Scaling up of a Self‐Confined Catalytic Hybridization Circuit for Robust microRNA Imaging

Abstract The precise regulation of cellular behaviors within a confined, crowded intracellular environment is highly amenable in diagnostics and therapeutics. While synthetic circuitry system through a concatenated chemical reaction network has rarely been reported to mimic dynamic self‐assembly system. Herein, a catalytic self‐defined circuit (CSC) for the hierarchically concatenated assembly of DNA domino nanostructures is engineered. By incorporating pre‐sealed symmetrical fragments into the preying hairpin reactants, the CSC system allows the hierarchical DNA self‐assembly via a microRNA (miRNA)‐powered self‐sorting catalytic hybridization reaction. With minimal strand complexity, this self‐sustainable CSC system streamlined the circuit component and achieved localization‐intensified cascaded signal amplification. Profiting from the self‐adaptively concatenated hybridization reaction, a reliable and robust method has been achieved for discriminating carcinoma tissues from the corresponding para‐carcinoma tissues. The CSC‐sustained self‐assembly strategy provides a comprehensive and smart toolbox for organizing various hierarchical DNA nanostructures, which may facilitate more insights for clinical diagnosis and therapeutic assessment.


Table of Contents
Table S1.Sequences

Schematic illustration of the CSC system
Profiting from the pre-blocked palindromic fragments in the stem domain, this compact CSC system allows reactant-to-template-mediated assembly.In the presence of the promotor (miRNA), the interconnecting catenated DNA reactant was generated to carry out proximal hybridization (Figure S3A), facilitating hierarchically concatenated DNA assembly.The DNA sequence of the CSC system is shown in Supporting Information

S-7
The essential role of the pre-blocked symmetrical fragments The indispensable role of the pre-blocked palindromic fragments in the CSC amplifier was extensively explored by the fluorescence assay.A dramatically enhanced fluorescence signal was observed for the miR-155-initiated intact CSC system (Figure S4A), while a relatively low fluorescence readout was revealed in the same miR-155-trigged nCSC system (Figure S4B).The enhanced fluorescence response of the CSC system suggested that the pre-blocked palindromic fragment is essential for the promoted templated assembly and the localization-intensified signal amplification.Supporting Information

S-8
The optimized incubation temperature The reaction temperature is an essential factor that affects the thermodynamics of the association/disassociation process of the DNA duplex, thus affecting the sensing performance of the CSC amplifier.The optimized reaction temperature of the CSC system was investigated and displayed the best signal-to-noise (S/N) ratio at 37 ℃ (Figure S5).Therefore, the optimum incubation temperature was chosen as 37 ℃ for the following experiments.

Stability of the CSC circuitry in diluted serum samples
The performance of the CSC system in diluted human serum was evaluated (Figure S9).The fluorescence readout even in 15% serum (ser) solution was comparable to that in ideal buffer solution, indicating high stability and acceptable accuracy of the CSC circuitry in complicated biological samples.Supporting Information

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The intracellular fluorescence signal of the miRNA-initiated CSC system The sensing performance of the CSC system was also investigated by quantitative flow cytometry assay.An intense fluorescence response was observed in CSC-treated MCF-7 cells as compared to that of the nCSC-treated MCF-7 cells and the CDC-

Figure S2 .
Figure S2.Construction of the CDC amplifier.(A) The detailed reaction process of the CDC system.(B) The DNA sequence of the CDC system.

Figure S3 .
Figure S3.Illustration of the working mechanism of the miR-155-responsive CSCinvolved sensing platform.

Figure S5 .
Figure S5.The signal-to-noise ratio (S/N) of the CSC system at different temperatures.The concentration of CSC probe was 200 nM, while the concentration of miR-155 is 0.5 nM.Error bars indicate the mean ± SD of three biological replicates.

Figure S6 .Figure S7A
Figure S6.The native PAGE characterization of different circuit systems.The concentration of DNA reactants is 200 nM, while the concentration of miR-155 is 10 nM.

Figure S8 .
Figure S8.Performance of the CDC for miR-155 analysis.(A) Time-dependent fluorescence changes of the CDC amplifier incubated with different concentrations of miR-155 : (a) 0 nM, (b) 0.5 nM, (c) 1 nM, (d) 5 nM, (e) 10 nM, (f) 25 nM, and (g) 50 nM.(B) The corresponding fluorescence spectra as shown in S8A at 180 min.Inset: the calibration curve as indicated by the fluorescence intensity change (=520) as a function of the miR-155 concentration.Error bars indicate the mean ± SD of three biological replicates.

Figure S10 .
Figure S10.Fluorescence spectra generated by the phosphorothioated CSC probes with (curve a') or without (curve a) 0.5 nM miR-155 and the unmodified CSC probes with (curve b') or without (curve b) 0.5 nM miR-155.

Figure S11 .
Figure S11.Cytotoxicity evaluation of the CSC system.(A) Cell viability of MCF-7 and MCF-10A cells incubated with various concentrations of the CSC reactants for 24 h, respectively.(B) Cell viability of MCF-7 and MCF-10A cells after various incubation times with 500 nM CSC reactants.Error bars indicate the mean ± SD of five biological replicates.
treated MCF-7 cells (FigureS13A), demonstrating the enhanced signal amplification features of the proposed CSC system inside living cells.However, faint fluorescence signal was observed in the miR-155 inhibitor-pretreated MCF-7 cells (FigureS13B), confirming that the fluorescence signal was indeed generated by the miR-155-initiated concatenated hybridization reaction and the designed CSC system is well-adapted for determinating miRNA of low concentration in living cells.The quantitative flow cytometry assay results were highly consistent with the CLSM observation, demonstrating that our proposed CSC amplifier is, indeed, suitable for in situ detection of less-abundantly expressed miRNAs.

Figure S13 .
Figure S13.(A) Flow cytometric analysis and corresponding statistical histogram analysis of the mean fluorescence intensity (MFI) of the four groups in MCF-7 cells with different treatments.one-way ANOVA test.Error bars indicate the mean ± SD of three biological replicates.

Table S1 .
Sequences of the oligonucleotides for miRNA stimulus-responsive system

Table S2 .
The oligonucleotide sequences of CSC amplifier for bioimaging