Use of surface plasmon resonance imaging to study viral RNA: protein interactions.

Surface plasmon resonance imaging (SPRi) is an emerging microarray technology that is label-free, rapid and extremely flexible. Here the capabilities of SPRi are demonstrated in results of proof-of-concept experiments detailing a method for studying viral genomic RNA:protein interactions in array format. The principal RNA is the well-characterized origin of assembly (OAS) containing region of Tobacco mosaic virus (TMV) RNA, whereas the principal protein is the primary subunit for TMV virion assembly, the 20S capsid protein aggregate. DNA probes complementary to TMV and non-TMV RNA fragments were covalently attached to a thin gold layer deposited on glass. These DNA probes were used to discreetly capture in vitro transcribed TMV and Red clover necrotic mosaic virus (RCNMV) RNA2 (used as a negative control for the subsequent protein binding). The 4S TMV capsid protein monomers were isolated from TMV particles purified from infected plants of Nicotiana tabacum L. and were induced to form 20S stacked disc aggregates. These 20S stacked disc aggregates were then injected onto the array containing the RNA fragments captured by the DNA probes immobilized on the microarray surface. The discrete and preferential binding of the 20S stacked disc aggregates to the array locations containing the TMV OAS RNA sequence was observed. The results demonstrate that SPRi can be used to monitor binding of large RNA molecules to immobilized DNA capture probes which can then be used to monitor the subsequent binding of complex protein structures to the RNA molecules in a single real-time, label-free microarray experiment. The results further demonstrate that SPRi can distinguish between RNA species that have or do not have an origin of assembly sequence specific for a particular viral capsid protein or protein complex. The broader implications of these results in virology research are found in other systems where the research goals include characterizing the specificity and kinetics of viral or host protein or protein complex interactions with viral nucleic acids.