Several bottlenecks in the design of current sensor technologies for small noncoding RNA must be addressed. The small size of the sensors and the large number of other nucleotides that may have sequence similarity makes selectivity a real concern. Many of the current sensors have one strand with an exposed region called a toehold. The toehold serves as a place for the analyte nucleic acid strand to bind and initiate competitive displacement of sensors' secondary strands. Since the toehold region is not protected, any endogenous oligonucleotide sequences that are similar or only different by a few nucleic acids will interact with the toehold and cause false signals. To address sensor selectivity, we investigated how the toehold location in the sensor impacts the sensitivity and selectivity for the analyte of interest. We will discuss the differences in sensitivity and selectivity for a miR-146a-5p biosensor in the presence of different naturally occurring mismatch sequences. We found that altering the toehold location lowered the rate of the false signal from off-analyte microRNA by upward of 20 percentage points. Detection limits as low as 56 pM were observed when the sensor concentration was 5 nM. The findings herein are broadly applicable to other small and large RNAs as well as other types of sensing platforms.
Keywords: fluorescence; microRNA; nanostructure; nucleic acids; toehold mediated strand displacement.