Programmable low-cost DNA-based platform for viral RNA detection

Lifeng Zhou; Arun Richard Chandrasekaran; Jibin Abraham Punnoose; Gaston Bonenfant; Stephon Charles; Oksana Levchenko; Pheonah Badu; Cassandra Cavaliere; Cara T. Pager; Ken Halvorsen

As a first proof of concept for detecting ZIKV, we designed DNA nanoswitches to target an already validated sequence in the ZIKV genome that has been used to bind primers in qPCR 28 (note: all oligo sequences are specified in Tables S1 to S9). We made the DNA nanoswitches by hybridizing singlestranded DNA (ssDNA) oligos to linearized single-stranded M13mp18 (M13) genomic DNA in a thermal annealing ramp for 1 hour 24 and then purified them by high-performance liquid chromatography (HPLC). 29 For our initial detection target, we in vitro transcribed RNA from the pFLZIKV infectious plasmid containing the full length genome of the Cambodia ZIKV isolate (FSS13025) (Fig. S1 ). 30 Previous results have shown robust nanoswitch detection of small DNA and RNA sequences (20-30 nucleotides) , however, the strong secondaty structures present in long viral RNA was expected to interfere with our detection. 31, 32 To overcome this, we used a chemical fragmentation method to segment the RNA into small pieces that are mostly shorter than 200 nt ( Fig. 1B-C and S2 ). By incubating with our nanoswitch in an annealing temperature ramp, we showed successful detection of the fragmented viral RNAs by gel electrophoresis, thus validating our approach (Fig. 1D) .