Specific Light-Up System for Protein and Metabolite Targets Triggered by Initiation Complex Formation

Gene regulation systems are mimicked by simple quantitative detection of non-nucleic acid molecular targets such as protein and metabolite. Here, we describe a one-tube, one-step real-time quantitative detection methodology for isothermal signal amplification of those targets. Using this system, real-time quantitative detection of thrombin and streptomycin, which were used as examples for protein and metabolite targets, was successfully demonstrated with detection limits of at most 50 pM and 75 nM, respectively. Notably, the dynamic range of target concentrations could be obtained for over four orders of magnitude. Thus, our method is expected to serve as a point-of-care or on-site test for medical diagnosis and food and environmental hygiene.

Potassium ion dependency on the detection reaction was more precisely analyzed ( Figure S4AB).
Potassium ion dependency on the detection reaction was more precisely analyzed ( Figure S4CD).

Correlation line and detection limits
The real-time quantitative analyses of thrombin and streptomycin in the abovementioned 21 and 22 different concentrations were conducted using a CFX96 real-time PCR detection system, respectively (Figures 4C, 5C, and S5). Respective relative rates of reaction (A) were obtained by fitting the time course data of fluorescence intensity from 0 to 100 min with equation 1, where I is the fluorescence intensity, t is the reaction time, A is the relative rate of reaction, and B is the y-intercept.
Three independent measurements (n = 3) were performed at a single concentration for all A-value determinations. Standard deviations (m) were calculated using equation 2, where Ā is the average of those measurements (A1, A2, and A3).
The logarithmic values of Ā (log Ā) with error bars that show the standard deviations (σ) were plotted versus the logarithm of target concentrations, as shown in Figures 4C and 5C. The standard deviations (σ) were calculated using equation 3.
The correlation lines, equations 4 for thrombin and 5 for streptomycin, could be plotted between = 0.500 + 3.49 . 5 The present system achieved detection limits of ca. 50 pM for thrombin and ca. 75 nM for streptomycin at the highest estimate, respectively. Namely, the present system provided signal intensities with small σ values at low target concentrations: log Ā ± σ was equal to -0.990 ± 0.050 and -0.174 ±0.071 at thrombin concentrations of 10 and 50 pM, respectively; while log Ā ± σ was equal to -0.329 ± 0.018 and -0.082 ± 0.012 at streptomycin concentrations of 50 and 75 nM, respectively.

Target detection in human serum
Target protein (thrombin; 10 nM, 2µL) was added to a mixture (18 µL) containing CS-thr (240 nM, at room temperature for 30 min and imaged in the same manner described above ( Figure S7).

Stabilities of the natural/modified oligonucleotides in human serum
Reactions (10 μL reaction volume) containing 1 µL of ϕ29 buffer (10×) and 1 μL of oligonucleotide (T26, P1-thr, or P1-thr-PS (4 μM)) were performed in 10% v/v human serum for all ODNs. In addition, experiments were conducted in 30% and 60% v/v human serum for T26. All reactions were incubated at 37 °C for 2 h. The reaction products were resolved by denaturing PAGE, and gel images were recorded with excitation of the 5′-labeled fluorophore at 488 nm ( Figure S6A for degradation of T26) or SYBR ® Gold at 488 ( Figure S8A for degradation of P1-thr and P1-thr-PS), and the band intensities were quantified by the Quantity One software ( Figures S6B and S8B). The decay curves of intact ODN were fitted from band intensities at appropriate intervals of reaction time (0, 10, 30, 60, and 120 min) by the least squares method using the OriginProver. 8.1 program.

Measurement of thrombin content in human serum
For the experiment to measure thrombin content in human serum, four tests were performed using 2 nM thrombin in ϕ29 buffer (1×), 20 nM thrombin in ϕ29 buffer (1×), 20% human serum in ϕ29 buffer (1×), and ϕ29 buffer (1×) for the blank. For each test, 40 μL of solution was mixed with an equal volume of ϕ29 buffer (1×) containing SPECTROZYME ® TH (1 mM) (each solution was warmed to 37 °C prior to mixing). Then, the four test samples were incubated at 37 °C and the degradation was monitored for 2000 s using a UV-2700 spectrophotometer (Shimadzu corporation, Tokyo, Japan). The changes in absorbance at 405 nm were recorded at 2 s intervals ( Figure S9).
FAM-TTTTTTTTTTTTTTTTTTTTTTTTTT Figure S1. Chemical structures of the compounds used in this study. S10 Figure S2. Effects of alkali metal ion concentration on RCA: a photograph of the aliquots containing ThT-HE (5 µM) captured under visible light irradiation at 410 nm. Each reaction mixture was incubated at 37 °C for 2 h. The aliquots in rows a and b are reaction mixtures with Na + and K + , respectively.