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

We then validated quality and function of each nanoswitch in the panel of 18 nanoswitches. All nanoswitches performed well with a molar excess of positive DNA controls, although they showed more signal variation with fragmented ZIKV RNA (Fig. S7) . We ranked the nanoswitches from strongest to weakest signal and made a series of equimolar nanoswitch mixtures. Using these mixtures, we validated our inherent signal multiplication strategy using a low concentration pool of equimolar DNA fragments to mimic the fragmented RNA. We observed that our detection signal increased steadily up to around 12 nanoswitches (Fig. 1E, F) , and then plateaued above that value. This plateau was not unexpected considering that the largest mixtures added lower performing nanoswitches that may contribute less to the overall sample. Since there was no significant change in performance between 12 and 18, we continued with using the 18 nanoswitches mix for our follow up experiments. High sensitivity is one of the key requirements for viral detection. Clinical levels of ZIKV RNA in body fluids of infected patients are often in the femtomolar range, 7,21,33 making amplification a prerequisite for most detection approaches.