Multiplex logic processing isothermal diagnostic assays for an evolving virus

Sanchita Bhadra; Miguel A. Saldaña; Hannah Grace Han; Grant L. Hughes; Andrew D. Ellington

The 4GO probe was composed of 5 oligonucleotides (S1 -S5) with a total of 19 degenerate loci that varied between two nucleobases (Supplementary Table T2 ). Oligonucleotides S1 -S4, designed based on validated degenerate OSD sequences, served as strand exchange probes for NS1, NS5, CA, and NS3 amplicons, respectively. The S5 strand acted as a scaffold for 4GO probe assembly ( Figure 1D) . Domain c at the 5'-end of S5 was complementary to domain c' in S1, while, domain f at the 3'-end of S5 was complementary to domain f' of the S4 strand. The intervening region of S5 contained two short target-independent sequences -domain d complementary to domain d' at the fluorophore-labeled 5'-end of S2 and domain e complementary to domain e' at the quencher-labeled 3'-end of S3. Concomitant hybridization of S2 and S3 to S5 would juxtapose the fluorophore and quencher leading to loss of signal. However, at temperatures ≥ 20 °C, pairing of S2 and S5 was contingent upon formation of a three-way junction with S1 via interactions between complementary S2 domain a' with S1 domain a and S5 domain c with S1 domain c'. Similarly, stable interaction of S3 and S5 was dependent upon a three-way hybridization with S4. As a result, strand exchange between the 4GO probe and any one or more of the ZIKV amplicons would separate the fluorophore-bearing S2 from quencher-labeled S3. For example, the NS5 LAMP amplicon loop would directly remove S2 from the 4GO probe. Meanwhile, by binding S1, the NS1 amplicons would destroy the three-way junction between S1, S2, and S5. Consequently, the 4GO probe would transduce the presence of one or any combination of the four ZIKV amplicons into a single-channel endpoint fluorescence that could be easily read at ambient temperature.