A probe based on the MNAzyme-AS1411 dual-signal amplification system and its application

Abstract

This invention discloses a probe based on the MNAzyme-AS1411 dual-signal amplification system and its applications. The probe comprises: Partzyme 1, Partzyme 2, and a linker strand. Partzyme 1 has the nucleotide sequence shown in SEQ ID NO: 1, and cholesterol is attached to its 5' end. Partzyme 2 has the nucleotide sequence shown in SEQ ID NO: 2. The linker strand has the nucleotide sequence shown in SEQ ID NO: 3, and an AS1411 aptamer sequence is attached to its 3' end. The AS1411 aptamer sequence has the nucleotide sequence shown in SEQ ID NO: 4 and is labeled with a FAM fluorescent group at its 3' end. A DNA strand partially complementary to the aptamer sequence and carrying a BHQ1 quencher group is also included. This probe can achieve specific recognition, in-situ assembly, and signal amplification output on the surface of tumor cells, possessing advantages such as high sensitivity, high specificity, and ease of operation. It is suitable for early tumor screening, liquid biopsy, and rapid intraoperative diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a probe based on the MNAzyme-AS1411 dual-signal amplification system and its application. Background Technology

[0002] Cancer has become a major global public health challenge, with early screening and accurate diagnosis of lung cancer being particularly crucial. Traditional detection methods, such as imaging, serum biomarkers, and tissue biopsies, suffer from low sensitivity, poor specificity, and high invasiveness, failing to meet clinical needs for early, non-invasive, and accurate detection. In recent years, emerging strategies such as liquid biopsy have attempted to achieve "non-invasive" sampling using biomarkers such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), or exosomes; however, their abundance is extremely low (only a few CTCs or trace amounts of ctDNA per milliliter of blood), placing higher demands on the sensitivity of detection technologies. Meanwhile, molecular probe technology is developing towards an "integrated recognition-amplification" approach, aiming to achieve in-situ, real-time, and high-contrast imaging of rare disease biomarkers in complex physiological environments.

[0003] Multicomponent nucleases (MNAzymes) have attracted attention due to their protease-free nature and ability to repeatedly catalyze cleavage under isothermal conditions. Representative existing technologies are as follows:

[0004] CN118667930 discloses a nucleic acid detection method using "asymmetric ligase chain reaction combined with MNAzyme": two Partzymes (A and B) are respectively equipped with a target nucleic acid recognition arm, a catalytic core, and a signal probe recognition arm. In the presence of the target nucleic acid, they assemble into an active MNAzyme and cleave the fluorescent signal probe, achieving secondary amplification after qPCR. However, this method is still limited to "nucleic acid recognition-nucleic acid output" and requires prior PCR amplification. The overall process is lengthy and instrument-dependent, making it unsuitable for direct in-situ imaging of low-abundance miRNAs in live cells.

[0005] The scheme disclosed in CN114958973A proposes to anchor MNAzyme components to the vertices of DNA tetrahedra via strand hybridization to form a framework nucleic acid probe. The target nucleic acid triggers the assembly of Partzyme on the tetrahedron and cleaves the fluorescent substrate, enabling single-cell level miRNA imaging. However, the recognition unit of this probe is still a nucleic acid hybridization arm, which can only detect nucleic acid targets; the tetrahedral framework is relatively large (approximately 10 nm), limiting intracellular diffusion and nuclear delivery, and it does not solve the problem of recognizing important disease biomarkers such as "membrane proteins".

[0006] Although the aforementioned existing technologies have achieved "one-to-many" catalytic amplification through MNAzyme, the recognition targets are limited to nucleic acids. If AS1411 is simply replaced with the nucleic acid recognition arm in MNAzyme, the following core defects still exist: 1. The system remains free in solution and cannot actively anchor to the cell membrane surface, resulting in low local concentration and poor assembly efficiency; 2. The lack of membrane localization leads to high background signal, and the signal-to-noise ratio does not meet the requirements for live cell imaging; 3. It cannot achieve the integrated design of "membrane protein recognition - in-situ catalysis - multiple signal output".

[0007] The *Chinese Journal of Physicians*, 2025, Vol. 4, pp. 622-626, reviewed the AS1411 aptamer: it can specifically bind to nucleolin (NCL) highly expressed on the surface of tumor cells, and has advantages such as low immunogenicity, easy chemical modification, and deep tissue penetration, and has been used for targeted delivery and molecular imaging. However, existing AS1411 probes are mostly in a "passive targeting" mode, lacking a signal amplification mechanism, and the signal generated by low-copy membrane proteins is weak, which is difficult to meet the sensitive detection requirements of sparse targets in the early tumor microenvironment. Although the membrane protein targeting unit AS1411 provided in the *Chinese Journal of Physicians* lacks an amplification strategy.

[0008] Therefore, how to achieve highly specific recognition of membrane proteins, efficient in-situ assembly on the cell membrane surface, and fluorescence / colorimetric dual-modal amplification output while maintaining the advantages of MNAzyme being enzyme-free, isothermal, and recyclable is a key problem that has not yet been solved by existing technologies, and it is also the core technical bottleneck that this invention aims to overcome. Summary of the Invention

[0009] The purpose of this invention is to provide a dual-signal amplification system probe based on MNAzyme-AS1411 and its application. This probe can achieve specific recognition, in situ assembly and signal amplification output on the surface of tumor cells. It has the advantages of high sensitivity, high specificity and simple operation, and is suitable for early tumor screening, liquid biopsy and rapid intraoperative diagnosis.

[0010] To achieve the objectives of this invention, the following implementation scheme is provided.

[0011] In one embodiment, a probe based on the MNAzyme-AS1411 dual-signal amplification system of the present invention comprises: Partzyme 1, Partzyme 2, and a linker. Partzyme 1 has a nucleotide sequence as shown in SEQ ID NO: 1 and cholesterol is attached to its 5' end. Partzyme 2 has a nucleotide sequence as shown in SEQ ID NO: 2. The linker has a nucleotide sequence as shown in SEQ ID NO: 3, and an AS1411 aptamer sequence is attached to its 3' end. The AS1411 aptamer sequence has a nucleotide sequence as shown in SEQ ID NO: 4 and is labeled with a FAM fluorescent group at its 3' end. A DNA strand (CP-AS1411) is also included, which is partially complementary to the aptamer sequence and carries a BHQ1 quencher group.

[0012] Preferably, the dual-signal amplification system probe of the present invention, wherein the DNA strand (CP-AS1411) carrying the BHQ1 quencher group has the nucleotide sequence shown in SEQ ID NO: 6.

[0013] The probe of the dual signal amplification system of the present invention has an AS1411 aptamer labeled with the FAM fluorescent group at the 3' end as GGTGGTGGTGGTTGTGGTGGTGGTGG-FAM (AS1411-FAM).

[0014] In the dual-signal amplification system probe of the present invention, the AS1411 aptamer sequence hybridizes with the DNA strand (CP-AS1411) carrying the BHQ1 quencher group to form a complex. In the complex, the molar ratio of AS1411-FAM to CP-AS1411 is 1:1 to 1:2.5, more preferably 1:2 to 1:2.5.

[0015] Furthermore, the dual-signal amplification system probe of the present invention comprises a substrate chain and a PS2.M sequence is attached to its 5' end, the substrate chain having a nucleoside sequence as shown in SEQ ID NO: 8 (attached to the PS2.M sequence), and the PS2.M having a nucleotide sequence as shown in SEQ ID NO: 9.

[0016] In another embodiment, the present invention also provides the application of the MNAzyme-AS1411 dual-signal amplification system probe of the present invention in the manufacture of reagents for detecting, diagnosing or screening cancer, such as detection reagents, diagnostic reagents or screening reagents.

[0017] In the application of the present invention described above, the cancer exhibits high nucleolarine expression. Preferably, the cancer is lung cancer.

[0018] The above-described application of the present invention further includes introducing a PS2.M sequence into a substrate chain, wherein the substrate chain comprises the nucleotide sequence shown in SEQ ID NO: 8, and the PS2.M has the nucleotide sequence shown in SEQ ID NO: 9.

[0019] In the application of the present invention described above, when the probe encounters cancer cells (substrate), AS1411 specifically binds to nucleolin on the cell surface, and its complementary strand CP-AS1411 detaches from AS1411, restoring the fluorescence signal. At the same time, the linker strand assembles with Partzyme 1 and Partzyme 2 to form a complete nuclease, which cleaves the lock-shaped substrate, releasing PS2.M from the substrate strand. PS2.M binds to Hemin and undergoes an allosteric transformation to form a G4 nuclease, catalyzing the decomposition of H2O2, oxidizing ABTS to green, and emitting a visual signal.

[0020] This invention utilizes a dual-signal amplification system probe based on MNAzyme-AS1411. It constructs a three-part MNAzyme system: Partzyme 1, Partzyme 2, and a linker strand. The linker strand extends with an AS1411 aptamer sequence (Linker strand-AS1411) to recognize nucleolin highly expressed on the surface of tumor cells. A cholesterol group is introduced at the 5' end of Partzyme 1, allowing it to anchor to the cell membrane surface via hydrophobic interactions, increasing local concentration and enhancing probe assembly efficiency. Simultaneously, a FAM fluorescent group is labeled at the 3' end of the Linker strand-AS1411, and a partially complementary DNA strand (CP-AS1411) carrying a BHQ1 quencher group is designed. The two hybridize to form a "fluorescence-off" state, with fluorescence restored only after the Linker strand-AS1411 binds to the target, achieving targeted activation fluorescence signal output. The PS2.M sequence is introduced into the substrate chain. After being cleaved and released by MNAzyme, this sequence can bind to Hemin to form a G-quadruplex / Hemin complex, which possesses peroxidase-like activity and can catalyze the ABTS colorimetric reaction to achieve colorimetric signal output. This probe system has an AND logic gating mechanism; AS1411 binds to nucleolin only in the presence of nucleolin on the surface of tumor cells, driving MNAzyme assembly and activating dual signal output, significantly reducing background interference.

[0021] The MNAzyme-AS1411 dual-signal probe system constructed in this invention has the following advantages:

[0022] 1. High specificity: It recognizes nucleolin through the AS1411 aptamer and combines the AND logic gating mechanism of MNAzyme to effectively reduce non-specific signals;

[0023] 2. High sensitivity: It can detect as few as 16 tumor cells in 200 μL of binding buffer, making it suitable for early screening;

[0024] 3. Multimodal output: Simultaneously provides fluorescence and colorimetric signals to meet different detection needs;

[0025] 4. Simple operation: No cell lysis, amplification, or complex equipment is required, making it suitable for use in areas with limited resources;

[0026] 5. Strong clinical applicability: It demonstrates good detection performance in real clinical samples and has translational potential. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the in-situ assembly and mechanism of action of the MNAzyme-AS1411 dual-signal probe.

[0028] Figure 2 The secondary structure diagram of the nucleoside sequence was predicted using NUPACK, where (A) MNAzyme, (B) MNAzyme-AS1411, and (C) substrate.

[0029] Figure 3The results show the construction and validation of the MNAzyme-AS1411 probes in Examples 1 and 2. (A) 3% agarose gel electrophoresis: 1, 8: DNA Marker; 2: Partzyme 1; 3: Partzyme 2; 4: Lk-AS1411; 5: substrate; 6: Successfully assembled MNAzyme-AS1411; 7: MNAzyme-AS1411 and the digested substrate product; (B) Control group: under tumor cell-free conditions, Partzyme 1, Partzyme... 2. Colorimetric reaction results obtained after incubation of Lk-AS1411 at room temperature. The MNAzyme+AS1411 group is the colorimetric reaction result of the same components and tumor cells incubated under the same conditions. (C) Fluorescence intensity of FAM-Lk-AS1411 and CP-AS1411 after hybridization at different molar ratios, with ratios of 1:0, 1:0.5, 1:1, 1:1.5, 1:2, and 1:2.5 (excitation wavelength: 480nm, emission wavelength: 520nm). (D) Construction verification of the MNAzyme-AS1411 probe, colorimetric reaction results of A549 incubated with Partzyme 1, Partzyme 2, Lk-AS1411, Partzyme 1+Partzyme 2, Partzyme 1+Lk-AS1411, Partzyme 2+Lk-AS1411 and MNAzyme+AS1411 under the same conditions.

[0030] Figure 4 This is a specificity recognition verification diagram of the NAzyme+AS1411 probe in Example 3, wherein (A) fluorescence imaging of different probes incubated with A549 cells and 16HBE; (B) flow cytometry analysis; (C) quantitative detection of absorbance after reaction with substrate, a: A549+Linker strand-random; b: 16HBE+MNAzyme-AS1411; c: A549+AS1411; d: A549+MNAzyme-AS1411.

[0031] Figure 5 The images show the identification and verification of the MNAzyme-AS1411 aptamer probe from Example 3 after incubation with six cell types: 16HBE, NCI-H1975, NCI-H446, RKO, Hep G2, and A549. (A) Fluorescence imaging; (B) Flow cytometry analysis.

[0032] Figure 6This is a graph showing the optimized recognition conditions of the MNAzyme+AS1411 probe in Example 3 and its ability to recognize cells in the mixed system. (A) Fluorescence imaging of A549 cells incubated with the MNAzyme-AS1411 probe for different times; (B) Flow cytometry analysis of A549 cells incubated with the MNAzyme-AS1411 probe for different times; (C) Quantitative absorbance detection of A549 cells after incubation with the MNAzyme-AS1411 probe for different times and reaction with the substrate (times of 0, 5, 10, 20, 30, and 60 minutes); (D) Flow cytometry analysis of A549 cells incubated with the MNAzyme-AS1411 probe at 4℃, 25℃, and 37℃; (E) Flow cytometry analysis of 16HBE and A549 cells mixed in ratios of 2:8, 5:5, 7:3, and 9:1, incubated with the MNAzyme-AS1411 probe.

[0033] Figure 7 This is a flow cytometry analysis of A549 cells from Example 4, incubated with different concentrations of AS1411, a cholesterol-free MNAzyme-AS1411 probe, and a cholesterol-containing MNAzyme-AS1411 probe.

[0034] Figure 8 This is a graph from Example 4 showing the quantitative analysis of A549 cells using the MNAzyme-AS1411 probe. (AE) represents the flow cytometry results of 160,000-11,000 A549 cells incubated with MNAzyme-AS1411 in 200 μL binding buffer; (F) shows the corresponding calibration curve, illustrating the linear relationship between cell counts from flow cytometry analysis and cell counts calculated by hemocytometer; and (G) shows the colorimetric reaction results after incubation with MNAzyme-AS1411 for different cell numbers.

[0035] Figure 9 These are representative images and colorimetric reaction results of the MNAzyme-AS1411 aptamer probe used in Example 5 to detect different clinical body fluid samples. Detailed Implementation

[0036] The present invention will be illustrated below with specific embodiments. It should also be understood that the following embodiments are only for illustrative purposes and should not be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are all within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0037] Reagents and materials:

[0038] DNA oligonucleotides (sequences shown in Table 1) were synthesized by Sangon Biotech (Shanghai, China), purified by HPLC to a purity ≥95%, and stored at -20℃ protected from light. Heme (Cat. A419195), 4S Gelred nucleic acid dye (Cat. A616697-0100), zinc sulfate heptahydrate (ZnSO4·7H4O, Cat. A602906-0500), and 50×TAE buffer (Cat. A548101-0500) were all purchased from Sangon Biotech. 6×Quadricolor loading buffer (Cat. 9171) was purchased from Takara Bio (Beijing, China). 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS, Cat. A188-1, HPLC ≥98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0039] Salmon sperm DNA (Cat.H1060) was purchased from Solarbio Science & Technology (Beijing, China).

[0040] RPMI-1640 medium (Cat. C11875500BT) and DMEM high-glucose medium (Cat. C11995500BT) were both obtained from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS, Cat. AUS-01S-02-S) was purchased from Cell-Box (Changsha, China). Penicillin-streptomycin-gentamicin solution (100×, Cat. C0223) was purchased from Beyotime Biotechnology (Shanghai, China). Trypsin (0.02% EDTA, Cat. PYG0015) and sterile PBS (Ca2+-free) were also used. 2+ Mg 2+ The cells (Cat. PYG0021) were purchased from BOSTRE (Wuhan, China). The A549 cells (Cat. FH0045) were purchased from FuHeng Biology (Shanghai, China).

[0041] The 16HBE, Hep G2, NCI-H1975, RKO, and NCI-H446 cell lines were kindly provided by the Oncology and Epigenetics Laboratory of Chongqing Medical University and tested negative for STR and mycoplasma.

[0042] The binding buffer (pH 7.4) was prepared with Milli-Q ultrapure water, containing 4.5 g L. -1 Glucose, 1 mg / mL -1 BSA, 0.1 mg / mL -1 salmon sperm DNA and 1 mmol L -1 MgCl2; after sterilization by 0.22μm filtration, store at 4℃ protected from light and use within 1 week.

[0043] The nucleotide sequences used in the following examples are shown in Table 1.

[0044] Table 1 Nucleotide Sequences

[0045]

[0046] * The substrate sequence contains the PS2.M sequence.

[0047] The principle of the MNAzyme-AS1411 probe for detecting target cells in this invention is as follows: Figure 1 As shown.

[0048] Cell line culture:

[0049] A549 (human lung adenocarcinoma epithelial cells), 16HBE (human normal bronchial epithelioid cells), and Hep G2 (human hepatocellular carcinoma cells) were cultured in DMEM medium (manufactured by Gibco, USA) containing 10% fetal bovine serum and 1% penicillin-streptomycin-gentamicin. NCI-H1975 (human non-small cell lung cancer cells), RKO (human colorectal cancer cells), and NCI-H446 (human small cell lung cancer cells) cell lines were cultured in RPMI 1640 medium (manufactured by Gibco, USA) containing 10% fetal bovine serum and 1% penicillin-streptomycin-gentamicin. All cells were cultured at 37°C and 5% carbon dioxide.

[0050] The flow cytometry analysis and detection method using probe systems is described in the following examples:

[0051] A549 and 16HBE cells in logarithmic growth phase were collected and digested with 0.02% EDTA solution at 37°C for approximately 2 minutes (the exact time was determined by the rounding of cell morphology and increase in intercellular spacing) to prepare a single-cell suspension. The cells were then washed three times with pre-chilled PBS. Finally, the cell pellet was resuspended in a specific volume of binding buffer, and the cell concentration was adjusted to 1 × 10⁻⁶ cells using a hemocytometer or automated cell counter. 5 cells / mL available for later use.

[0052] In this study, 'MNAzyme-AS1411 probe' refers to an equimolar mixture of its constituent DNA strands. Unless otherwise specified, this mixture is co-incubated with cells to allow for in-situ assembly into a catalytically active probe structure.

[0053] To optimize cell recognition conditions, A549 cells were incubated with the MNAzyme-AS1411 probe (200 nM) in binding buffer at room temperature for different times (0, 5, 10, 20, 30, and 60 minutes) and then analyzed by flow cytometry. Additionally, A549 cells were incubated with 200 nM M MNAzyme-AS1411 at 4°C, 25°C, and 37°C for 10 minutes each before flow cytometry analysis to determine the optimal incubation temperature.

[0054] To verify the ability of the MNAzyme-AS1411 probe to recognize target cells, a random sequence (named Linker strand-random) was introduced at the 3' end of the linker strand. A549 cells were incubated with Linker strand-random, AS1411, and MNAzyme-AS1411 probes in binding buffer at 37°C in the dark for 10 minutes, respectively; 16HBE incubation with the MNAzyme-AS1411 probe served as a negative control. After incubation, all samples were washed three times with PBS and resuspended in PBS for flow cytometry analysis.

[0055] To verify the specificity of the probe, the MNAzyme-AS1411 probe (200 nM) was incubated with a series of cell lines (16HBE, NCI-H1975, NCI-H446, HepG2, RKO and A549) in binding buffer at 37°C for 10 minutes, washed with PBS and analyzed by flow cytometry.

[0056] To assess the affinity of the probes for cell recognition, different concentrations of AS1411 (0, 25, 50, 100, 125, 200, 300, 500 nM), MNAzyme-AS1411 (cholesterol-free) (0, 25, 50, 100, 125, 200, 300, 500 nM), and MNAzyme-AS1411 probe (0, 5, 25, 50, 75, 100, 150, 200, 250, 300 nM) were incubated with A549 cells in binding buffer at 37°C for 10 minutes, followed by flow cytometry analysis.

[0057] To evaluate the recognition ability of the probe in the mixed system, A549 cells and 16HBE cells were mixed at different ratios and incubated with 200 nM MNAzyme-AS1411 probe at 37°C for 10 minutes. After washing three times with PBS, flow cytometry analysis was performed.

[0058] Quantitative analysis of the sensitivity of probe to cell recognition was performed by incubating different numbers of A549 cells with a 200 nM MNAzyme-AS1411 probe, washing them, and then performing flow cytometry analysis.

[0059] All flow cytometry samples were analyzed using a FACScanto flow cytometer (BD Biosciences, Inc., USA), with at least 10,000 cell events collected for each sample. Data analysis was performed using FlowJo software (version 10.5.3).

[0060] The colorimetric detection method using the probe system is described in the following examples:

[0061] To verify the feasibility of this probe system, A549 cells were incubated with all DNA strands of MNAzyme-AS1411 (i.e., an equimolar mixture of Partzyme 1, Partzyme 2, and Lk-AS1411 strands) in binding buffer for a certain period of time, allowing the probe to assemble in situ on the cell surface. After incubation, the supernatant was removed by centrifugation, and the cells were washed three times with PBS before the substrate strand and Zn were added. 2+ Incubation (20 µM) for a specified time allows successfully assembled MNAzyme on the cell surface to cleave substrate chains. After the reaction, centrifugation was performed again, and the supernatant was collected. An equal volume of HEPES buffer (50 mM HEPES, 40 mM KCl, 400 mM NaCl, 0.1% Triton X-100, 2% DMSO) was added, and heme was added to a final concentration of 1 μM. Incubation was carried out at room temperature for 1 h. Subsequently, 5 mM ABTS and 0.75% H2O2 were added, and the mixture was allowed to stand at room temperature for 3 minutes. The absorption spectrum was recorded at a wavelength of 420 nm using a Varioskan™ LUX multifunction reader.

[0062] To verify whether the MNAzyme-AS1411 probe successfully assembled on the cell surface, A549 cells were incubated with individual components such as Partzyme 1 chain, Partzyme 2 chain, and Lk-AS1411 chain, as well as with the MNAzyme-AS1411 component chains for a certain period of time. Subsequent steps were the same as before, and a colorimetric reaction was performed, with absorption spectra detected in the 420 nm wavelength range. To further explore the assembly mechanism, different combinations of MNAzyme components (part1, part2) and Lk-AS1411 (e.g., Part1 + Lk-AS1411; Part2 + Lk-AS1411) were additionally incubated with A549 cells, and their ability to cleave substrate chains was detected.

[0063] To determine the optimal detection performance of the MNAzyme-AS1411 probe system, we optimized its detection conditions, including substrate concentration and enzyme digestion time. A549 cells were incubated with the probe, washed three times with PBS, and then incubated with different concentrations of substrate before colorimetric detection. For time optimization, the solution of A549 cells incubated with the MNAzyme-AS1411 probe was centrifuged, washed, and then incubated with the substrate for different times. MNAzyme components (such as part1, part2, and Lk-AS1411) were directly incubated with the substrate for different times as negative controls, and colorimetric detection was performed.

[0064] To verify the probe's specificity for target cells and optimize recognition time, the aforementioned colorimetric detection method was followed. The absorption spectra of all samples were recorded using a Varioskan™ LUX multifunction reader in the 420 nm wavelength range.

[0065] Fluorescence microscopy imaging:

[0066] A549 and 16HBE cells were incubated with the MNAzyme-AS1411 probe at 37°C for 10 minutes, followed by washing with PBS to remove unbound probe. Cell nuclei were stained with Hoechst 33342 (blue) for 10 minutes, washed three times with PBS, and 10 μL of cell suspension was transferred to a glass slide, covered with a coverslip, and observed and imaged under a fluorescence microscope.

[0067] Clinical specimen processing methods and applications:

[0068] This study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (Approval No.: 2025-909-01). A total of 44 fresh cytological specimens from patients at the hospital between September 2024 and December 2025 were collected, including 23 cases of lung adenocarcinoma, 1 case of small cell lung cancer, 6 cases of other types, and 14 negative controls. All specimens were obtained from the Cytopathology Laboratory of the Department of Respiratory Medicine, First Affiliated Hospital of Chongqing Medical University. Specimen inclusion criteria: (1) sufficient specimen volume, not less than 200 μL; (2) no agglutination in the specimen; (3) no hemolysis in the specimen; (4) a clear clinical pathological diagnosis. Specimens were uniformly numbered after collection, and experiments were conducted without knowing the diagnostic results to avoid bias.

[0069] After centrifugation, the supernatant was discarded, and the precipitate was washed three times with physiological saline. A suitable amount of cell pellet was then resuspended in 200 μL of binding buffer. 200 nM MNAzyme-AS1411 probe fraction was added, and the mixture was thoroughly mixed and incubated at room temperature in the dark for 10 minutes. After incubation, the staining results were first observed under a fluorescence microscope, followed by colorimetric detection (420 nm).

[0070] Statistical analysis:

[0071] All data in this study were statistically analyzed and graphically plotted using GraphPad Prism 10.1.2 software. Results are expressed as mean ± standard deviation (x ± s), and data were obtained from three independent replicates. Student's test was used for comparisons between two groups, with P < 0.05 considered statistically significant. Pathological diagnosis was used as the gold standard, and the sensitivity and specificity of the probe were calculated as follows: Sensitivity: number of true positives / (number of true positives + number of false negatives); Specificity: number of true negatives / (number of true negatives + number of false positives). Wherein, a true positive refers to a sample pathologically confirmed as malignant and with a positive probe test result; a false negative refers to a sample pathologically confirmed as malignant but with a negative probe test result; a true negative refers to a sample pathologically confirmed as benign and with a negative probe test result; and a false positive refers to a sample pathologically confirmed as benign but with a positive probe test result. All combination experiments were independently replicated three times.

[0072] Example 1: Construction and Validation of the MNAzyme-AS1411 Probe

[0073] MNAzyme catalytic core design, using a Zn 2+Using the 17E DNAzyme-dependent catalytic core as the structural backbone, we modified its catalytic core. Referring to the reported 17E DNAzyme catalytic core sequence in existing literature, we split it at its central site and introduced specific binding arms at the split site that do not interfere with the activity of the catalytic core, designing it as two independent nucleic acid strands (named Partzyme 1 and Partzyme 2, respectively). Simultaneously, we designed a specific linker strand, which can precisely bind to the binding arms of Partzyme 1 and Partzyme 2 through base complementarity pairing. When Partzyme 1, Partzyme 2, and the linker strand are co-incubated, the linker strand can mediate the assembly of the two MNAzyme fragments through hybridization to form a complete MNAzyme functional structure. To verify the thermodynamic stability of this assembly, we performed theoretical simulations using the NUPACK online analysis platform (http: / / www.nupack.org). Simulation results show that the free energy of the MNAzyme structure assembled from the three chains reaches -52.83 kcal / mol. This significant negative free energy value indicates that the complex possesses excellent thermodynamic stability, providing a structural basis for its subsequent biological functions. See [see results below]. Figure 2 A.

[0074] Introduction of Targeting Elements: After obtaining the core functional modules of the controllable assembly described above, to endow the probe with the ability to specifically target tumor cells, we integrated the AS1411 nucleic acid aptamer sequence into the 3' end of the original Linker strand using a sequence extension strategy, naming it Linker strand-AS1411 (Lk-AS1411). The AS1411 aptamer is a known nucleic acid sequence capable of specifically binding to nucleolin, which is highly expressed on the surface of tumor cells. To assess the impact of the introduction of the AS1411 sequence on the thermodynamic stability of the assembly, we used the NUPACK online analysis platform (http: / / www.nupack.org) to simulate and predict the interaction between Partzyme 1, Partzyme 2, and Lk-AS1411. The results showed that the free energy of the MNAzyme-AS1411 complex did not change significantly after the integration of the AS1411 aptamer sequence. Figure 2 B) indicates that the introduction of the target sequence did not disrupt the thermodynamic stability of the assembly.

[0075] Construction and Validation of MNAzyme-AS1411: Next, we validated whether the assembly ability and catalytic activity of Lk-AS1411 for the probe were retained. We assembled three nucleic acid strands (Partzyme 1, Partzyme 2, and Lk-AS1411) by incubation at 95°C for 5 minutes, followed by incubation at 25°C for 2 hours. Subsequently, they were combined with the substrate strand and Zn. 2+ Incubate at 37°C for 1 hour. Verify the cut product; 3% agarose gel electrophoresis results show (…). Figure 3 A), adding Zn 2+ Under the specified conditions, clear substrate cleavage bands were observed, consistent with the expected cleavage products of the intact MNAzyme structure. This result demonstrates that, with the introduction of the AS1411 aptamer strand, the three nucleic acid strands can still successfully assemble into a catalytically active MNAzyme structure via the linker strand.

[0076] To further achieve targeted recognition and signal output at the cellular level, we introduced a labeling and modification strategy for the probe. First, a FAM fluorescent group was added to the 3' end of Lk-AS1411 to output a fluorescent signal for observation. Second, a cholesterol group was attached to the 5' end of Partzyme 1, allowing it to anchor to the cell membrane surface through hydrophobic interactions, thereby locally increasing the probe concentration around the cell. This enables more effective triggering of MNAzyme assembly by utilizing the proximity effect when the target cell is present. Since Lk-AS1411 carries the AS1411 aptamer sequence that specifically recognizes nucleolin highly expressed in cancer cells, when nucleolin is highly expressed on cancer cells, the aptamer can bind to it and promote the assembly and activation of the MNAzyme-AS1411 probe.

[0077] To determine whether the MNAzyme-AS1411 probe could achieve targeted activation and visualized signal output in the presence of specific cells (such as tumor cells), a colorimetric signal was used. To achieve visual signal output, the colorimetric signal was integrated into the substrate strand design: a PS2.M sequence was introduced at the 5' end of the substrate strand. PS2.M is an 18-base guanine-rich oligonucleotide sequence that can form a stable G-quadruplex structure. The substrate strand containing PS2.M was designed as a lock-type structure, and its secondary structure was predicted using the NUPACK website. Figure 2C). Once MNAzyme-AS111 is successfully assembled and active, it cleaves the substrate chain, releasing the PS2.M sequence. The released PS2.M is coupled with hemin chloride to form a complex with peroxidase-like activity. This complex catalyzes the oxidation of ABTS by hydrogen peroxide (H2O2), resulting in a color change (producing the green oxidation product ABTS+), thereby enabling visual signal output.

[0078] In the following verification experiments, the substrates used all incorporated the PS2.M sequence.

[0079] To verify the feasibility of the dual-signal system, simple tests were conducted at the in vitro and cellular levels:

[0080] Partzyme 1, Partzyme 2, and Lk-AS1411 were incubated with tumor cells. A control group was established by incubating Partzyme 1, Partzyme 2, and Lk-AS1411 in a tumor-free environment. After incubation, both groups were incubated with substrate and Zn. 2+ After incubation at 37°C for 1 hour, colorimetric detection was performed, and the results are shown below. Figure 3 B.

[0081] Colorimetric results ( Figure 3 B) shows that the binding efficiency of the three chains is not ideal in the absence of a target in solution; that is, no color change is observed in the control group, while significant color changes are seen in the group with tumor cells. The results demonstrate that MNAzyme can only assemble efficiently in the presence of tumor cells.

[0082] Construction and verification of the MNAzyme-AS1411 probe: Under the same conditions, Partzyme 1, Partzyme 2, Lk-AS1411, Partzyme 1+Partzyme 2, Partzyme 1+Lk-AS1411, Partzyme 2+Lk-AS1411, and MNAzyme+AS1411 were constructed on the substrate chain and Zn. 2+ In the presence of these cells, they were co-incubated with tumor cells (A549), and colorimetric detection was performed after incubation (see [link to relevant documentation]). Figure 3 (D) The results showed that a significant color change was only detected when all three nucleic acid chains were present, i.e., when MNAzyme was fully assembled, which preliminarily proved the functionality of the system in a cell recognition environment.

[0083] Example 2: Fluorescence quencher of the probe

[0084] To further improve the specificity of the MNAzyme-AS1411 probe in recognizing cancer cells, we introduced an activation-based design strategy based on structure transition. This strategy utilizes the Förster resonance energy transfer principle to form a "fluorescence-quenching" pair between the FAM fluorescent group and the BHQ1 quencher. Specifically, a DNA strand complementary to the AS1411 aptamer sequence (named CP-AS1411) was designed, and a BHQ1 quencher group was labeled at its 5' end. This strand was then hybridized with the Lk-AS1411 strand labeled with FAM at its 3' end (Lk-AS1411-FAM) to form a double-stranded complex (Lk-AS1411 complex). In this state, the FAM and BHQ1 are spatially close, and the fluorescence is quenched due to efficient resonance energy transfer, putting the probe in an "off" state.

[0085] In cell experiments, the key component used is the Lk-AS1411 complex. The activation mechanism of this complex involves competitive binding. When the complex comes into contact with target cells that highly express nucleolin on their surface, the AS1411 sequence in the Lk-AS1411 chain interacts more strongly with nucleolin, thereby driving the dissociation of the CP-AS1411 quencher chain, restoring FAM fluorescence, and achieving targeted activation and "signal activation".

[0086] Optimization of fluorescence quenchers:

[0087] To obtain the optimal signal-to-noise ratio, we first optimized the assembly ratio of the complex. Lk-AS1411-FAM and BHQ1-CP-AS1411 were hybridized at different molar ratios (1:0, 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5). Subsequently, the fluorescence intensity of FAM was quantitatively detected using a Varioskan™ LUX multi-functional microplate reader under the conditions of excitation wavelength 490 nm and emission wavelength 520 nm. The fluorescence quenching efficiency was calculated according to formula (1): Quenching Efficiency (%) = [1 - (F / F0)] × 100%, where F0 and F represent the fluorescence intensity of the system before and after the addition of the quenching chain, respectively. By comparing the quenching efficiency at different ratios, the optimal molar ratio with the lowest background signal was screened. The fluorescence quenching efficiency was calculated using formula (1): Quenching Efficiency (%) = [1 - (F / F0)] × 100%, where F0 and F represent the fluorescence intensity of the system before and after the addition of the quenching chain, respectively. The optimal molar ratio with the lowest background signal was selected by comparing the quenching efficiencies at different ratios. The results are shown below. Figure 3 C. The results showed that the quenching efficiency was optimal when the molar ratio of Lk-AS1411-FAM to BHQ1-CP-AS1411 was 1:2-1:2.5, with 1:2 being the preferred ratio based on cost.

[0088] Example 3: Specificity of flow cytometry and fluorescence imaging analysis for tumor cell recognition

[0089] In this experiment, 'MNAzyme-AS1411 probe' refers to an equimolar mixture of its constituent DNA strands. Unless otherwise specified, this mixture is co-incubated with cells to allow for in-situ assembly into a catalytically active probe structure.

[0090] Specific recognition and functional verification of the probe: A549 cells (human lung adenocarcinoma cells) with high nucleolin expression were used as positive target cells, and 16HBE cells (human normal bronchial epithelial-like cells) with low nucleolin expression were used as negative control cells for specific recognition experiments. Two control groups were also set up: A549 cells incubated with a single AS1411 aptamer, and A549 cells incubated with a linker strand-random (Lk-random) sequence. After incubation with different probes, cells were analyzed by fluorescence microscopy and flow cytometry. Fluorescence microscopy results showed that no obvious FAM green fluorescence was observed in any of the cells incubated with the Lk-random probe, or in the 16HBE cells incubated with the MNAzyme-AS1411 probe. A weak green fluorescence was observed in the cells incubated with the single AS1411 aptamer. However, the A549 cells incubated with the MNAzyme-AS1411 aptamer probe showed a significant green fluorescence signal. Figure 4 A). Flow cytometry analysis results ( Figure 4 B) Consistent with fluorescence microscopy imaging results, the average fluorescence intensity measured in the A549+MNAzyme-AS1411 aptamer probe group was significantly higher than that in the other three control groups. This result demonstrates that the MNAzyme-AS1411 aptamer probe can specifically recognize A549 cells with high nucleolin expression. ABTS staining assays were performed simultaneously to verify the probe's cleavage activity on the cell surface; the results showed that only the A549+MNAzyme-AS1411 aptamer probe group exhibited a visible color change. Figure 4 (C) The absorbance values ​​of this group were also significantly higher than those of the other three control groups. This result indicates that the MNAzyme-AS1411 aptamer probe can not only specifically bind to tumor cells with high nucleolin expression, but also successfully assemble on the surface of target cells and efficiently catalyze the cleavage of the substrate chain, releasing PS2.M to initiate the colorimetric reaction.

[0091] To verify the broad-spectrum recognition of the MNAzyme-AS1411 aptamer probe for different tumor cell lines, we selected five nucleolin-high expressing tumor cell lines (NCL-H1975, NCL-H446, RKO, Hep G2, and A549) and 16HBE cells with low nucleolin expression as negative controls. The MNAzyme-AS1411 probe (200 nM) was incubated with a series of cell lines (16HBE, NCI-H1975, NCI-H446, HepG2, RKO, and A549) in binding buffer at 37°C for 10 minutes. After washing with PBS, fluorescence microscopy and flow cytometry were performed for analysis. Flow cytometry analysis (…) Figure 5 A) shows that the fluorescence intensity of all five positive cells was significantly higher than that of the negative control cells, and the microscopic fluorescence results ( Figure 5 B) Consistent with flow cytometry analysis. However, the fluorescence intensity of the five tumor cell types was inconsistent, which may be related to the differences in the expression levels of nucleolin on the surface of each cell line. Among them, A549 cells showed the most obvious fluorescence under the microscope and the highest flow cytometry fluorescence intensity, indicating that their surface nucleolin expression level was relatively high. Therefore, A549 cells were selected for subsequent condition optimization experiments.

[0092] Optimization of probe detection conditions:

[0093] To achieve optimal detection performance, we systematically optimized the incubation temperature and incubation time of the probe.

[0094] In binding buffer, MNAzyme-AS1411 probe (200 nM) was incubated with A549 cells at room temperature for different times (0, 5, 10, 20, 30, 60 minutes), followed by flow cytometry analysis. Alternatively, 200 nM MNAzyme-AS1411 was incubated with A549 cells at 4°C, 25°C, and 37°C for 10 minutes each before flow cytometry analysis to determine the optimal incubation temperature. Results are shown below. Figure 6 AD.

[0095] The results showed that, for optimal incubation time, A549 cells were incubated with the probe for different times (0, 5, 10, 20, 30, and 60 minutes) under the same conditions, and the results were analyzed by fluorescence microscopy and flow cytometry. Fluorescence imaging results ( Figure 6 A) showed that weak fluorescence could be observed after 5 minutes of incubation with the probe using A549, and the fluorescence intensity basically stabilized after 10 minutes. Flow cytometry analysis ( Figure 6 B) The results were consistent with the fluorescence imaging results. Further colorimetric reactions were performed and absorbance was measured (…). Figure 6(C) The results showed that the absorbance changed significantly after 5 minutes. Therefore, 10 minutes was selected as the optimal reaction time for subsequent experiments. The incubation temperature was optimized by incubating A549 cells with the MNAzyme-AS1411 probe at 4℃, 25℃, and 37℃ for 10 minutes, respectively, followed by flow cytometry analysis. Results ( Figure 6 D) shows that there was no significant difference in incubation at the three different temperatures. Considering that cell culture conditions are usually set at 37℃, 37℃ was selected as the reaction temperature for subsequent experiments.

[0096] To investigate the target cell recognition ability of this probe in a mixed cell system and to study its recognition ability in more complex biological environments, we constructed a mixed cell system by incubating positive A549 cells and negative 16HBE cells together. The total cell count was fixed at 102. 5 The ratios of positive to negative cells were 8:2, 5:5, 3:7, and 1:9, respectively. A549 cells and 16HBE cells were mixed at different ratios and incubated with 200 nM MNAzyme-AS1411 probe at 37°C for 10 minutes. After washing three times with PBS, flow cytometry analysis was performed. The mixed groups of cells were then incubated with the MNAzyme-AS1411 aptamer probe, and flow cytometry analysis was performed after incubation. The flow cytometry results (…) Figure 6 E) shows that as the proportion of positive cells in the mixed system gradually increases, the proportion of cells in the high fluorescence intensity region also expands. This result indicates that the MNAzyme-AS1411 aptamer probe can maintain high specificity and targeting in mixed cell samples, accurately identifying and distinguishing target tumor cells.

[0097] Figure 6 In the study, (A) fluorescence imaging of A549 cells incubated with the MNAzyme-AS1411 probe for different times; (B) flow cytometry analysis of A549 cells incubated with the MNAzyme-AS1411 probe for different times; (C) quantitative absorbance detection of A549 cells after incubation with the MNAzyme-AS1411 probe for different times followed by reaction with the substrate (times were 0, 5, 10, 20, 30, and 60 minutes); (D) flow cytometry analysis of A549 cells incubated with the MNAzyme-AS1411 probe at 4℃, 25℃, and 37℃; and (E) flow cytometry analysis of 16HBE and A549 cells mixed at ratios of 2:8, 5:5, 7:3, and 9:1, and incubated with the MNAzyme-AS1411 probe. The results showed that as the proportion of positive cells in the mixed system gradually increased, the proportion of cells in the high fluorescence intensity region also expanded. The MNAzyme-AS1411 aptamer probe maintains high specificity and targeting in mixed cell samples, accurately identifying and distinguishing target tumor cells.

[0098] Example 4: Evaluating the sensitivity of the probe

[0099] To investigate the effect of probe concentration on recognition efficiency, we selected A549 cells and incubated them with different concentrations of AS1411 (concentration range: 0 to 500 nM), MNAzyme-AS1411 (concentration range: 0 to 500 nM), and MNAzyme-AS1411 aptamer probe (concentration range: 0 to 300 nM) under optimal conditions, followed by flow cytometry analysis.

[0100] result( Figure 7 The results showed that as the probe concentration increased, the fluorescence intensity of FAM on the cell surface increased accordingly, and the increase in FAM value on the cell surface of cells incubated with the MNAzyme-AS1411 aptamer probe was significantly stronger than that of the other two groups. Furthermore, a significant fluorescence signal generated by cell-specific binding was observed when the MNAzyme-AS1411 aptamer probe concentration reached 50 nM, and within a certain concentration range, the fluorescence intensity was positively correlated with the probe concentration. These results determined the optimal working concentration range of the probe for subsequent experiments.

[0101] Quantitative detection of cell count and detection limit

[0102] Quantitative analysis of the sensitivity of probe to cell recognition was performed by incubating different numbers of A549 cells with a 200 nM MNAzyme-AS1411 probe, washing them, and then performing flow cytometry analysis.

[0103] All flow cytometry samples were analyzed using a FACScanto flow cytometer (BD Biosciences, Inc., USA), with at least 10,000 cell events collected for each sample. Data analysis was performed using FlowJo software (version 10.5.3).

[0104] Samples containing varying numbers of A549 cells (cell counts ranging from 16 to 11,000 in 200 μL buffer) were incubated with the MNAzyme-AS1411 aptamer probe at 37°C for 10 minutes, followed by flow cytometry analysis. Results are shown below. Figure 8 Through streaming scatter plot ( Figure 8 The number of cells appearing in the high-fluorescence region of the MNAzyme-AS1411 aptamer probe was used to achieve quantitative detection of target cells. Linear regression analysis was performed between the cell number detected by the MNAzyme-AS1411 aptamer probe (Y-axis) and the cell number measured by a hemocytometer (X-axis). The results showed a significant linear relationship, with the regression equation being Y = 0.9702X + 12.8 and a correlation coefficient R² reaching 0.9998. Figure 8F). In 200 μL buffer, this probe system achieved efficient linear detection of A549 cells, with a detection range of 16 to 11,000 cells. According to linear regression results, the limit of detection of the MNAzyme-AS1411 aptamer probe was approximately 16 cells. Further colorimetric experiments with the same number of cells showed that the absorbance increased with increasing cell number. Figure 8 G).

[0105] Example 5: Application of the MNAzyme-AS1411 probe in clinical sample monitoring

[0106] To evaluate the clinical application value of the MNAzyme-AS1411 probe, we conducted an in-depth study on its performance in clinical samples. Using clinicopathological diagnostic results as the gold standard, the detection performance indicators of the MNAzyme-AS1411 aptamer probe in these clinical samples were: specificity 83.3%, sensitivity 91.3%, and accuracy 87.8%. Furthermore, we incubated the clinical samples with the probe at 37°C for 10 minutes and then performed microscopic imaging observation. Results (see...) Figure 9 This indicates that the probe can successfully identify tumor cells in the sample. Subsequent colorimetric experiments further confirmed the probe's effectiveness, with all tumor sample solutions showing a clear green signal. These experimental results demonstrate the promising application potential of the MNAzyme-AS1411 aptamer probe of this invention in clinical sample diagnosis.

[0107] The MNAzyme-AS1411 dual-signal probe constructed in this invention fully utilizes the high expression of nucleolin (NCL) in the cell membrane of non-small cell lung cancer (NSCLC) cells. By combining aptamer-targeted recognition with in-situ catalytic amplification of MNAzyme, it achieves rapid, sensitive, and visualized detection of lung cancer cells. Experimental results show that the probe can complete detection within 10 minutes, with a detection limit as low as 16 cells, and accurately identifies target cells in a mixed cell model, demonstrating good specificity and sensitivity.

[0108] In clinical sample validation, the probe demonstrated a sensitivity of 91.3%, a specificity of 83.3%, and an accuracy of 87.8% in 41 cases of pleural effusion (27 malignant and 14 benign), indicating its potential clinical application value in lung cancer liquid biopsy. Furthermore, this probe requires no cell lysis, nucleic acid amplification, or complex instruments; it can be interpreted with the naked eye using only 200 μL of sample, making it simple to operate, low in cost, and suitable for rapid screening in resource-limited areas.

[0109] In summary, the MNAzyme-AS1411 dual-signal probe, as a novel liquid biopsy tool, offers a rapid, sensitive, and visualized solution for early detection and postoperative monitoring of lung cancer, demonstrating promising translational applications. Future research will focus on large-scale clinical validation and multi-cancer differentiation to advance its clinical application.

Claims

1. A probe based on MNAzyme-AS1411 dual signal amplification system, characterized in that, The product comprises: Partzyme 1, Partzyme 2, a linker strand, a DNA strand CP-AS1411, and a substrate strand. Partzyme 1 has the nucleotide sequence shown in SEQ ID NO: 1, with cholesterol attached to its 5' end. Partzyme 2 has the nucleotide sequence shown in SEQ ID NO:

2. The linker strand has the nucleotide sequence shown in SEQ ID NO: 3, with an AS1411 aptamer sequence attached to its 3' end. The AS1411 aptamer sequence is the nucleotide sequence shown in SEQ ID NO: 4, and a FAM fluorescent group is labeled at its 3' end. The DNA strand CP-AS1411 is partially complementary to the AS1411 aptamer sequence and carries a BHQ1 quencher group. The substrate strand has a PS2.M sequence attached to its 5' end. The substrate strand has the nucleotide sequence shown in SEQ ID NO: 8, and PS2.M is the nucleotide sequence shown in SEQ ID NO:

4. The nucleotide sequence shown in NO:9 indicates that when the probe encounters cancer cells, the AS1411 aptamer sequence specifically binds to nucleolin on the cell surface. Its complementary strand CP-AS1411 detaches from the AS1411 aptamer sequence, restoring the fluorescence signal. Simultaneously, the linker strand assembles with Partzyme 1 and Partzyme 2 to form a complete nuclease, which cleaves the lock-shaped substrate strand, releasing PS2.M from the substrate strand. PS2.M binds to Hemin, undergoes an allosteric transformation to form a G4 nuclease, catalyzes the decomposition of H2O2, oxidizes ABTS to turn green, and emits a visual signal.

2. The probe as described in claim 1, characterized in that, The DNA strand CP-AS1411 carrying the BHQ1 quencher group has the nucleotide sequence shown in SEQ ID NO:

6.

3. The probe as described in claim 1, characterized in that, The AS1411 aptamer sequence hybridizes with the CP-AS1411 portion of the DNA strand carrying the BHQ1 quencher group to form a complex, wherein the molar ratio of the AS1411 aptamer sequence to CP-AS1411 is 1:1 to 1:2.

5.

4. The probe as described in claim 3, characterized in that, The molar ratio of the AS1411 aptamer sequence to CP-AS1411 is 1:2 to 1:2.

5.

5. The use of any probe of claims 1-4 in the manufacture of a reagent for detecting, diagnosing or screening for cancer, wherein the cancer is lung cancer.

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