Polymerase Chain Reaction on a Viral Nanoparticle

Abstract

The field of synthetic biology includes studies that aim to develop new materials and devices from biomolecules. In recent years much work has been carried out using a range of biomolecular chassis including α-helical coiled coils, β-sheet amyloids and even viral particles. In this work we show how hybrid bionanoparticles can be produced from a viral M13 bacteriophage scaffold through conjugation to DNA primers that can template a polymerase chain reaction (PCR). This unprecedented example of a PCR on a virus particle has been studied by flow aligned linear dichroism spectroscopy, which gives information on the structure of the product as well as a new protototype methodology for DNA detection. We propose that this demonstration of PCR on the surface of a bionanoparticle is a useful addition to ways in which hybrid assemblies may be constructed using synthetic biology.

Combining bacteriophage engineering and linear dichroism spectroscopy to produce a DNA hybridisation assay

“…7b The two stage method of conjugating the precursor DNA strands to lysine residues on the pVIII protein of the M13, and their subsequent purification by size-exclusion chromatography, were carried out as described previously (see ESI † for further information). 8 In the case of the bacterial probes, the average number of DNA strands per M13 nanoparticle was calculated to be 15, giving a conjugation efficiency of 15%, almost double the value reported previously for AMPR-P1. 8 Scheme 1 Gold nanoparticle sandwich assay described by Mirkin et al 5e The left-hand side shows DNA-modified AuNPs in the absence of the DNA target.…”

“…8 In the case of the bacterial probes, the average number of DNA strands per M13 nanoparticle was calculated to be 15, giving a conjugation efficiency of 15%, almost double the value reported previously for AMPR-P1. 8 Scheme 1 Gold nanoparticle sandwich assay described by Mirkin et al 5e The left-hand side shows DNA-modified AuNPs in the absence of the DNA target. The right-hand side shows their aggregation in the presence of complementary DNA, which gives rise to a change in absorbance (SPR band) in the visible spectrum.…”

“…Previously we established the chemical method whereby DNA oligomers are attached to the pVIII protein on the surface of the M13 bacteriophage to give an M13–DNA conjugate. 8 Our new sandwich assay would involve the production of two DNA–M13 conjugates, with one complementary to the 5′ end of the target and the other complementary to the 3′ end, as shown in Scheme 2 . The hypothesis was that in the absence of target, both conjugates would be able to align in shear flow and hence produce an LD signal.…”

“…Next these strands were to be conjugated to the M13 coat protein using the methods that we established previously. 8 Then the ability of these modified bionanoparticles to bind DNA targets would be tested using fluorescence anisotropy. Finally, the scope of this new LD sensing approach would be assessed by targeting DNA from both bacterial and viral pathogens.…”

“…amino acid residues on its protein coat and nucleobases in its genome) absorb strongly when subjected to linearly polarised light. In previous M13 conjugation experiments, 7,8 we demonstrated that by attaching antibodies or DNA primers to the coat of the M13, LD sensors for bacteria ( E. coli ) 7 a or genes 8 could respectively be generated. This was a result of M13 alignment in shear flow being altered by target binding, which in turn altered the LD signal, providing a concentration dependent read-out for the presence of the analyte.…”