BEACONX

The development of over-the-counter methods for viral RNA detection could revolutionize human health care. It was hypothesized that we could create a robust biosensor material, and amplify otherwise weak signals, by concentrating molecular sensors into porous crystalline arrays with ~nanoLiter volume. As a proof of principle, this year’s BIOMOD team designed a biosensor for West Nile virus that features design on the molecular and materials scale.

ViPR was used to detect an area of the genome unique to West Nile virus. mFold was used to assess for any secondary structures forming in the target sequence. NUPACK was then used to analyze secondary structure in the beacon, probability of folding, and melting temperatures of candidate beacon designs. Once the design was optimized, molecular dynamic simulations were ran to prove folding of the beacon and hybridization of the target sequence.

Molecular beacon performance was assessed in solution using complementary target strands. Meanwhile, a Campylobacter jejuni protein with a cysteine mutation was expressed, purified, and assembled into highly porous crystals. These "host" crystals were then crosslinked. The resulting crystals have a high affinity for DNA adsorption. Confocal microscopy was used to monitor the subsequent installation of the DNA beacon components within the host crystals. Basic loading experiments involved covalent installation of thiolated single-stranded DNA with fluorophores and quenchers. Loading experiments were repeated using the beacon and target strands. 

​

Preliminary data suggests that the beacon was able to load into the crystal and fluorescence was intensified in the presence of target DNA strands.