Space-limited double-foot DNA nano-motor with enhanced dynamics and preparation method and application of space-limited double-foot DNA nano-motor

Abstract

The invention discloses a space-limited double-foot DNA nano-motor with enhanced dynamics and a preparation method and application thereof. The preparation method comprises the following steps: annealing a specific DNA single chain to form a Cy5-substrate probe, a Cy3-substrate probe, a double-foot walker 1 and a double-foot walker 2; and respectively assembling the two substrate probes and the gold nanoparticles through a freezing method to obtain the DNA nano motor 1 and the DNA nano motor 2. The nano motor, a corresponding biped walker and a fuel chain form a detection system which is used for detecting messenger RNA (Ribonucleic Acid). The space confinement effect of the gold nanoparticles and the proximity effect of the biped walker are utilized, entropy-driven catalytic reaction kinetics is remarkably accelerated, ultra-high-sensitivity detection of survivin mRNA and TK1 mRNA is achieved, and the detection limits reach 501 aM and 314 aM respectively. And rapid and high-contrast in-situ imaging can be carried out on double mRNA in living cells.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical technology, and specifically relates to a spatially confined bipedal DNA nanomotor with enhanced kinetics and its preparation method, as well as the application of the spatially confined bipedal DNA nanomotor with enhanced kinetics for rapid imaging of dual mRNAs in living cells. Background Technology

[0002] Cancer progression is a major threat to human health and is closely associated with the aberrant expression of specific biomarkers. Messenger RNA, transcribed from a DNA template, carries genetic instructions that are decoded by the ribosome to guide the synthesis of functional proteins. Endogenous tumor-associated mRNAs are considered key biomarkers for a variety of major diseases, including cancer, cardiovascular disease, diabetes, and neurodegenerative diseases. In cancer, dysregulation of these mRNAs (originating from abnormalities in their processing, nuclear export, or translation) can trigger tumorigenesis. Therefore, quantifying the levels of these mRNAs in cells and tissues provides crucial information for understanding tumor behavior and directly guides the development of diagnostic and targeted drug delivery strategies. For example, survivin mRNA is frequently found to be upregulated in colon cancer, prostate cancer, lung cancer, pancreatic cancer, and breast cancer. Overexpression of survivin mRNA has been considered a poor prognostic indicator for malignancies such as soft tissue sarcoma and neuroblastoma. Overexpression of TK1 mRNA disrupts cell cycle control, promoting abnormal division and proliferation. Therefore, TK1 mRNA levels are considered an important biomarker for monitoring tumor progression. Therefore, developing efficient and reliable in situ mRNA analysis platforms for live cells and tissues is crucial for advancing modern medicine through precision diagnostics. A range of established methods, including Northern blotting, fluorescence in situ hybridization, microarray analysis, and real-time polymerase chain reaction (PCR), are currently fundamental technologies for in vitro mRNA detection. However, because these methods typically rely on analyzing RNA isolated from lysed cells, they are inherently limited to capturing gene expression at fixed time points. Therefore, they cannot provide real-time information on the dynamics of mRNA expression in live cells or intact tissues. In vivo detection and imaging of tumor-associated mRNAs remains a significant technological bottleneck. This challenge stems primarily from the typically low abundance of mRNA targets and the highly complex and interfering intracellular environment. Fluorescence assays based on nucleic acid probes have become the primary strategy for tracking mRNA in live cells due to their real-time imaging capabilities and excellent biocompatibility. However, traditional free nucleic acid probes have inherent limitations, including low cellular uptake efficiency and poor stability to nucleases, which severely hinder their wider application.

[0003] Leveraging the programmability and predictability of Watson-Crick base pairing, DNA nanomachines have become an attractive platform for biosensing, molecular computing, and smart materials. Their appeal in these fields stems from their inherent advantages, such as excellent biocompatibility, powerful signal amplification capabilities, and precise control over molecular structures. Precise molecular motors are ubiquitous in living systems and perform essential functions. Inspired by these natural phenomena, researchers are dedicated to developing artificial molecular motors that provide precise control and function at both the molecular and macroscopic scales. A variety of DNA-based motor devices have been developed, including tweezers, gears, cranes, robots, and walkers. A typical DNA motor consists of three basic components: a walking chain, a track chain, and a driving force. The walking chain is driven by mechanisms such as nucleases, DNAzymes, or chain displacement, moving stepwise along a track. This process disrupts cyclic equilibrium by consuming energy, enabling the performance of multiple tasks. Early DNA walkers were primarily designed to operate on one-dimensional linear or two-dimensional planar tracks. In contrast, recently developed three-dimensional walkers navigating on spherical substrates exhibit significantly improved walking efficiency and signal amplification capabilities, attributed to the high specific surface area and greater load capacity of the three-dimensional track. Much research interest has focused on three-dimensional DNA machines employing DNA-functionalized gold nanoparticles as orbitals, where thiol-modified oligonucleotides are bound to the AuNP surface via stable Au-S bonds. A notable example is a monopodial DNAzyme nanomachine activated by a specific intracellular target and autonomously walking on AuNPs, enabling miRNA imaging in live cells for the first time. However, the movement of such monopodial walkers is inherently confined to the vicinity of their anchor points, and this spatial limitation severely hinders the overall walking range and amplification efficiency. In contrast, bipodial DNA walkers capable of randomly traversing three-dimensional surfaces within live cells remain largely unexplored territory. Furthermore, most existing DNA walkers are designed for single-target detection only, lacking the multiplex detection capabilities required for complex biomedical analyses. Therefore, there is an urgent need to develop a universal three-dimensional DNA walker with high specificity and efficient movement for advanced cellular bioimaging applications. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention aims to provide a spatially confined bipedal DNA nanomotor with enhanced dynamics, its preparation method, and its application. This nanomotor is powered by intramolecular entropy-driven catalysis and is used for rapid, sensitive, and reliable detection and simultaneous imaging of various mRNAs in living cells.

[0005] This invention, based on the entropy-driven principle, provides a promising strategy for detecting low-abundance biomarkers using DNA amplifiers. Compared to catalytic hairpin assembly and hybridization chain reactions, this entropy-driven catalytic strategy is a thermodynamic process driven by the increase in net entropy rather than the free energy of forming new base pairs. However, the practical application of this EDC technology faces major limitations, including low stability to heat and nucleases, and the inability of the DNA amplifier to autonomously enter cells. Gold nanoparticles possess a range of properties beneficial for biosensing, such as inherent cell permeability (primarily through endocytosis), strong affinity for nucleic acids, high resistance to degradation, and distance-dependent fluorescence quenching. These properties make AuNPs an ideal platform for developing diverse fluorescent nanosensors that respond to specific endogenous biomolecules. In this work, by integrating the EDC strategy with gold nanoparticles, a bipedal DNA nanomotor was designed, allowing for high-contrast imaging of multiple mRNAs. We demonstrate that the bipedal DNA nanomotor exhibits superior sensitivity (501 aM for survivin mRNA and 314 aM for TK1 mRNA) and faster kinetics. The developed bipedal DNA nanomotor can achieve accurate intracellular quantification and reliable differentiation of the levels of multiple mRNAs in breast cancer tissue and its normal counterpart tissue, and perform multiplex mRNA imaging in living cells.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing a spatially confined bipedal DNA nanomotor with enhanced dynamics.

[0008] A method for preparing a spatially confined bipedal DNA nanomotor with enhanced kinetics includes the following steps:

[0009] Step 1: Anneal three single-stranded DNA linkers 1, AS1-1, and AS1-2 to form a Cy5-substrate probe; anneal three single-stranded DNA linkers 2, AS2-1, and AS2-2 to form a Cy3-substrate probe; anneal three single-stranded DNA blockers-1, triggers 1-1, and triggers 1-2 to form a bipedal walker 1; and anneal three single-stranded DNA blockers-2, triggers 2-1, and triggers 2-2 to form a bipedal walker 2.

[0010] Step 2: Mix Cy5-substrate probe with AuNPs solution and assemble DNA nanomotor 1 by freezing method; mix Cy3-substrate probe with AuNPs solution and assemble DNA nanomotor 2 by freezing method.

[0011] Preferably, in step one, the single-stranded DNA linker 1, AS1-1, AS1-2, linker 2, AS2-1, AS2-2, blocker-1, trigger 1-1, trigger 1-2, blocker-2, trigger 2-1, and trigger 2-2 are first dissolved in 1×Tris-EDTA buffer to prepare stock solutions, and then diluted to 10 μM with 1× hybridization buffer. Then, the three single-stranded DNA linkers 1, AS1-1 and AS1-2, the three single-stranded DNA linkers 2, AS2-1 and AS2-2, the three single-stranded DNA blockers 1, trigger 1-1 and trigger 1-2, and the three single-stranded DNA blockers 2, trigger 2-1, and trigger 2-2 are heated at 95°C for 5 minutes, and then slowly cooled to 25°C.

[0012] Preferably, in step two, the freezing assembly involves rotating the mixed solution on a drum mixer for 10 minutes, followed by freezing at -20°C overnight.

[0013] Preferably, in step one, the sequence of the DNA single-stranded linker 1 is 5′-SH-TTT TTT TTT TGGATG CAG AGG TTG ATT GAA TGC CGG GAT TAG GCA TGT AGA GAT GCG GT-3′;

[0014] In DNA single-stranded linker 1, a thiol group (SH) is modified at the 5′ end.

[0015] Preferably, in step one, the sequence of the single-stranded DNA AS1-1 is 5′-ATC TCT ACA TGC CTA ATCCC-3′.

[0016] Preferably, in step one, the sequence of the single-stranded DNA AS1-2 is 5′-GGC ATT CAA TCA ACC TCTGCA TCC-Cy5-3′;

[0017] In the single-stranded DNA AS1-2, a fluorophore Cy5 is modified at the 3′ end.

[0018] Preferably, in step one, the sequence of the DNA single-stranded linker 2 is 5′-SH-TTT TTT TTT TGCTGA GAT GAA CAA GAA GCG GGA TAT CAA CAT CAG CGA GTG TCT TTG-3′;

[0019] In the DNA single-stranded linker 2, a thiol (SH) group is modified at the 5′ end.

[0020] Preferably, in step one, the sequence of the single-stranded DNA AS2-1 is 5′-CAC TCG CTG ATG TTG ATATCC C-3′.

[0021] Preferably, in step one, the sequence of the single-stranded DNA AS2-2 is 5′-GCT TCT TGT TCA TCT CAGC-Cy3-3′;

[0022] In the single-stranded DNA AS2-2, a fluorophore Cy3 is modified at the 3′ end.

[0023] Preferably, in step one, the sequence of the DNA single-stranded blocker-1 is 5′-GGC ATG TAG AGA TGCGGT CCT TGA GA-3′.

[0024] Preferably, in step one, the sequence of the DNA single-stranded trigger 1-1 is 5′-ACC GCA TCT CTACAT GCC TAA TTT TTT TTT TTT TTT AAT CCG ATG CAC AGT T-3′.

[0025] Preferably, in step one, the sequence of the DNA single-stranded trigger 1-2 is 5′-ACC GCA TCT CTACAT GCC TAA TTT TTT TTT TTT TTT AAC TGT GCA TCG GAT T-3′.

[0026] Preferably, in step one, the sequence of the DNA single-stranded blocker-2 is 5′-TCA GCG AGT GTC TTTGGC ATA CTT G-3′.

[0027] Preferably, in step one, the sequence of the DNA single-stranded trigger 2-1 is 5′-CAA AGA CAC TCGCTG ATG TTG ATT TTT TTT TTT TTT TAA TCC GAT GCA CAG TT-3′.

[0028] Preferably, in step one, the sequence of the DNA single-stranded trigger 2-2 is 5′-CAA AGA CAC TCGCTG ATG TTG ATT TTT TTT TTT TTT TAA CTG TGC ATC GGA TT-3′.

[0029] Secondly, the present invention provides a spatially confined bipedal DNA nanomotor with enhanced dynamics.

[0030] A spatially confined bipedal DNA nanomotor with enhanced kinetics prepared by the method described above.

[0031] Thirdly, the present invention provides an application of a spatially confined bipedal DNA nanomotor with enhanced dynamics.

[0032] The application of the spatially confined bipedal DNA nanomotor with enhanced kinetics in the preparation of reagents or kits for detecting mRNA.

[0033] Preferably, the reagent or kit is used for in situ imaging of mRNA in living cells.

[0034] Preferably, the mRNA is survivin mRNA and / or TK1 mRNA;

[0035] The survivin mRNA sequence is 5′-UCU CAA GGA CCA CCG CAU CUC UAC A-3′;

[0036] The TK1 mRNA sequence is 5′-CAA GUA UGC CAA AGA CAC UCG C-3′.

[0037] Preferably, the method of using the reagent or kit includes:

[0038] The reaction solution was prepared and then incubated at 37°C for 50 minutes. The reaction solution contained: DNA nanomotor 1 with a working concentration of 300 nM, DNA nanomotor 2 with a working concentration of 300 nM, bipedal walker 1 with a working concentration of 60 nM, bipedal walker 2 with a working concentration of 60 nM, fuel chain 1 with a working concentration of 400 nM, fuel chain 2 with a working concentration of 400 nM, 1×TH buffer, and the sample to be tested.

[0039] Preferably, the fuel chain F1 is a single-stranded DNA with the sequence 5′-ATC TCT ACA TGC CTA ATC CCG GCATTC AAT CAA CCT CTG CAT CC-3′;

[0040] Preferably, the fuel chain F2 is a single-stranded DNA with the sequence 5′-CAC TCG CTG ATG TTG ATA TCC CGCTTC TTG TTC ATC TCA GC-3′;

[0041] The 1×TH buffer solution comprises 100 mM NaCl, 10 mM MgCl2, and pH 8.0.

[0042] Preferably, different concentrations of survivin mRNA and TK1 mRNA are added to 20 μL of reaction solution, and then incubated at 37°C for 50 minutes.

[0043] Preferably, the reagent or kit has a limit of detection of 501 aM for survivin mRNA and a limit of detection of 314 aM for TK1 mRNA.

[0044] Beneficial Effects: This invention provides a spatially confined bipedal DNA nanomotor with enhanced kinetics for rapid imaging of dual cancer-associated coding RNAs (mRNAs). Compared with reported mRNA detection methods, this DNA nanomotor has unique advantages: (1) Probe design is significantly simplified by using only single-stranded DNA, thus avoiding the need for complex engineered hairpin probes; (2) Nucleic acid-functionalized AuNPs can be directly internalized by cells through endocytosis without the need for external transfection reagents; (3) The synergistic combination of the proximity effect of the bipedal trigger strand and the spatial confinement provided by the DNA nanomotor significantly accelerates the reaction kinetics, resulting in more effective signal amplification than traditional EDC systems; (4) The inherent nuclease resistance of the DNA nanomotor prevents the degradation of the reaction probe, thereby significantly enhancing the signal output; (5) The combination of the DNA nanomotor and single-molecule detection promotes ultrasensitive measurement of low-expression survivin mRNA and TK1 mRNA in complex biological matrices; (6) This DNA nanomotor system can complete the determination in one step at physiological temperature without the need for precise temperature control or cumbersome washing and separation steps. With its enhanced biostability, superior cellular uptake capacity, high imaging contrast, rapid reaction kinetics, and efficient signal amplification, this DNA nanomotor represents a universal platform for simultaneously imaging intracellular survivin mRNA and TK1 mRNA. As a versatile platform, this DNA nanomotor can accurately quantify survivin mRNA and TK1 mRNA in MCF-7 cells, distinguish breast cancer patients from healthy individuals, and track mRNA dynamics in living cells in real time. By simply modifying the recognition sequence of the bipedal trigger chain, the modular design of this DNA nanomotor can be further adapted to image a variety of intracellular targets, such as non-coding RNA, small molecules, and enzymes, thus highlighting its broad potential in precision medicine and clinical diagnostics. Attached Figure Description

[0045] Figure 1A represents the design and assembly of the blocked bipedal trigger and three-dimensional DNA nanomotor; B represents the working principle of the EDC catalytic cycle; and C represents the operating principle of the bipedal DNA nanomotor platform for live-cell multiplex mRNA imaging.

[0046] Figure 2 Construction of intelligent DNA nanomotors; A shows the stepwise assembly of Cy5-AuNPs and Cy3-AuNPs verified by 2% agarose gel electrophoresis analysis, with lanes from left to right representing naked AuNPs, Cy5-AuNPs, and Cy3-AuNPs; B shows the UV-Vis absorption spectra of naked AuNPs, substrate probes, and Cy5-AuNPs; C shows the Zeta potential values ​​of AuNPs, Cy5-AuNPs, and Cy3-AuNPs in Tris-HCl; DF shows TEM images of naked AuNPs, Cy5-AuNPs, and Cy3-AuNPs, scale bar: 10 nm; GI shows the DLS characterization of naked AuNPs, Cy5-AuNPs, and Cy3-AuNPs; J and K show high-angle annular dark-field scanning TEM images of Cy5-AuNPs and Cy3-AuNPs and corresponding elemental mapping analysis, scale bar: 10 nm. nm, error bars: mean ± standard deviation (n=3).

[0047] Figure 3A: Validation of target-induced bipedal DNA trigger chain activation using 12% non-denaturing PAGE analysis. Lane M: marker; Lane 1: trigger chain 1-1; Lane 2: blocking chain-1; Lane 3: survivin mRNA; Lane 4: blocked bipedal trigger chain; Lane 5: activated bipedal trigger chain; Lane 6: survivin mRNA + blocking chain-1; Lane 7: survivin mRNA + blocked bipedal trigger chain; Lane 8: blocked bipedal trigger chain; B: Feasibility assessment of the bipedal DNA trigger chain-induced EDC circuit using 12% non-denaturing PAGE. Lane M, marker; Lane 1, auxiliary strand 1-1; Lane 2, auxiliary strand 1-2; Lane 3, fuel strand; Lane 4, survivin mRNA + blocking strand -1; Lane 5, blocked bipedal trigger strand; Lane 6, Cy5-AuNPs; Lane 7, survivin mRNA + Cy5-AuNPs + blocked bipedal trigger strand + fuel strand; Lane 8, Cy5-AuNPs + blocked bipedal trigger strand + fuel strand; C and E are the fluorescence emission spectra of Cy5 and Cy3 with and without target mRNA, respectively; D and F are the fluorescence intensities of Cy5 and Cy3 with and without target mRNA, respectively; G is the single-molecule imaging map of the response control, survivin mRNA, TK1 mRNA, and survivin mRNA + TK1 mRNA, respectively, scale bar = 5 μm; H and I are the fluorescence intensity trajectories of a Cy5 point and a Cy3 point over time, respectively. Error bars represent the mean ± standard deviation (n = 3).

[0048] Figure 4 A represents the reaction kinetics detection diagrams for the bipedal system, 2-monopaline system, monopedal system, bipedal system without AuNPs, and monopedal system without AuNPs; BF represents the real-time monitoring of FAM fluorescence signals with or without survivin mRNA added to the bipedal system, 2-monopaline system, monopedal system, bipedal system without AuNPs, and monopedal system without AuNPs, respectively; GK represents the calibration curves of Cy5 counts against different concentrations of survivin mRNA obtained using the bipedal system, 2-monopaline system, monopedal system, bipedal system without AuNPs, and monopedal system without AuNPs, respectively; LP represents the real-time monitoring of FAM fluorescence signals with or without TK1 mRNA added to the bipedal system, 2-monopaline system, monopedal system, bipedal system without AuNPs, and monopedal system without AuNPs, respectively; QU represents the Cy3 counts against different concentrations of TK1 mRNA obtained using the bipedal system, 2-monopaline system, monopedal system, bipedal system without AuNPs, and monopedal system without AuNPs, respectively. Calibration curve of mRNA.

[0049] Figure 5 A and D represent Cy5 and Cy3 counts generated under different conditions: target mRNA mixed with all interfering RNAs, target mRNA alone, interfering RNA group, and control group without any RNA; B and E represent Cy5 and Cy3 counts induced by survivin mRNA, TK1 mRNA, and their respective mismatched mRNAs; C and F represent Cy5 and Cy3 counts generated by survivin mRNA and TK1 mRNA alone and their mixture with all interfering RNAs, ***p < 0.001; G and J represent the expression levels of survivin mRNA and TK1 mRNA in different cell lines detected by bipedal DNA nanomotors and qRT-PCR, ***p < 0.001; H and K represent the correlation analysis between the proposed DNA nanomotors and qRT-PCR detection of survivin mRNA and TK1 mRNA in different human cells; I and L represent the linear correlation between Cy5 and Cy3 counts and the number of cancer cells, with error bars representing mean ± standard deviation (n = 3).

[0050] Figure 6 A is a schematic diagram of the procedure for quantifying survivin mRNA and TK1 mRNA in breast tissue using bipedal DNA nanomotors; B and E are heatmaps of Cy5 and Cy3 counts generated in breast cancer tissue and healthy control tissue, respectively; C and F are analyses of survivin mRNA and TK1 mRNA levels in breast cancer tissue and healthy control tissue measured by DNA nanomotors and qRT-PCR, respectively; D and G are box plots of survivin mRNA and TK1 mRNA levels in breast cancer tissue and healthy control tissue measured by the proposed DNA nanomotors and qRT-PCR, respectively; H and J are ROC curves for distinguishing breast cancer patients from healthy individuals using the developed DNA nanomotor system and qRT-PCR to detect survivin mRNA alone, TK1 mRNA alone, and the combination of both; K and M are analyses based on the confusion matrix to evaluate the performance of the developed DNA nanomotor system in distinguishing breast cancer patients from healthy individuals when using survivin mRNA alone, TK1 mRNA alone, and the combined detection of survivin mRNA and TK1 mRNA. Error bars represent mean ± standard deviation (n = 3).

[0051] Figure 7Imaging of multiplex mRNAs in live cells; where: A shows fluorescence images and corresponding average fluorescence intensity quantification of MCF-10A and MCF-7 cells after incubation with non-quenched fluorescent nanomotors; B shows fluorescence images and MFI quantification of survivin mRNA and TK1 mRNA in MCF-7, HeLa, HepG-2, A549, and MCF-10A cells, scale bar: 50 μm; C and D are flow cytometry analyses of the fluorescence intensity of Cy5 and Cy3 in the cell lines shown, respectively, with error bars representing mean ± standard deviation (n = 3).

[0052] Figure 8 A shows fluorescence images and corresponding mean fluorescence intensity quantification of cells treated with survivin mimics, PBS, and survivin inhibitors; B shows in situ fluorescence images and MFI quantification of TK1 mRNA in cells treated with β-estradiol, PBS, and tamoxifen; C shows fluorescence images and MFI quantification of cells treated with bipedal, 2-monopal, monopal, AuNP-free bipedal, or AuNP-free monopal systems. Scale bar: 50 μm; D and E are flow cytometry analyses of Cy5 and Cy3 fluorescence intensities corresponding to A; F and G are flow cytometry analyses of Cy5 and Cy3 fluorescence intensities corresponding to B; H and I are flow cytometry analyses of Cy5 and Cy3 fluorescence intensities corresponding to C. Error bars represent mean ± standard deviation (n = 3). Detailed Implementation

[0053] The present invention will be further explained below with reference to the accompanying drawings and embodiments.

[0054] Reagents and materials:

[0055] All DNA oligonucleotides (linker 1, AS1-1, AS1-2, linker 2, AS2-1, AS2-2, blocker-1, trigger 1-1, trigger 1-2, blocker-2, trigger 2-1, trigger 2-2, survivin mRNA, TK1 mRNA, F1, and F2) were synthesized and purified by Shanghai Sangon Biotech Co., Ltd. 10 nm AuNPs were purchased from Ruixi Biotechnology Co., Ltd. (Xi'an, Shaanxi). Dithiothreitol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Hoechst 33342 nuclear dye was purchased from Nanjing Kaiji Biotechnology Development Co., Ltd. Opti-MEM, fetal bovine serum, and cell culture medium were purchased from Gibco. Lipofectamine-3000 was purchased from Thermo Fisher Scientific. Human breast cancer cell lines (MCF-7 cells), human lung adenocarcinoma cell lines (A549 cells), human cervical cancer cell lines (HeLa cells), human liver cancer cell lines (HepG-2 cells), and normal human breast cell lines (MCF-10A cells) were purchased from the Cell Bank of the Chinese Academy of Sciences. Phosphate-buffered saline was purchased from Shanghai Pasteur Technology Co., Ltd. SYBR Gold was purchased from Life Technologies. All other chemical reagents were of analytical grade and used directly without further purification. Paraffin-embedded tissue samples were obtained from Nanjing Drum Tower Hospital, and this study was approved by the Ethics Committee of Nanjing Drum Tower Hospital. All experiments used ultrapure water obtained through a Millipore filtration system.

[0056] Single-molecule detection and actual sample measurement:

[0057] The reaction product was diluted 1000-fold with imaging buffer. A 10 μL sample was directly added to a coverslip for measurement. TIRF imaging was performed using an inverted Olympus IX71 microscope, with Cy5 molecules excited by a 640 nm laser and Cy3 molecules excited by a 580 nm laser. Photons from Cy5 and Cy3 molecules were collected through a 100x oil immersion objective and imaged onto two halves of an Andor Ixon EMCCD camera (DU897) at an exposure time of 500 ms. Each sample was imaged 15 times, and 15 images were randomly selected. The Analyze Particles function in ImageJ software was used to process single-molecule images to determine the number of Cy5 and Cy3 spots, with the particle size set to 2–10 pixels to reduce false positives caused by noise. Cy5 and Cy3 spots within the 400×400 pixel imaging area were counted.

[0058] MCF-7, HeLa, HepG-2, and A549 cells were cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C under a humidified atmosphere of 5% CO2. MCF-10A cells were cultured in MCF 10A complete medium. Cell counts were performed using a Countstar automated cell counter prior to extraction. One million cells were transferred to pre-chilled 1.5 mL EP tubes and washed twice with ice-cold PBS buffer. Total RNA was extracted from the cells using the SteadyPure Rapid RNA Extraction Kit. Total RNA from tissues was prepared using the miRNeasy FFPE kit. Total RNA concentration was determined using a NanoDrop 2000 spectrophotometer.

[0059] Detection by gel electrophoresis and fluorescence spectroscopy:

[0060] DNA products were analyzed using the Bio-Rad ChemiDoc MP imaging system. To analyze target-mediated release of the bipedal trigger chain, we performed 12% non-denaturing polyacrylamide gel electrophoresis in 1× TBE buffer at a constant voltage of 110 V for 45 minutes at room temperature. To investigate the feasibility of entropy-driven amplifiers induced by the bipedal trigger chain, we performed 14% PAGE in 1× TBE buffer at a constant voltage of 110 V for 70 minutes at room temperature. After electrophoresis, the gels were stained with SYBR Gold and observed using the ChemiDoc™ MP imaging system. DNA nanomotors and naked AuNPs were characterized using 1% agarose gel electrophoresis in 1× TAE buffer at a constant voltage of 110 V for 40 minutes.

[0061] Fluorescence emission spectra of Cy5 and Cy3 were measured at room temperature using an FLS 1000 photoluminescence spectrometer. For quantification of survivin mRNA, the emission spectrum of Cy5 was recorded at an excitation wavelength of 635 nm, ranging from 650 to 750 nm. The fluorescence intensity at 664 nm was used for analysis. For quantification of TK1 mRNA, the emission spectrum of Cy3 was recorded at an excitation wavelength of 535 nm, ranging from 550 to 650 nm. The fluorescence intensity at 566 nm was used for analysis.

[0062] A highly efficient bipedal DNA nanomotor was developed by spatially organizing an EDC circuit on AuNPs. In this system, an mRNA-triggered bipedal walker moves stepwise across the AuNP surface via a fuel-assisted EDC cascade, resulting in an amplified fluorescence signal for sensitive imaging of survivin mRNA and TK1 mRNA in live cells. Compared with reported mRNA detection methods, this DNA nanomotor has unique advantages: (1) It significantly simplifies probe design by using only single-stranded DNA, thus avoiding the need for complex engineered hairpin probes; (2) Nucleic acid-functionalized AuNPs can be directly internalized by cells through endocytosis without the need for external transfection reagents; (3) The synergistic combination of the proximity effect of the bipedal trigger strand and the spatial confinement provided by the DNA nanomotor significantly accelerates the reaction kinetics, resulting in more effective signal amplification than traditional EDC systems; (4) The inherent nuclease resistance of the DNA nanomotor prevents the degradation of the reaction probe, thereby significantly enhancing the signal output; (5) The combination of the DNA nanomotor and single-molecule detection promotes ultrasensitive measurement of low-expression survivin mRNA and TK1 mRNA in complex biological matrices; (6) This DNA nanomotor system can complete the determination in one step at physiological temperature without the need for precise temperature control or cumbersome washing and separation steps. With its enhanced biostability, superior cellular uptake capacity, high imaging contrast, rapid reaction kinetics, and efficient signal amplification, this DNA nanomotor represents a universal platform for simultaneously imaging intracellular survivin mRNA and TK1 mRNA. As a versatile platform, this DNA nanomotor can accurately quantify survivin mRNA and TK1 mRNA in MCF-7 cells, distinguish breast cancer patients from healthy individuals, and track mRNA dynamics in living cells in real time. By simply modifying the recognition sequence of the bipedal trigger chain, the modular design of this DNA nanomotor can be further adapted to image a variety of intracellular targets, such as non-coding RNA, small molecules, and enzymes, thus highlighting its broad potential in precision medicine and clinical diagnostics.

[0063] Example 1: Mechanism of simultaneous imaging of multiple mRNAs

[0064] like Figure 1As shown in Figure A, the bipedal DNA catalyst comprises a bridging strand connecting two identical catalytic domains. Before activation, this bridging strand hybridizes with the blocking strand to form a blocked bipedal triggering strand. A triple-stranded DNA substrate is prepared by thermally annealing a mixture of the linker strand, auxiliary strand-1, and auxiliary strand-2. This triple-stranded DNA substrate serves as both a signal probe and a stepping site for the bipedal triggering strand. This three-dimensional nanomotor structure is constructed by immobilizing the triple-stranded DNA substrate on the surface of gold nanoparticles via Au-S bonds. In this configuration, the fluorescence of Cy5 and Cy3 is effectively quenched by AuNPs through a nanometal surface energy transfer mechanism. Subsequently, the blocked bipedal triggering strand and the three-dimensional nanomotor are delivered to the cytoplasm of cancer cells via liposome transfection. In the cytoplasm, endogenous survivin mRNA and TK1 mRNA hybridize with the blocking strand immobilized on the blocked bipedal triggering strand via a strand displacement reaction, thereby activating the bipedal triggering strand. Figure 1 As shown in Figure B, the activated bipedal trigger chain interacts with the linker strand on the triple-stranded DNA substrate via a sticky-end-mediated strand displacement. This interaction induces the dissociation of helper strand 1 and the formation of a new triple-stranded intermediate, while simultaneously exposing a new sticky-end region in the middle of the linker strand. Subsequently, an additional fuel strand hybridizes further with the newly exposed sticky-end region on the linker strand, thereby inducing the release of helper strand 2 and the bipedal trigger chain, while generating a linker strand / fuel strand double-stranded complex. The released bipedal trigger chain then diffuses and binds to an intact triple-stranded DNA substrate, thereby triggering a new round of EDC catalytic cycling. This self-driven EDC circuit promotes the dissociation of the triple-stranded DNA substrate while releasing large quantities of helper strand 1 and helper strand 2. Specifically, helper strands 1-2 and 2-2 are labeled with Cy5 and Cy3 at their 3' ends, respectively. Figure 1As shown in Figure C, an EDC catalytic cycle initiated by a bipedal trigger chain progressively separates the Cy5 and Cy3-modified auxiliary chains from the AuNP surface. Increasing distance weakens the quenching effect, leading to the recovery of amplified Cy5 and Cy3 fluorescence signals. This mechanism enables highly selective and sensitive imaging of survivin mRNA and TK1 mRNA in live cells. Notably, the separate emission spectra of Cy5 and Cy3 result in negligible crosstalk, enabling precise and simultaneous quantification of multiple mRNA targets without spectral interference. In contrast, in the absence of target survivin or TK1 mRNA, the bipedal trigger chain remains inactive, preventing the initiation of the sticky-end-mediated chain displacement cascade reaction, ultimately resulting in undetectable Cy5 or Cy3 fluorescence signals. Compared to monopedal systems, the bipedal design enables more mobile walking and faster kinetics on a single DNA nanomotor, resulting in superior amplification efficiency. To our knowledge, this work is the first to realize an EDC-driven bipedal DNA nanomotor in live cells. This system provides a powerful platform for intracellular sensing, with enhanced sensitivity and faster response kinetics, and holds great promise for detecting low-abundance biomarkers in basic cell biology and clinical diagnostics.

[0065] Example 2: Engineering Construction and Characterization of Bipedal DNA Nanomotors

[0066] DNA nanomotors were constructed by immobilizing substrate probes onto 10 nm AuNPs via gold-sulfur bonds using a cryo-assisted labeling method. We first characterized the resulting DNA nanomotors. Figure 2 AuNPs were modified with Cy5-labeled substrate probes (linker / helper strand 1-1 / helper strand 1-2) and Cy3-labeled substrate probes (linker / helper strand 2-1 / helper strand 2-2) to generate DNA nanomotors A (Cy5-AuNPs) and B (Cy3-AuNPs), respectively. The DNA nanomotors were characterized by agarose gel electrophoresis. Figure 2 (A). Compared to bare AuNPs aggregated at the starting position ( Figure 2 Middle A, Lane 1), Cy5-AuNPs ( Figure 2 Middle A, lane 2) and Cy3-AuNPs ( Figure 2 Lane A, lane 3, showed a faster electrophoretic migration. UV-Vis absorption spectra were also collected to explore the modification of AuNPs with DNA oligonucleotides. Figure 2 (B). Compared to naked AuNPs, the maximum absorption peak of DNA-modified AuNPs is lower than that of naked AuNPs (520 nm). Figure 2 (Middle B, green curve) redshifted to 524 nm ( Figure 2(Middle B, red curve) This is because the assembly of the substrate probe caused a change in the dielectric constant of the environment surrounding the AuNPs. Furthermore, the absorption peak of the substrate probe at 260 nm was observed (…). Figure 2 (Middle B, purple curve) and the characteristic DNA absorption peak of DNA-modified AuNPs at 260 nm ( Figure 2 (Middle B, red curve). UV-Vis absorption spectroscopy confirmed the successful modification of the substrate probe onto AuNPs. Figure 2 As shown in Figure C, zeta potential measurements were used to characterize the changes in electrostatic potential of naked AuNPs and DNA nanomotors. The zeta potential of naked AuNPs was -7.83 mV ( Figure 2 (C, green column), while DNA nanomotors showed a reduced potential: Cy5-AuNPs were -17.53 mV ( Figure 2 (C in the middle, pink column), Cy3-AuNPs is -19.11 mV ( Figure 2 (C, blue column). This difference can be attributed to the electrical properties of the oligonucleotides, which significantly enhance the negative charge density of the nanomotors. This result further demonstrates the attachment of thiolated DNA substrate probes to AuNPs. Transmission electron microscopy images of naked AuNPs and DNA-modified AuNPs show that they possess spherical morphology and good dispersion, without aggregation (…). Figure 2 (middle DF). TEM images show that the average diameter of bare AuNPs is 10 nm ( Figure 2 (D), while the average diameters of Cy5-AuNPs and Cy3-AuNPs are both greater than 10 nm ( Figure 2 In Figures E and F, halos were observed around AuNPs in the DNA nanomotors, indicating the successful construction of the DNA nanomotors. The size distribution of the bare AuNPs and DNA nanomotors was evaluated using dynamic light scattering measurements. Figure 2 (Medium GI). After oligonucleotide modification, the diameter decreased from 10.48 nm ( Figure 2 The wavelength of G increased to 14.14 nm. Figure 2 (H) and 14.91 nm ( Figure 2 (I), which is similar to the size distribution in the TEM image ( Figure 2 (Middle DF). Additional elemental spectral results ( Figure 2 The results (J and K) show that the DNA nanomotors are composed of the characteristic elements Au, P and S, indicating that the AuNPs are coated with thiolated DNA substrate probes.

[0067] Example 3: Feasibility verification and single-molecule imaging of bipedal DNA nanomotors

[0068] The feasibility of using this bipedal DNA nanomotor for in vitro messenger RNA detection was verified. We analyzed the target-mediated release of the bipedal trigger chain using 12% non-denaturing PAGE. Figure 3 (A) In Figure 3 In diagram A, lanes 1-3 represent trigger strand 1-1, blocking strand-1, and survivin mRNA, respectively. When trigger strand 1-1, trigger strand 1-2, and blocking strand-1 are mixed and annealed simultaneously, a high molecular weight band is observed. Figure 3 (Channel A, Lane 4) indicates that trigger strands 1-1 and 1-2 are locked by blocking strand-1 to form a closed bipedal trigger strand. When survivin mRNA is present, two distinct strand substitution product bands are observed ( Figure 3 Middle A, lane 7), respectively with the activated bipedal trigger chain ( Figure 3 (Channel A, 5th) and double-stranded closed-strand 1--survivin mRNA ( Figure 3 Size matching (A, lane 6). However, in the absence of survivin mRNA, only one distinct closed bipedal trigger chain band was observed ( Figure 3 (Chapter A, No. 8) indicates that survivin mRNA can mediate efficient activation of the bipedal trigger chain. Furthermore, we investigated the feasibility of entropy-driven amplifiers induced by the bipedal trigger chain using 12% non-denaturing PAGE. In the absence of survivin mRNA, we observed a corresponding effect on the closed bipedal trigger chain (…). Figure 3 Middle B, lane 5), Cy5-AuNPs ( Figure 3 (Channel B, Lane 6) and fuel chain ( Figure 3 Characteristic bands of (Channel B, Lane 3) Figure 3 (Medium B, Question 8). Conversely, when survivin mRNA is present, the corresponding AS1-2 ( Figure 3 (Middle B, second channel) and double-stranded closed-strand 1--survivin mRNA ( Figure 3 The characteristic bands of (Channel B, Track 4) are accompanied by the disappearance of fuel chain bands. Figure 3 (Medium B, lane 3). Furthermore, a distinct band was detected that migrated faster than the closed bipedal trigger chain band ( Figure 3 (Channel B, Channel 5), which corresponds to the smaller molecular weight activated bipedal trigger strand. These results indicate that only survivin mRNA can induce a strand displacement reaction to release the bipedal trigger strand, thereby initiating an entropy-driven DNA catalysis (EDC) circuit to generate an amplified signal.

[0069] We performed fluorescence measurements to verify the feasibility of this bipedal DNA nanomotor. Figure 3 C and E). When survivin mRNA is missing ( Figure 3(C, green curve) and TK1 mRNA ( Figure 3 When the fluorescence signal is in the middle (orange curve), a very low fluorescence signal is obtained, but when survivin mRNA is present (…), a very low fluorescence signal is obtained. Figure 3 (C, red curve) and TK1 mRNA ( Figure 3 At point E (blue curve), a significant fluorescence signal was observed, which was 6.22 times stronger than the control lacking survivin mRNA. Figure 3 The levels of D and the control group lacking TK1 mRNA were enhanced by 8.94 times (D). Figure 3 These results clearly demonstrate that survivin mRNA and TK1 mRNA can catalyze the unlocking of the bipedal trigger chain, and the activated bipedal trigger chain can initiate the EDC catalytic cycle, ultimately inducing the recovery of Cy5 and Cy3 fluorescence signals. Single-molecule fluorescence imaging has significant advantages over bulk fluorescence measurement, allowing for visualization and sensitive quantification of target biomolecules with minimal interference and low sample consumption. We further performed TIRF-based fluorescence imaging to detect multiplex mRNAs at the single-molecule level (…). Figure 3 (G). When survivin mRNA and TK1 mRNA were absent, no Cy5 spots were observed ( Figure 3 No Cy3 spots were observed (Ga), nor were any observed in Cy3 spots. Figure 3 The presence of Ge in the middle layer indicates that neither the bipedal trigger chain nor the EDC circuit was activated. Conversely, Cy5 spots were detected only in response to survivin mRNA. Figure 3 (Medium Gb), but no Cy3 spots were observed ( Figure 3 Gf). Cy3 spots were observed only in response to TK1 mRNA ( Figure 3 (Gg), no Cy5 spots were produced ( Figure 3 Gc). When survivin mRNA and TK1 mRNA are present simultaneously, Cy5 ( Figure 3 Gd and Cy3 Figure 3 The Cy5 (Gh) spots appeared simultaneously. We further measured the single-step photobleaching trajectories of the Cy5 and Cy3 fluorescent spots. Notably, the Cy5 (Gh) spots in the single-molecule image... Figure 3 (H) and Cy3 ( Figure 3 The spots in the middle (I) all showed single-step photobleaching, confirming that the observed spots represent individual Cy5 / Cy3 molecules.

[0070] Example 4: Reaction Kinetics and Detection Sensitivity

[0071] We tested bipedal DNA nanomotors for the in vitro detection of survivin mRNA and TK1 mRNA under optimal conditions. We designed five different systems to study the kinetics: an AuNP-modified bipedal DNA nanomotor system (bipedal system) (…). Figure 4 Bi-monopod DNA nanomotor system modified with Aa) and AuNP (2-monopod system) Figure 4 Monopodial DNA nanomotor systems modified with Ab and AuNP (monopodial systems) Figure 4 (Ac), bipedal EDC system without AuNPs (n bipedal system) Figure 4 (Ad) and monopodial EDC systems without AuNPs (n monopodial systems) Figure 4 The bipedal system utilizes the inherent spherical structure of gold nanoparticles and the superior efficiency of the bipedal triggering chain, thus exhibiting the fastest reaction kinetics and the most significant fluorescence response in the tested system. Figure 4 (A). Time-dependent fluorescence analysis was performed to investigate the reaction kinetics of bipedal, 2-monopal, monopal, n-bipedal, and n-monopal systems. In the absence of survivin mRNA and TK1 mRNA, negligible Cy5 and Cy3 fluorescence signals were observed in the bipedal, 2-monopal, monopal, n-bipedal, and n-monopal systems, respectively. Figure 4 BF is survivin mRNA. Figure 4 LP is TK1 mRNA. The presence of survivin mRNA and TK1 mRNA induced the enhancement of Cy5 and Cy3 fluorescence signals in bipedal, 2-monopalal, monopalal, n-bipedal, and n-monopal systems. Figure 4 BF is survivin mRNA. Figure 4 LP is TK1 mRNA. Notably, the presence of survivin mRNA and TK1 mRNA caused a rapid increase in Cy5 and Cy3 fluorescence in the bipedal system. Figure 4 (B and L). For survivin mRNA detection, the rate constant measured by the bipedal system was 1.36 ( Figure 4 The value of B was significantly higher than the 0.70 observed in the 2-monopod system. Figure 4 (C), 0.50 observed in the monopodial system Figure 4 The 0.29 observed in the bipedal system (D) and n-type systems Figure 4 The 0.23 observed in the n-monopod system (E) and the n-monopod system Figure 4 (F). Similarly, for TK1 mRNA detection, the rate constant measured by the bipedal system was 1.09 (F). Figure 4The value of L was significantly higher than the 0.57 observed in the 2-monopod system. Figure 4 The 0.46 observed in the monopodial system (M) Figure 4 The 0.34 observed in the bipedal system (N) and n-type systems Figure 4 The 0.19 observed in the n-monopod system (O) and n-monopod system Figure 4 (P). For survivin mRNA detection, the maximum fluorescence intensity of Cy5 in the bipedal system was 1.31 times, 1.91 times, 2.59 times, and 3.26 times that of the induced intensity of the 2-monopedal system, monopedal system, n-bipedal system, and n-monopedal system, respectively. Figure 4 (BF). For TK1 mRNA detection, the maximum fluorescence intensity of Cy3 in the bipedal system was 1.39 times, 2.37 times, 3.20 times, and 3.57 times that of the induced intensity of the 2-monopedal system, monopedal system, n-bipedal system, and n-monopedal system, respectively. Figure 4 (LP). These results clearly demonstrate that the designed bipedal system can significantly accelerate the kinetics of the EDC reaction by increasing the local concentration of reactants and restricting the movement of the bipedal trigger chain, thus endowing the nanomotor with high-efficiency scale-up capability.

[0072] In vitro detection of target survivin mRNA and TK1 mRNA was performed by measuring fluorescence recovery. The amplification efficiency of the bipedal system was first compared with that of the 2-monopalal, monopedal, n-bipedal, and n-monopalal systems. Survivin mRNA and TK1 mRNA induced a dose-responsive increase in Cy5 and Cy3 counts, and the counts (N) showed a significant linear correlation with the logarithm (C) of the target concentration. The regression equations were: survivin mRNA: N = 29.45 log 10 C + 501.26 (R 2 =0.9979) Figure 4 (G) and TK1 mRNA: N = 30.21 log 10 C + 505.13 (R 2 = 0.9991) Figure 4 The LOD values ​​were 501 aM for survivin mRNA and 314 aM for TK1 mRNA, respectively. For comparison, we examined the sensitivity of the 2-monopod system, monopod system, n-bipod system, and n-monopod system. For survivin mRNA detection, the sensitivity of the bipod system (…) Figure 4 The value of G in the middle is slightly higher than that obtained by the 2-monopod system. Figure 4 (H), compared to the value obtained by the monopodary system ( Figure 4 The value obtained by the bipedal system (I) is one order of magnitude higher than that obtained by the bipedal system (I). Figure 4The value of J is two orders of magnitude higher than that obtained by the n-legged system. Figure 4 The TK1 mRNA level was three orders of magnitude higher than that of the bipedal system. Similarly, in TK1 mRNA detection, the bipedal system ( Figure 4 The sensitivity shown by the Q-test is slightly higher than that obtained by the 2-monopod system. Figure 4 R), compared to a monopodial system ( Figure 4 The value of S is one order of magnitude higher than that of the n-bipedal system. Figure 4 The T-value is two orders of magnitude higher than that of the n-legged system ( Figure 4 The difference in performance is three orders of magnitude greater than that in the middle (U). In summary, these results demonstrate that our designed bipedal system achieves optimal detection performance.

[0073] Example 5: In vitro selectivity of bipedal DNA nanomotors and analysis of survivin mRNA and TK1 mRNA levels in different human cell lines

[0074] To investigate the specificity of bipedal DNA nanomotors, we used Egr-1 mRNA, miRNA 210, MALAT1, and piRNA 932 as interfering agents. Figure 5 As shown in Figures A and D, the target survivin mRNA and TK1 mRNA can induce high Cy5 and Cy3 signals, but the Cy5 and Cy3 signals generated by these four interfering RNAs are very weak, indicating that the constructed bipedal DNA nanomotor has good selectivity for survivin mRNA and TK1 mRNA. Furthermore, the addition of interfering RNA did not cause significant changes in the specific Cy5 and Cy3 signals targeting survivin mRNA and TK1 mRNA, respectively. Figure 5 (Columns A and D, purple bars) demonstrate the high specificity and interference resistance of the bipedal system. Notably, single-base, dibase, and triple-base mismatched RNAs (i.e., mis-1, mis-2, and mis-3) and the control only produced lower Cy5 and Cy3 signals. Figure 5 (B and E in the middle). For survivin mRNA detection, the interference values ​​(the influence of other interfering RNAs on target detection) of mis-1, mis-2, and mis-3 were 1.67 × 10⁻⁶. -10 2.65 × 10 -12 and 1.29 × 10 -12 Similarly, for the detection of the target TK1 mRNA, the interference values ​​of mis-1, mis-2, and mis-3 were 2.36 × 10⁻⁶. -7 , 1.78 × 10 -10 , and 4.59 × 10 -11These results confirm that bipedal DNA nanomotors exhibit high specificity and can effectively distinguish single-base mismatches. Next, we used different concentrations of survivin mRNA (… Figure 5 (C) and TK1 mRNA ( Figure 5 The anti-interference ability of DNA nanomotors was evaluated by mixing survivin (F) with all interfering RNAs. The concentrations of both survivin and TK1 mRNA were 10... -7 M, 10 -11 M or 10 -14 M, and Cy5 signaling generated by survivin mRNA alone ( Figure 5 (C, red column) and Cy3 signaling generated by TK1 mRNA alone ( Figure 5 Compared to the blue bar (F), adding interfering RNA did not cause Cy5 ( Figure 5 (C, green bar) and Cy3 signal ( Figure 5 Significant changes in the middle F (purple column).

[0075] We then evaluated the practical application of bipedal DNA nanomotors for multiplex mRNA detection in different human cell lines. This method successfully quantified survivin mRNA and TK1 mRNA in extracts from HeLa, MCF-7, HepG-2, A549, and non-tumorigenic MCF-10A cells. Figure 5 As shown in G and J, MCF-10A cells produced weak Cy5 and Cy3 signals. In contrast, HeLa, MCF-7, HepG-2, and A549 cells produced significantly enhanced Cy5 and Cy3 signals. The expression order of survivin mRNA was HeLa > MCF-7 > HepG-2 > A549 cells, and the expression order of TK1 mRNA was MCF-7 > HepG-2 > HeLa > A549 cells (t-test, P < 0.001), indicating the upregulation of survivin mRNA and TK1 mRNA in human cancer cell lines. We further quantified the relative expression of survivin mRNA and TK1 mRNA in the above human cell lines using standard qRT-PCR, and the results obtained were ( Figure 5 The results for G and J in the study were consistent with those of the proposed DNA nanomotor measurements. Furthermore, a high linear correlation was achieved between the proposed DNA nanomotor and the qRT-PCR method, with a Pearson coefficient of 0.9989 for survivin mRNA detection. Figure 5 The TK1 mRNA detection result was 0.9999 (H), Figure 5(Middle K). Furthermore, both Cy5 and Cy3 counts (N) showed good linear correlations with the logarithms (X) of the number of cancer cells ranging from 10 to 100,000. The linear equations were as follows: survivin mRNA detection: N = 73.59 log 10 X -16.55 (R 2 =0.9944) Figure 5 Detection of TK1 mRNA and TK1: N = 66.57 log 10 X – 27.07 (R 2 = 0.9985) Figure 5 (L). The measured LOD was obtained from survivin mRNA in 6 cells and TK1 mRNA in 6 cells, indicating that the constructed DNA nanomotor can accurately measure survivin mRNA and TK1 mRNA in cancer cells.

[0076] Example 6: Quantitative analysis of Survivin mRNA and TK1 mRNA in human breast tissue

[0077] To assess the potential of this bipedal DNA nanomotor in clinical applications, we quantified the levels of survivin mRNA and TK1 mRNA in breast cancer tissues (n=8) and healthy control tissues (n=8). Figure 6 (A) For example Figure 6 As shown in Figures B and E, the Cy5 and Cy3 counts from breast cancer tissue were significantly higher than those from healthy control tissue, indicating upregulation of survivin mRNA and TK1 mRNA in breast tissue from cancer patients. Furthermore, we used qRT-PCR to measure the relative expression of survivin mRNA and TK1 mRNA in the aforementioned 16 breast tissue samples. Figure 6 C and F). The qRT-PCR results are consistent with those of the proposed bipedal DNA nanomotor (C and F). Figure 6 (B and E). Box plots showed that the mean levels of survivin mRNA and TK1 mRNA in breast cancer tissues were higher than those in healthy control tissues (t-test, P < 0.001). Figure 6 (D and G). Notably, the p-value obtained for the proposed DNA nanomotor was less than 0.001, consistent with the results obtained by qRT-PCR (P < 0.001). Figure 6The presence of D and G indicates that the proposed DNA nanomotors can effectively distinguish between breast cancer tissue and healthy tissue. Furthermore, we analyzed receiver operating characteristic (ROC) curves to evaluate the detection accuracy of the DNA nanomotors. For survivin mRNA detection, the area under the curve (AUC) was determined to be 0.95 (…). Figure 6 The AUC value obtained by qRT-PCR was 0.92 (in H), while the AUC value obtained by qRT-PCR was 0.92 (in H). Figure 6 (H). Similarly, for TK1 mRNA detection, the area under the curve (AUC) was determined to be 0.97 (H). Figure 6 The AUC value obtained by qRT-PCR was 0.95 (I), while that obtained by qRT-PCR was 0.95 (I). Figure 6 (I). However, when two targets are detected simultaneously, the area under the curve (AUC) is determined to be 1 ( Figure 6 The AUC values ​​obtained from qRT-PCR were consistent with those obtained from qRT-PCR, confirming that the DNA nanomotor can effectively distinguish between healthy individuals and breast cancer patients. Quantitative analysis of the confusion matrix showed that for survivin mRNA detection, the DNA nanomotor achieved 100.0% sensitivity, 88.89% specificity, 93.75% accuracy, and 87.50% precision (J). Figure 6 Similarly, quantitative evaluation of the confusion matrix showed that, in TK1 mRNA detection, DNA nanomotors exhibited 100.0% sensitivity, 80.00% specificity, 87.50% accuracy, and 75.00% precision (TK1 mRNA). Figure 6 (L). When simultaneously detecting two targets (survivin mRNA and TK1 mRNA), quantitative assessment of the confusion matrix showed that the sensitivity, specificity, accuracy, and precision of the DNA nanomotor were all improved to 100%. Figure 6 (M). These findings demonstrate that simultaneous detection of dual targets significantly improves the accuracy and precision of DNA nanomotor detection compared to single-target detection. Therefore, this bipedal DNA nanomotor exhibits superior accuracy in distinguishing breast cancer tissue from healthy control tissue, highlighting its promising potential for clinical diagnostic applications.

[0078] Example 7: In situ real-time imaging of survivin mRNA and TK1 mRNA in living cells

[0079] To evaluate the intracellularization and in situ surveillance capabilities of bipedal DNA nanomotors, we used MCF-7 cells, which highly expressed survivin mRNA and TK1 mRNA, as a model cell line. Gold nanoparticles (AuNPs) could enter various cell types without the need for transfection reagents. The linker chain was modified with a thiol group at the 5' end and a Cy5 or Cy3 fluorophore at the 3' end. The linker chain was then covalently coupled to the gold nanoparticles via Au-S bonds, thus constructing a non-quenched fluorescent nanomotor. To assess the intracellular delivery efficiency of the nanomotors in cancer cells and normal cells, we imaged MCF-7 and MCF-10A cells using the non-quenched fluorescent nanomotors. Figure 7 As shown in Figure A, enhanced Cy5 and Cy3 fluorescence signals were observed in both MCF-10A and MCF-7 cells. Statistical analysis revealed no significant difference in fluorescence intensity between MCF-10A and MCF-7 cells (t-test, p > 0.05), indicating that the nanomotors have the same delivery efficiency in cancer cells and normal cells. We then evaluated the specificity of the DNA nanomotors for imaging intracellular survivin and TK1 mRNA. Figure 7 As shown in Figure B, significant Cy5 and Cy3 fluorescence signals were detected in MCF-7, HeLa, HepG-2, and A549 cells, respectively, while no significant signals were observed in MCF-10A cells. This result can be attributed to the high expression of survivin mRNA and TK1 mRNA in cancer cells, while their expression levels are quite low in normal cells. Notably, for survivin mRNA detection, the average fluorescence intensity per cell decreased in the order of HeLa > MCF-7 > HepG-2 > A549 > MCF-10A cells. Figure 7 (B), consistent with results obtained from in vitro measurements and qRT-PCR analysis ( Figure 5 (Middle A). Similarly, for TK1 mRNA detection, the mean fluorescence intensity per cell decreased in the order of MCF-7 > HepG-2 > HeLa > A549 > MCF-10A cells ( Figure 7 (B), consistent with results obtained from in vitro measurements and qRT-PCR analysis ( Figure 5 These findings collectively demonstrate that DNA nanomotors can perform high-contrast, specific imaging of target mRNAs in a variety of living cells. To quantify intracellular survivin mRNA and TK1 mRNA levels, we performed flow cytometry analysis (…). Figure 7 (C and 7D). The results showed that Cy5 (Cy5) was detected in cancer cells. Figure 7 C) and Cy3 ( Figure 7The fluorescence signals in both cells were significantly stronger than those in normal cells. Therefore, these results confirm that the developed strategy can accurately visualize target mRNAs in cancer cells.

[0080] Example 8: Intracellular mRNA Expression Differences and Response Kinetics Analysis

[0081] Intracellular survivin mRNA was upregulated and downregulated by transfection with survivin mimics and anti-survivin, respectively. To regulate TK1 mRNA levels, β-estradiol was used to induce TK1 mRNA upregulation, and tamoxifen was used to induce TK1 mRNA downregulation. Figure 8 As shown in Figure A, compared to the PBS-treated control, the Cy5 fluorescence intensity decreased in MCF-7 cells treated with anti-survivin and increased after treatment with a survivin mimic. In contrast, no significant change was observed in the Cy3 fluorescence signal in cells treated with PBS, anti-survivin, or a survivin mimic. We subsequently performed flow cytometry analysis to validate this finding. Figure 8 As shown in Figure D, MCF-7 cells exposed to the survivin mimicry exhibited a strong Cy5 fluorescence signal compared to the PBS-treated control, while cells treated with anti-survivin showed a significantly weaker signal. Conversely, no significant change in Cy3 fluorescence signal was observed in cells treated with PBS, anti-survivin, or the survivin mimicry. Figure 8 (Middle E). Similarly, compared to the PBS-treated control, MCF-7 cells exposed to β-estradiol showed a strong Cy3 fluorescence signal, while cells treated with tamoxifen showed a significantly weaker signal (Middle E). Figure 8 (Middle B). In contrast, no significant changes in Cy5 fluorescence signal were observed in cells treated with PBS, β-estradiol, or tamoxifen. These results were confirmed by corresponding quantitative flow cytometry data (Middle B). Figure 8 (F and G in the middle). For example Figure 8 As shown in Figure G, compared to the PBS-treated control, the Cy3 fluorescence intensity decreased in MCF-7 cells treated with tamoxifen, while the Cy3 fluorescence intensity increased in cells treated with β-estradiol. In contrast, no significant changes in Cy5 fluorescence signal were observed in cells treated with PBS, β-estradiol, or tamoxifen. Figure 8 (Middle F). To demonstrate the enhanced amplification and faster reaction kinetics of bipedal DNA nanomotors in sensing multiple intracellular mRNAs, we treated MCF-7 cells with bipedal, 2-monopegal, monopegal, n-bipedal, and n-monopegal systems, respectively. Figure 8As shown in Figure C, MCF-7 cells treated with the bipedal system exhibited superior Cy5 and Cy3 fluorescence intensity compared to cells treated with the 2-monopod system, monopod system, n-bipod system, or n-monopod system, in the following order: bipod system > 2-monopod system > monopod system > n-bipod system > n-monopod system. The bipod system utilizes the inherent spherical structure of gold nanoparticles and the superior efficiency of the bipod triggering chain, thus exhibiting the most significant fluorescence response. This observation was confirmed by quantitative flow cytometry analysis. Figure 8 (H and I in the middle). For example Figure 8 As shown in Figures H and 8I, MCF-7 cells incubated with the bipedal system exhibited higher Cy5 and Cy3 fluorescence intensities than cells treated with the 2-monopegiac, monopegiac, n-bipedal, or n-monopegiac systems, in descending order: bipedal system > 2-monopegiac system > monopegiac system > n-bipedal system > n-monopegiac system. This observation is consistent with cellular fluorescence imaging (CFI). Figure 8 (C) and in vitro assay ( Figure 4 The results obtained are consistent.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for the preparation of a spatially confined bipedal DNA nanomotor with enhanced dynamics, characterized in that: The method comprises the following steps: Step one, annealing three DNA single strands linker 1, AS1-1, AS1-2 to form Cy5-substrate probe, annealing three DNA single strands linker 2, AS2-1, AS2-2 to form Cy3-substrate probe, annealing three DNA single strands blocker-1, trigger 1-1, trigger 1-2 to form biped walker 1, annealing three DNA single strands blocker-2, trigger 2-1, trigger 2-2 to form biped walker 2; Step two, mixing the Cy5-substrate probe with AuNPs solution to assemble DNA nanomotor 1 by freezing method; mixing the Cy3-substrate probe with AuNPs solution to assemble DNA nanomotor 2 by freezing method.

2. The method of claim 1, wherein: In step one, first, DNA single strands linker 1, AS1-1, AS1-2, linker 2, AS2-1, AS2-2, blocker-1, trigger 1-1, trigger 1-2, blocker-2, trigger 2-1, trigger 2-2 are respectively dissolved with 1×Tris-EDTA buffer to prepare stock solution, then they are respectively diluted to 10 μM with 1×hybridization buffer, and then three DNA single strands linker 1, AS1-1 and AS1-2, three DNA single strands linker 2, AS2-1 and AS2-2, three DNA single strands blocker-1, trigger 1-1 and trigger 1-2, three DNA single strands blocker-2, trigger 2-1 and trigger 2-2 are respectively heated at 95℃ for 5 minutes, and then slowly cooled to 25℃.

3. The method of claim 1, wherein: In step two, the freezing method assembly is that the mixed solution is rotated on a roller mixer for 10 minutes, and then frozen at-20℃ overnight.

4. The preparation method of claim 1, wherein: The sequence of the DNA single strand linker 1 is 5'-SH-TTT TTT TTT TGG ATG CAG AGG TTG ATT GAA TGC CGG GAT TAG GCA TGT AGA GAT GCG GT-3'; In the DNA single strand linker 1, one thiol group (SH) is modified at the 5' end; The sequence of the DNA single strand AS1-1 is 5'-ATC TCT ACA TGC CTA ATC CC-3'; The sequence of the DNA single strand AS1-2 is 5'-GGC ATT CAA TCA ACC TCT GCA TCC-Cy5-3'; In the DNA single strand AS1-2, one fluorophore Cy5 is modified at the 3' end; The sequence of the DNA single strand linker 2 is 5'-SH-TTT TTT TTT TGG ATG CAG AGG TTG ATT GAA TGC CGG GAT TAG GCA TGT AGA GAT GCG GT-3'; 5′-SH-TTT TTT TTT TGC TGA GAT GAA CAA GAA GCG GGA TAT CAA CAT CAG CGA GTGTCT TTG-3′; In the DNA single-stranded linker 2, a thiol (SH) group is modified at the 5′ end; The sequence of the single-stranded DNA AS2-1 is as follows: 5′-CAC TCG CTG ATG TTG ATA TCC C-3′; The sequence of the single-stranded DNA AS2-2 is as follows: 5′-GCT TCT TGT TCA TCT CAG C-Cy3-3′; In the single-stranded DNA AS2-2, a fluorophore Cy3 is modified at the 3′ end; The sequence of the DNA single-stranded blocker-1 is as follows: 5′-GGC ATG TAG AGA TGC GGT CCT TGA GA-3′; The sequence of the DNA single-stranded trigger 1-1 is as follows: 5′-ACC GCA TCT CTA CAT GCC TAA TTT TTT TTT TTT TTT AAT CCG ATG CAC AGT T-3′; The sequences of the DNA single-stranded triggers 1-2 are as follows: 5′-ACC GCA TCT CTA CAT GCC TAA TTT TTT TTT TTT TTT AAC TGT GCA TCG GAT T-3′; The sequence of the DNA single-stranded blocker-2 is as follows: 5′-TCA GCG AGT GTC TTT GGC ATA CTT G-3′; The sequence of the DNA single-stranded trigger 2-1 is as follows: 5′-CAA AGA CAC TCG CTG ATG TTG ATT TTT TTT TTT TTT TAA TCC GAT GCA CAGTT-3′; The sequence of the DNA single-stranded trigger 2-2 is as follows: 5′-CAA AGA CAC TCG CTG ATG TTG ATT TTT TTT TTT TTT TAA CTG TGC ATC GGATT-3′.

5. A spatially confined bipedal DNA nanomotor with enhanced kinetics prepared by the method of claim 1.

6. The use of the spatially confined bipedal DNA nanomotor with enhanced kinetics as described in claim 5 in the preparation of reagents or kits for detecting mRNA.

7. Use according to claim 6, characterized in that: The reagents or kits are used for in situ imaging of mRNA in living cells.

8. Use according to claim 6, characterized in that: The mRNA is survivin mRNA and / or TK1 mRNA; The survivin mRNA sequence is 5′-UCU CAA GGA CCA CCG CAU CUC UAC A-3′; The TK1 mRNA sequence is as follows: 5'-CAAGUAUGC CAAAGACAC UCGC-3'.

9. Use according to any one of claims 6 to 8, characterized in that: The method for using the reagent or kit comprises: preparing a reaction solution, and then incubating at 37 DEG C for 50 minutes; wherein the reaction solution comprises: DNA nanomotor 1 with a working concentration of 300 nM, DNA nanomotor 2 with a working concentration of 300 nM, biped walker 1 with a working concentration of 60 nM, biped walker 2 with a working concentration of 60 nM, fuel chain 1 with a working concentration of 400 nM, fuel chain 2 with a working concentration of 400 nM, 1×T-H buffer, and a sample to be tested; the fuel chain F1 is a DNA single strand, and the sequence is 5'-ATC TCT ACA TGC CTA ATC CCG GCA TTC AAT CAA CCT CTG CAT CC-3'; the fuel chain F2 is a DNA single strand, and the sequence is 5'-CAC TCG CTG ATG TTG ATA TCC CGC TTC TTG TTC ATC TCA GC-3'; the 1×T-H buffer comprises 100 mM NaCl, 10 mM MgCl2, pH 8.

0.

10. Use according to claim 8, characterized in that: The reagent or kit has a minimum detection limit of 501 aM for survivin mRNA and 314 aM for TK1 mRNA.

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