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Electronic Supplementary Material (ESI) for Analytical Methods. This journal is ? The Royal Society of Chemistry 2015 Supplementary Information Fluorescent microRNA Biosensors: A comparison of signal generation to quenching C. Kyle Almlie, Nicholas E. Larkey, and Sean M. Burrows* 153 Gilbert Hall, Corvallis, OR 97331, United States of America Email: sean.burrows@oregonstate.edu Comparison of baseline fluorescent signals of the RQ hairpin Figure S1. Demonstration of the difference in fluorescence intensity from the hairpin configuration of 500 nM and 1 μM RQ. Signal arises from incomplete quenching by Iowa Black Red Quencher and equilibrium between open and closed hairpin states during signal acquisition. From thermodynamic predictions about 1.3 and 2.6 nM of RQ are open for the 500 nM and 1 μM RQ solutions, respectively. Having fewer fluorescent dyes in solution allows for a lower background, which correlates to a higher sensitivity. (N = 3) Homodimer considerations Figure S2. Comparison of probabilities27-29 for (A) RQ and (B) MB hairpins as well as homodimers for (C) RQ and (D) MB (color and size of boxes represent probability, axis numbering starts from 5 prime end of strand). The stem binding of MB hairpin is greater than RQ (compare A and B). The probability of MB stem base pairing is greater than the MB homodimer base pairing (compare B and D). For the RQ, there is equal probability for base pairing in either the homodimer or the hairpin (compare A to C). From a probability stand point the RQ will probably have more homodimers than MB. The probability analysis coupled with greater thermodynamic stability of homodimers for both MB and RQ supports the argument that an equilibrium may exist