Entropy‐Driven Strand Displacements Around DNA Tetrahedron for Sensitive Detection and Intracellular Imaging of mRNA

Messenger RNA (mRNA) is the molecule which carries genetic information from DNA and guides the synthesis of protein. It participates in many cellular cascades and exhibits substantial associations with certain diseases. The development of advanced approaches for mRNA sensing and imaging is of utmost importance for biological studies and early clinical diagnosis. Herein, a novel fluorescent method is demonstrated for highly sensitive analysis of mRNA based on entropy‐driven strand displacements around three‐dimensional DNA nanostructures. Tetrahedral DNA with four pendant linear sequences provides excellent molecular scaffold loading at multiple sites for target mRNA‐initiated cascade toehold strand displacement reactions. The variations of fluorescence resonance energy transfer states can be used to indicate the presence and level of target mRNA. Besides, MnO2 nanosheets are introduced for intracellular transportation of the reaction system for bioimaging. The operation is relatively simple and the developed approach provides a promising tool for mRNA‐related biological researches and clinical applications.

suitable reaction scaffold for biosensing and bioimaging. [23,24]specially, TDNA has been widely utilized as the module with well size-controllability and programmability.The structural or functional information encoded in TDNA can be used to reveal the level of target. [25]Here, we have carefully designed TDNA nanostructures for amplified mRNA assay and employed MnO 2 nanosheets as excellent carriers to transport the reaction system inside cells. [26]Abundant glutathione (GSH) molecules degrade the nanosheets and the loaded DNA strands are released. [27]After target mRNA-initiated strand displacement cycles around TDNA, fluorescence resonance energy transfer state of the probe is shifted from "off" to "on".The recovered fluorescence response indicates the level and position of target mRNA inside cells.This method can precisely probe the information of intracellular mRNA, which holds intriguing potential for biological and clinical applications.

Working Mechanism
The procedures of the strand displacement and imaging principle are illustrated in Scheme 1.The sequences of all designed oligonucleotides are listed in Table S1, Supporting Information.TDNA with excellent controllability and high precision is constructed by bottom-up annealing of four single-stranded DNA (Probes TA, TB, TC, and TD).In previous biosensing studies, TDNA usually employs the amplifying element on one vertex of tetrahedron. [28,29]In this study, four pendant single-stranded regions with the same sequence are positioned at all vertexes.These spatial sites are fully utilized for enzyme-free strand displacement, which promises the high sensitivity.The three-dimensional DNA nanostructure also facilitates the cell endocytosis. [30]Due to the influx of Cl À and water from cytoplasm, TDNA could be escaped from lysosomes, which is beneficial for the reaction with target mRNA. [31]In contrast, a duplex is formed by three single-stranded DNAs (Probes S, B1, B2), which contain a toehold for strand displacement.Probe S is labeled with Cy3 at the 3 0 terminal and Probe B1 is labeled with BHQ2 at the 5 0 terminal.In the duplex of Probes S/B1/B2, Cy3, and BHQ2 are adjacent to each other, leading to the quenching of fluorescent dye.In the presence of target mRNA, entropy-driven strand displacement starting from the toehold occurs between Probe S and mRNA.Probe B2 is released and a new toehold is generated in the middle of the duplex (Probes S/B1/mRNA).The pendant linear sequences around TDNA tetrahedron (termed as Probe TE) recognize the toehold, which then displaces mRNA and Probe B1 simultaneously.mRNA is thus recycled.A single TDNA is able to localize four strands of Probe S. Without the quenching effect of Probe B1, significant fluorescence emission is recovered, which is related to the initial level of mRNA.The sensing system can be facilely transported inside cells for bioimaging.Both of the elements of TDNA and Probe S/B1/B2 duplex contain single-stranded segments, which can be absorbed on the surface of the nanocarrier of MnO 2 nanosheets.During Scheme 1. Illustration of the entropy-driven strand displacements around TDNA for mRNA assay.Probes S and B1 are labeled with Cy3 and BHQ2, respectively.
this process, MnO 2 nanosheets further quench the fluorescence emission of Cy3 and protect these DNA strands from enzymatic degradation.Due to the existence of abundant GSH inside cells, MnO 2 nanosheets are reduced to Mn 2þ , and the DNA nanostructures are released to cytoplasm.The sensing elements are thus activated by the mRNA.Bright emission of Cy3 labeled at the end of Probe S could light the cells and indicate the level of intracellular target mRNA.This sensing and imaging approach can be modified to suit other targets by the design of TDNA and duplex sequences.In addition, the four pendant single-stranded regions on TDNA can be adjusted to recognize different targets.Therefore, multiplex imaging can be achieved by further incorporating more fluorescence dyes.

Characterizations of the Nanocarrier
The prepared MnO 2 nanosheets are observed directly by transmission electron microscopy (TEM).As shown in Figure 1A, ultrathin lamellar morphology is clearly observed and the nanosheets are well-dispersed.Single-stranded DNA can be attached on the surface of nanosheets via the synergistic physisorption of nucleobases on the basal plane of MnO 2 and co-ordination of phosphate.Due to the DNA backbone, the zeta potential of DNA-loaded MnO 2 nanosheets becomes more negative.In addition, the hydrodynamic diameter is also increased, demonstrating successful conjugation of MnO 2 and DNA (Figure 1B).UV-vis absorption spectra of MnO 2 nanosheets after different reactions are then studied.As shown in Figure 1C, a large molar extinction coefficient around 350 nm is observed, which is ascribed to d-d transitions of manganese ions in the nanosheets.After mixing DNA and the nanosheets, a characteristic peak at 260 nm emerges, which is the evidence of DNA loading event.
With the reaction of GSH, the nanosheets can be digested, leading to the disappearance of the absorption peak at 350 nm.However, the peak at 260 nm is retained, which is the released DNA from the surface of nanosheets.The fluorescence quenching and recovery of Cy3 at Probe S are then checked.As shown in Figure 1D, a significant fluorescence emission peak at 580 nm is observed.After conjugated with the nanosheets, the fluorescence is quenched with remarkably declined peak intensity.Nevertheless, after the treatment with GSH, fluorescence can be recovered and the intensity is nearly the same as the initial level of Probe S.These fluorescence performances further demonstrate the excellent carrier capability of MnO 2 nanosheets.Toxicity has always been an important factor of nanomaterials for live-cell applications.The potential cytotoxicity of the used MnO 2 nanosheets is checked by Cell Counting Kit-8 (CCK-8) assay.It is clear that even after the treatment with nanosheets of a high concentration (200 μg mL À1 ) for a long incubation time (15 h), the cell viabilities are still higher than 80% (Figure 1E,F).Thus, good biocompatibility of the nanomaterials is demonstrated, which is suitable for sensing and imaging applications.

Feasibility Confirmation of Strand Displacements
Entropy-driven strand displacements are first theoretically analyzed.The free energy of the duplex structure of Probes S/B1/B2 is about À78 kcal mol À1 (NUPACK).In the presence of mRNA, the duplex of Probes S/B1/mRNA shows a more negative value, demonstrating the thermodynamic tendency of strand displacement reaction.Similarly, after further introduction of Probe TE, the formation of duplex of Probes S/TE is predicted (Figure S1, Supporting Information).The direct evidence of the DNA assembly and strand displacement reactions are found in polyacrylamide gel electrophoresis (PAGE) images.Briefly, after blending the four fuel strands of TDNA, a band with much larger molecule weight appears, which is the formed three-dimensional nanostructure (Figure S2A, Supporting Information).The assembly of DNA duplex and subsequent strand displacements are also checked by PAGE.The bands of Probes B1, B2 and the duplex of Probes S/B1/B2 can be clearly observed in lanes a-c (Figure S2B, Supporting Information).In lane d, the bands are the products after the reaction between Probes S/B1/B2 and mRNA.Compared with lane c, a band with similar position is shown, which is ascribed to duplex of Probes S/B1/mRNA.Besides, a new band appears and the position is in good accordance with the displaced Probe B2, demonstrating successful entropy-driven strand displacement.In contrast, the downstream strand displacements of Probe B1 and mRNA are shown by the bands observed in lanes e and f.
We have then utilized fluorescence and electrochemical techniques to confirm the feasibility of reactions, separately.As shown in Figure 2A, the emission of Cy3 labeled at the end of Probe S could be quenched by BHQ2 after the hybridization with Probes B1 and B2.After directly incubating the duplex with TDNA, the segment of TE cannot initiate strand displacement since the middle toehold is still hidden.Therefore, the fluorescence emission is inactivated.Only in the presence of mRNA, cascade strand displacement reactions occur in succession and the fluorescence can be recovered.The feasibility of fluorescence analysis of mRNA is thus demonstrated.Next, the TDNA nanostructure is modified with triple thiols at the bottom (Probes TA/TBS/TCS/TDS), which facilitates its immobilization at the gold electrode surface.The pendant linear sequence (TE) is then applied to trigger strand displacement of Probes SS/B1/B2.Probes SS is labeled with electrochemical species instead of Cy3.The DNA immobilization and reaction processes are first characterized by electrochemical impedance spectroscopy (EIS).As shown in Figure 2B, a straight line is observed for bare gold electrode.After modified with thiolated TDNA, a semicircle domain is reflected in the Nyquist diagram, which represents a significant charge transfer resistance caused by the negatively charged DNA monolayer.In the absence of mRNA, Probes SS/B1/B2 cannot interact with TDNA on the electrode, thus the Nyquist diagram barely changes.In the presence of mRNA, Probe SS can be immobilized after the displacement reaction.The DNA layer becomes more negative, which repels the electrochemical species significantly and leads to the increase of semicircle domain.We then carried out square wave voltammetry (SWV) to study the electrochemical response from Probe SS.As expected, only with mRNA-mediated strand displacement, a significant current peak can be observed (Figure 2C).These electrochemical results further confirm the feasibility of the strand displacement principle.Besides, we have stored the TDNA nanostructures at 4 °C for different time durations, which are then used for the reactions with mRNA.The recorded electrochemical and fluorescence intensities of the peaks are quite stable in two weeks, demonstrating good stability of the DNA materials and excellent reproducibility of the sensing strategy (Figure S3, Supporting Information).

In Vitro Fluorescent Quantification of mRNA
The designed TDNA (Probes TA/TB/TC/TD) contains four single-stranded regions for entropy-driven strand displacements, which are supposed to exhibit higher fluorescence response than TDNA (Probes TA/TBS/TCS/TDS).We have thus compared the performances of TDNAs with one and four pendant reaction sites directly.In the absence of target mRNA, Probes S/B1/B2 are not changed and no emission can be observed.After spiking 1 pM mRNA, toehold strand displacement reaction occurs and Cy3 can be loaded at the TDNA.With recycled mRNA, TDNA with more reaction sites enriches more strands labeled with Cy3, which are reflected by the significantly increased fluorescence.The result demonstrates excellent amplification efficiency of the designed three-dimensional DNA scaffold (Figure S4A, Supporting Information).To achieve the best analytical performances, some critical parameters are optimized.First, different concentrations of MnO 2 nanosheets are prepared and blended with Probe S. With the increase of MnO 2 amount, the fluorescence peak intensity of Cy3 decreases, indicating more DNA strands are attached at the surface of nanosheets.The decline reaches a saturation value with 200 μg mL À1 of MnO 2 , which is then utilized as the optimal value (Figure S4B, Supporting Information).We have also checked GSH-mediated release of Probe S by monitoring the fluorescence recovery.With the concentration larger than 0.9 mM, most MnO 2 nanosheets can be reduced, reflected by the significant increased fluorescence emission (Figure S4C, Supporting Information).GSH levels in cells are sufficient to release the loaded DNA strands.Besides, the kinetics of entropy-driven strand displacements around TDNA is studied by in vitro measurement of the fluorescence response.In the absence of mRNA, the duplex of Probes S/B1/B2 is stable and fluorescence is persistently low.On the contrary, with mRNA-initiated reactions, Probe S can be assembled on TDNA and the fluorescence peak intensity grows rapidly with longer time.An optimal value of 90 min is selected for following experiments (Figure S4D, Supporting Information).
Under these optimized conditions, we have prepared a series of concentrations of mRNA and triggered strand displacement reactions on Probe S/B1/B2/TDNA with different degrees.The fluorescence emission spectra are compared in Figure 3A.As expected, with larger concentrations of mRNA, the emission peak intensity is increased, indicating the degree of fluorescence recovery of Cy3 increases.The detailed relationship between the peak intensity and mRNA level is depicted in Figure 3B.A linear range is established from 1 fM to 1 pM, which is rather wide (Figure 3C).The equation is fitted according to the spectra information as follows: in which, y stands for the fluorescence peak intensity, and x is the logarithmic concentration of mRNA.The limit of detection is calculated to be 0.33 fM (S/N = 3), which is superior to most recently reported isothermal amplification methods (Table S2, Supporting Information).The amplifier does not involve any other external materials, which is highly dependent on entropy-driven strand displacements on framework DNAs.Therefore, a highly robust analysis is promised.By taking full use of the target recycle and four pendant regions of TDNA, enriched fluorescence response can be recorded for the determination of trace mRNA.The sensitivity is comparable with that of RT-PCR, while the reaction time of the isothermal and enzyme-free process is quite short, demonstrating potential practical utility.

Selectivity Investigation and Serum Assay
Selectivity assessment is then carried out by introducing six RNA strands with one or two mismatched sites.These strands are incubated with the DNA duplex and TDNA for strand displacement reactions.The fluorescence peak intensities are summarized in Figure 3D.The results reflect that these mismatched sequences cannot trigger the reactions since the emissions are weak.After spiking with target mRNA, the fluorescence peaks are increased significantly, which are comparable with original response of target mRNA case (Figure 3E).Together, these findings demonstrate good selectivity of the proposed biosensor.Subsequently, the reaction system is challenged with real biological samples.Human serum samples are directly taken as examples of complicated biological fluids.After diluted with phosphate-buffered saline (PBS) for 10-folds, different levels of mRNA are added before the strand displacement reactions and subsequent fluorescence measurements.The recorded peak intensities are compared in Figure 3F, which are consistent with the performances in PBS conditions.The recoveries are between 95.32% and 106.36%.It is concluded that the proposed method is highly antijamming even with complicated biological samples.

Intracellular Imaging
Having demonstrated the low toxicity of nanocarrier, the feasibility for mRNA imaging is investigated.Previous studies already proved that the MnO 2 nanosheets enter cells through scavenger receptor-mediated phagocytosis. [32]In the absence of the nanocarrier of MnO 2 nanosheets, TDNA can be delivered into the cells within 5 h, [33] but Probes S/B1/B2 cannot be effectively transfected.Although A549 expresses high levels of TK1 mRNA, the substrate fluorescence donors are insufficient and the recovered fluorescence is limited.After utilizing 200 μg mL À1 of MnO 2 nanosheets as the nanocarrier, all components including TDNA and duplex of Probes S/B1/B2 are synchronously transported into the living cells.Therefore, bright Cy3 fluorescence is observed, which is provided by the immobilized Probe S around TDNA inside cells (Figure S5, Supporting Information).To demonstrate the response is activated by target mRNA, we have introduced BEAS-2B cells with low TK1 mRNA as negative controls.The mRNA expression levels are first validated by the proposed fluorescent method and standard RT-PCR.The calculated values are in good accordance with each other (Figure 4A).Both of the two approaches verify the low expression of TK1 mRNA in BEAS-2B.In addition, the obtained confocal image of BEAS-2B does not reflect significant fluorescence emission as expected.For A549 with abundant TK1 mRNA, bright emission can be observed.Their results demonstrate the close relationship between the fluorescence response and target mRNA expression (Figure 4B).The proposed strategy provides a powerful tool for not only highly sensitive quantification, but also intracellular imaging of disease-related mRNA.The involved sequence design is simple and materials are of low cost for clinical use.

Conclusion
In summary, based on the entropy-driven strand displacements and subsequent DNA assembly events, a highly sensitive enzyme-free fluorescent biosensor is constructed for the detection of target mRNA.Benefiting from the thermodynamic favorable reactions and the full use of TDNA scaffold, the biosensor exhibits the limit of detection as low as 0.33 fM.The cascade strand displacement reactions are also highly sequencedependent, endowing the assay with excellent anti-interference capability.After coupling with MnO 2 nanocarrier, the reaction system is successfully transported inside cells and an intracellular bioimaging protocol is established.Cells with different mRNA expressions can be easily distinguished and the results are highly consistent with RT-PCR.It is anticipated that the pendant linear sequences on TDNA can be adjusted and three more fluorescence dyes can be incorporated to analyze four target mRNAs simultaneously.The sensitivity is sacrificed to a certain degree but the multiplex imaging may have broad application prospects.

Figure 1 .
Figure 1.A) TEM image of freshly prepared MnO 2 .B) Zeta potentials and hydrodynamic diameters of MnO 2 before and after DNA loading.C) UV-vis spectra of MnO 2 before and after incubation with DNA and GSH (separately and simultaneously).D) Fluorescence spectra of Probe S, Probe S adsorbed on MnO 2 before and after GSH digestion.Cytotoxicity tests of MnO 2 with different E) concentrations and F) incubation times.Error bars indicate the standard deviation of triplicate tests.Statistical analysis is performed using Student's t-test.**p < 0.01, ***p < 0.001.

Figure 2 .
Figure 2. A) Fluorescence spectra of Probe S and Probes S/B1/B2 after the interaction with TDNA in the absence and presence of mRNA.B) Nyquist diagrams and C) square wave voltammograms of bare electrode, TDNA-modified electrode, after interaction with Probes SS/B1/B2 in the absence and presence of mRNA.

Figure 3 .
Figure 3. A) Fluorescence spectra for the detection of mRNA with the concentrations from 1 fM to 10 pM.B) Calibration curve reflecting relationship between peak intensity and logarithmic mRNA concentration.C) The linear range.D) Histogram of the obtained fluorescence peak for target mRNA assay against various mismatched sequences.E) Histogram of the fluorescence responses for mismatched mRNAs after the spiking of target mRNA.F) Fluorescence peak intensities for the detection of spiked mRNA in PBS and serum samples.Error bars indicate the standard deviation of four tests.Statistical analysis is performed using Student's t-test.***p < 0.001.

Figure 4 .
Figure 4. A) mRNA expression levels in A549 and BEAS-2B cells determined by the proposed fluorescent method and standard RT-PCR.B) Confocal laser scanning microscopy images of mRNA in A549 and BEAS-2B cells.Error bars indicate the standard deviation of four tests.Statistical analysis is performed using Student's t-test.***p < 0.001.