A Molecular Beacon of Hope

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Victor U. Weiss and Günter Allmaier from TU Vien discuss how combining molecular beacons with chip capillary electrophoresis (CE) offers scope to investigate viral RNA replication and improve gene therapy delivery systems.

Interview by Lewis Botcherby

Q. The use of molecular beacons and chip capillary electrophoresis (CE) is a very interesting methodology. Could you explain in the simplest terms how it works?

Günter Allmaier: During chip capillary zone electrophoresis (CZE), analytes are separated according to their specific migration velocity in an electric field applied to an electrolyte solution. Analyte detection on the instrument used is based on laser-induced fluorescence. The analyte of our study (that is, the viral RNA) does not exhibit an intrinsic fluorescence, it is only detectable if it is tagged by a fluorescent molecule or marker. It is for this particular reason that molecular beacons get involved. They are oligonucleotide probes modified by a fluorophore and a fluorescence quencher at their 3’ and 5’ ends. In the absence of a complementary DNA or RNA target sequence, the terminal nucleotides of a molecular beacon form a helix stem. This in turn leads to spatial proximity between the attached fluorophore and quencher resulting in only very low intensity fluorescence. However, in the presence of a respective target, the molecular beacon hybridizes to the target, the stem opens up, and an increase in fluorescence is recorded. Hence, in our setup we are able to specifically detect the genome or our virus serotype with good sensitivity due to fluorescence detection after separation of sample components via electrophoresis in the chip format.

Q. What other uses could you envision for this methodology other than investigating viral RNA?

Victor U. Weiss: Chip CE itself was, for example, already successfully used by us for the analysis of fluorescently labelled intact virions, virus-like particles, gelatin nanoparticles, or liposomes. The investigation of a gene therapy delivery system would be a future field of application of our methodology or to study the blocking of RNA release by other small molecule drugs.

Q. Desalting the virus preparations before the analysis increased the recorded signal. Why did this occur?

GA: Virus uncoating but also molecular beacon folding and the binding of the probe to the viral RNA can be influenced by sample components, for example, divalent cations. Removal of these sample constituents significantly improves detection of the viral RNA by molecular beacons in our setup.

Q. You also touched on the difficulties of storing the noncovalent MB-RNA complex. Do you have any advice to minimize degradation?

VUW: Performing experiments under RNase‑free conditions on a sterile work bench and using RNase inhibitors minimizes degradation. However, in the latter case, the impact of these additives on chip CE separation and possibly on viral RNA release itself have to be considered. Also, the addition of large quantities of junk RNA not detectable by the MB used but prone to RNase digestion can prolong the time in which the complex of the MB and the intact viral RNA can be detected.

Q. Currently your hypothesis is that the viral RNA is released from the capsid in the form of a completely unfolded single strand. Is there any evidence to support this?

GA: The exact mechanism of RNA release from a rhinovirus in vivo from inside the protective virus capsid and through the lipid membrane of endosomes is at the moment still not completely understood and hence the question of RNA folding during cell infection cannot be fully answered. However, release of a completely unfolded single strand is to allow the genome to fit through one of the pores formed in the capsid during viral uncoating.

Q. Would the release of RNA from a virus differ in vivo compared to in vitro?

VUW: Actually that depends on your in vitro setup. If you know all necessary components for the in vivo viral RNA release process, you can in principle adjust your in vitro setup accordingly to perfectly mimic the in vivo case. However, if you only want to have the genome molecule to be outside the capsid, simple heating of the sample for a particular time and certain temperature might be sufficient - although the genome release process itself is in this case triggered by conditions that are not physiologic.

Reference

• V.U. Weiss et al., Anal. Bioanal. Chem.408, 4209–4217 (2016).

Victor U. Weiss is an assistant professor with tenure track in the research group Bio- and Polymer Analysis at TU Wien (Vienna University of Technology), Institute of Chemical Technologies and Analytics, Vienna, Austria. He obtained his Ph.D. in analytical chemistry with Prof. Ernst Kenndler (University of Vienna) and Prof. Dieter Blaas (Medical University of Vienna). In his research he focuses on the characterization and detection of bionanoparticles as viruses, liposomes, and virus‑like-particles by application of electrophoretic techniques (liquid phase electrophoresis in the capillary and the chip format) and hyphenation of these to other techniques.

Günter Allmaier is managing director of the Institute of Chemical Technologies and Analytics at TU Wien (Vienna University of Technology) and full professor of analytical chemistry, and also heads the Bio- and Polymer Analysis research group at the institute. He studied pharmaceutical sciences acquiring a masters degree and obtained a Ph.D. degree in analytical chemistry from the University of Vienna in 1983 followed by postdoctoral work at MIT (Cambridge, Massachusetts, USA) with Prof. Klaus Biemann and at the University of Southern Denmark (Odense, Denmark) with Prof. Peter Roepstorff. He has been working since the beginning of the 1980s in the field of mass spectrometry and the analysis of bioactive compounds with emphasis on proteins, oligosaccharides, and lipids. During the last 15 years a significant part of his research activities have been in the field of electrophoretic separation in the liquid and gas phase of various classes of biopolymers and nano(bio)particles as viruses or engineered nanoparticles.