RNA Detection

Fig. 1Designing effective sequences of ECHO probes and in vitro characterization. (A) Transcript-specific
ECHO probes (black lines) generated against U3 snoRNA, 28S rRNA, and poly(A) RNA (gray lines). Known
functional motifs are labeled along the transcripts (masked areas). (B) Kinetics of hybridization-dependent
fluorescence activation measured with stopped-flow technique. Fluorescence intensity of 50 nM probes (y) vs.
the lapsed time (sec) after mixing with 1400 nM target oligonucleotides (x).Gray lines: 10 trials of
measurements;green line: fitted curve (y¼ce-Kx + A). Fluorescence activation occurs within tens of
milliseconds in the presence of target oligonucleotides. (C) K value representing effective affinity plotted
against the concentrations of target oligonucleotides. K¼Kon[target oligo] + Koff,KD¼Koff/Kon. (D) A list of
RNA targets and probe sequences; (E) Spectral measurement (535–700 nm) of poly(A) and U3 snoRNA probes
(0.2μM) in the absence (gray) or presence (green) of complimentary DNA oligonucleotide solutions (0.2μM).
D514-random was mixed with d(A) 23 where no fluorescence activation was observed (on–off ratio of 1.1).
(Reproduced from [24] with permission from Oxford University Press)