpn I/Pac I sites of the pACYC-MS2 vector [14,15] to form the recombinant plasmids, pACYC-MS2-upE and pACYC-MS2-ORF 1ab/N, respectively. These two kinds of MS2 virus-like particles (VLPs) packaging specific sequences of MERS-CoV were expressed and purified according to previously published protocols of our laboratory [14][15][16]. Then, they were identified by transmission electron microscopy (TEM), enzymatic digestion test, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and RT-PCR.…”

High molecular weight DNA assembly in vivo for synthetic biology applications

“…Bacteriophages can be also used to mobilize large pre-assembled and integrated DNA fragments. Flanking DNA fragments with a phage packaging recognition signal (pac site) [97][98][99] may speed up the building of large genomes. Recently developed or improved systems for manipulation of high molecular weight DNA, such as BGM vector or iREX described above have many advantages over the traditional tools, such as BACs and YACs.…”