A modified nucleic acid aptamer and its application

Abstract

The present invention provides a modified nucleic acid aptamer and its application. In a first aspect, the present invention provides a modified nucleic acid aptamer, wherein the nucleic acid aptamer is nucleic acid aptamer Sgc8, and the nucleotide sequence of the nucleic acid aptamer Sgc8 is SEQ ID NO: 1; one end of the nucleic acid aptamer is extended by a polynucleotide fragment, and the extended end of the polynucleotide fragment is modified with a fluorescent molecule; the nucleotide sequence of the polynucleotide fragment is TTTTT. By adding a base sequence of a certain length between the fluorescent molecule and the nucleic acid aptamer, the present invention can effectively avoid interference of the fluorescent molecule with the nucleic acid aptamer recognition domain, thereby improving the efficiency of recognition and imaging.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a modified nucleic acid aptamer and application thereof. Background Art

[0002] With the continuous acceleration of the aging process of the population, the incidence of bladder cancer has shown a continuous upward trend. 1 Bladder cancer is easy to detect in its early stages. Most patients are still in the non-muscle invasive bladder cancer (NMIBC) stage when diagnosed, making treatment relatively easy. 2 However, bladder cancer has a high recurrence rate and requires repeated examinations and treatments, which brings a heavy economic burden to patients and society and also aggravates the pain of patients. 2 Early-stage bladder cancer is usually treated with electrosurgical resection under white light guidance. However, due to the physiological limitations of the human eye, there are factors such as small flat lesions that cannot be found or the surgical margins of the lesions are not cleaned up. It is impossible to ensure that these missed lesions are completely removed, and there is a high risk of recurrence of bladder cancer. 3 Therefore, developing a highly specific and sensitive surgical auxiliary method to assist in clearing the tumor as much as possible during surgery while avoiding excessive treatment of normal tissues is of great significance for the surgical treatment of bladder tumors.

[0003] Endoscopic detection methods based on fluorescence technology are advanced tumor detection and diagnosis methods based on fluorescence imaging systems. 3 Compared with traditional white light observation, the fluorescence system enhances the visibility of tumor tissue by using specific fluorescent imaging perfusion fluids, such as indocyanine green (ICG) and 5-aminolevulinic acid (5-ALA), thereby improving the ability to identify cancerous areas. 4 These fluorescent imaging perfusion solutions will be selectively absorbed by the tumor tissue after perfusion and emit fluorescence under the illumination of a light source of a specific wavelength. 5 Using an endoscopic detection system with fluorescence, doctors can clearly observe the fluorescence contrast between tumor tissue and normal tissue, making it easier to detect tiny or hidden tumor lesions. 5 In practical applications, the fluorescence system can not only accurately detect small or flat lesions that are difficult to find, but also accurately identify tumor boundaries during surgery, helping doctors to remove tumor tissue as thoroughly as possible and reduce the risk of recurrence. 6 This technology not only improves the ability to identify small lesions, but is also easy to operate. It only requires adding fluorescence imaging function to the existing imaging system, without the need for complex additional equipment. 5 However, the currently used fluorescent imaging perfusion solution only reflects the difference by the relative absorption of the tumor, and has no histological specificity. Therefore, there are still deficiencies in specificity and sensitivity, and the efficiency of tumor identification is still unsatisfactory, thus affecting the treatment effect.3 .

[0004] The nucleic acid aptamer Sgc8 was screened and obtained in 2006, and its target was identified as protein tyrosine kinase 7 (PTK7) in 2008. PTK7 is highly expressed in various tumor cells and has been widely used in tumor targeted imaging and other fields. 7,8 Currently, in tumor-targeted imaging, fluorescent molecules are usually directly linked to the 3' or 5' end of the aptamer. This approach may cause the fluorescent molecules to interfere with the recognition domain of the aptamer, thereby reducing its recognition ability.

[0005] References:

[0006] (1) PRC, NHC o. Chinese guidelines for diagnosis and treatment of urothelial carcinoma of bladder 2018 (English version). Chin. J. CancerRes. 2019, 31 (1), 49-66.

[0007] (2) Bray, F.; Laversanne, M.; Sung, H.; Ferlay, J.; Siegel, RL;Soerjomataram, I.; Jemal, A. Global cancer statistics 2022: GLOBOCANestimates of incidence and mortality worldwide for 36 cancers in 185countries. CA-Cancer J. Clin. 2024, 74 (3), 229-263.

[0008] (3) Pang, C.; Guan, Y.; Li, H.; Chen, W.; Zhu, G. Urologic cancer in China. Jpn. J. Clin. Oncol. 2016, 46 (6), 497-501.

[0009] (4) Dyrskjøt, L.; Hansel, D.E.; Efstathiou, J.A.; Knowles, MA;Galsky, MD; Teoh, J.; Theodorescu, D. Bladder cancer. Nat. Rev. Dis.Primers 2023, 9 (1), 58.

[0010] (5) Teoh, JY; Kamat, A.M.; Black, P.C.; Grivas, P.; Shariat, S.F.; Babjuk, M. Recurrence mechanisms of non-muscle-invasive bladder cancer - a clinical perspective. Nat. Rev. Urol. 2022, 19 (5), 280-294.

[0011] (6) Babjuk, M.; Böhle, A.; Burger, M.; Capoun, O.; Cohen, D.; Compérat, E.M.; Hernández, V.; Kaasinen, E.; Palou, J.; Rouprêt, M.; et al. EAUguidelines on non-muscle-invasive urothelial carcinoma of the bladder: update2016. Eur. Urol. 2017, 71 (3), 447-461.

[0012] (7) Shangguan, D.; Li, Y.; Tang, Z.W.; Cao, Z.C.; Chen, H.W.;Mallikaratchy, P.; Sefah, K.; Yang, C.J.; Tan, W. H. Aptamers evolved from live cells as effective molecular probes for cancer study. Proc. Natl. Acad.Sci. USA 2006, 103 (32), 11838-11843.

[0013] (8) Shangguan, D.; Cao, CH; Meng, L.; Mallikaratchy, P.; Sefah, K.; Wang, H. Li, Y.; Tan, WH Cell-specific aptamer probes for membraneprotein elucidation in cancer cells. J. Proteome Res. 2008, 7 (5), 2133-2139. Summary of the Invention

[0014] The present invention provides a modified nucleic acid aptamer and a method for modifying the nucleic acid aptamer, which are used to solve the interference of fluorescent molecules on the ability of the nucleic acid aptamer to recognize target molecules.

[0015] The first aspect of the present invention provides a modified nucleic acid aptamer, wherein the nucleic acid aptamer is nucleic acid aptamer Sgc8, and the nucleotide sequence of the nucleic acid aptamer Sgc8 is SEQ ID NO: 1;

[0016] One end of the nucleic acid aptamer is extended by a polynucleotide fragment, and one end of the polynucleotide fragment away from the nucleic acid aptamer is modified with a fluorescent molecule;

[0017] The nucleotide sequence of the polynucleotide fragment is TTTTT.

[0018] In the aforementioned nucleic acid aptamer, one end of the nucleic acid aptamer is the 5' end or the 3' end. The end of the polynucleotide fragment distal to the nucleic acid aptamer is the extended end of the polynucleotide fragment. When the polynucleotide fragment is located at the 5' end of the nucleic acid aptamer, the fluorescent molecule is modified at the 5' end of the polynucleotide fragment; when the polynucleotide fragment is located at the 3' end of the nucleic acid aptamer, the fluorescent molecule is modified at the 3' end of the polynucleotide fragment, i.e., the nucleic acid aptamer and the fluorescent molecule are connected via the polynucleotide fragment.

[0019] In the nucleic acid aptamer described above, the first nucleotide at the 5' end or 3' end of the nucleic acid aptamer is linked to the first nucleotide at the 3' end or 5' end of the polynucleotide fragment through a phosphodiester bond.

[0020] In the nucleic acid aptamer described above, the fluorescent molecule refers to a substance that, upon absorbing light of a given wavelength, re-emit light at a longer wavelength, thereby enabling visualization and analysis. The fluorescent molecule can be an organic chemical molecule or a bioluminescent molecule. The fluorescent molecule is covalently linked to the first nucleotide at the 5' or 3' end of the polynucleotide fragment.

[0021] It is understood that, in some cases, the fluorescent molecule can be modified and then linked to the first nucleotide at the 5' end or 3' end of the polynucleotide fragment.

[0022] In a specific embodiment, one end of the nucleic acid aptamer is the 3' end. When the 3' end of the nucleic acid aptamer is subjected to polynucleotide segment extension and fluorescent molecule modification, the structural formula of the modified nucleic acid aptamer is as shown in Formula 1:

[0023] 5'-n(a)- n(b)-Y-3' formula 1;

[0024] In formula 1, n(a) is a nucleic acid aptamer, n(b) is a polynucleotide fragment, and Y is a fluorescent group.

[0025] In a specific embodiment, the fluorescent molecule is selected from one or more of ICG or its derivatives, 5-ALA or its derivatives, FAM or its derivatives.

[0026] A second aspect of the present invention provides a method for modifying a nucleic acid aptamer, comprising: the nucleic acid aptamer is nucleic acid aptamer Sgc8, and the nucleotide sequence of the nucleic acid aptamer Sgc8 is SEQ ID NO: 1; extending a polynucleotide fragment at one end of the nucleic acid aptamer, and modifying the end of the polynucleotide fragment away from the nucleic acid aptamer with a fluorescent molecule to obtain a modified nucleic acid aptamer;

[0027] The nucleotide sequence of the polynucleotide fragment is TTTTT.

[0028] As described above, the extension of the polynucleotide fragment and the modification of the fluorescent molecule can be performed according to conventional techniques in the art.

[0029] The third aspect of the present invention provides a composition comprising any of the modified nucleic acid aptamers described above.

[0030] The composition as described above further comprises at least one of physiological saline and PBS buffer.

[0031] In the composition described above, the concentration of the modified nucleic acid aptamer in the composition is 250 nM-1000 nM; specifically, it can be 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1000 nM, or any two thereof. Furthermore, the concentration of the modified nucleic acid aptamer in the composition is not less than 400 nM.

[0032] The fourth aspect of the present invention provides a product comprising any of the modified nucleic acid aptamers described above or any of the compositions described above.

[0033] The product as described above can be specifically a reagent for target molecule fluorescence imaging. In a specific embodiment, the reagent is an irrigation solution for cystoscopy.

[0034] A fifth aspect of the present invention provides use of any of the modified nucleic acid aptamers described above or any of the compositions described above in the preparation of a product for target molecule fluorescence imaging, wherein the nucleic acid aptamer specifically recognizes the target molecule.

[0035] In the application described above, the target molecule may be protein tyrosine kinase 7 (PTK7); further, the target molecule may be cells, tissues, etc. that highly express protein tyrosine kinase 7 (PTK7); further, the target molecule may be derived from or located in bladder cancer cells, tissues, etc.

[0036] The applications described above can include the precise identification and imaging of bladder tumors. Specifically, a perfusion solution containing the modified nucleic acid aptamer can be injected into the bladder. The nucleic acid aptamer Sgc8 can accurately identify tumor cells. After the perfusion solution is incubated in the bladder for 10-40 minutes, the precise identification and imaging of the bladder tumor can be completed by detecting the fluorescence signal within 10-12 hours. In addition, the bladder can be flushed with solutions such as physiological saline, PBS buffer, and 5% glucose water to avoid interference with imaging caused by excessive perfusion solution.

[0037] Furthermore, the perfusion volume of the perfusion fluid may be 50 mL.

[0038] The present invention provides a method for modifying nucleic acid aptamers. The method involves extending one end of the nucleic acid aptamer with a polynucleotide fragment and modifying the extended end of the polynucleotide fragment with a fluorescent molecule. The polynucleotide fragment increases the spatial distance between the fluorescent molecule and the nucleic acid aptamer recognition domain, reducing the interference of the fluorescent molecule on the nucleic acid aptamer's recognition of the target molecule, thereby improving the recognition ability of the nucleic acid aptamer. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A schematic structural diagram of the modified nucleic acid aptamer provided in Example 1 of the present invention;

[0040] Figure 2 The figure shows the comparison of relative fluorescence intensity after different lengths of nucleotides were extended at the 3' end of the nucleic acid aptamer Sgc8;

[0041] Figure 3The statistical results of fluorescence intensity on the surface of T24 cells after co-incubation of different concentrations of nucleic acid aptamer Sgc8 without polynucleotide fragment modification with T24 cells;

[0042] Figure 4 The statistical results of fluorescence intensity on the surface of T24 cells after co-incubation of different concentrations of nucleic acid aptamer Sgc8 modified with a polynucleotide fragment of 5 Ts with T24 cells;

[0043] Figure 5 The statistical results of fluorescence intensity on the surface of T24 cells after co-incubation of different concentrations of nucleic acid aptamer Sgc8 modified with 10 T polynucleotide fragments with T24 cells;

[0044] Figure 6 To test the recognition ability of nucleic acid aptamer Sgc8 modified with a polynucleotide fragment of 5 T to 5637 cells;

[0045] Figure 7 To detect the recognition ability of nucleic acid aptamer Sgc8 modified with a polynucleotide fragment of 5 Ts on SCaBER cells;

[0046] Figure 8 To detect the recognition ability of the nucleic acid aptamer Sgc8 modified with a polynucleotide fragment of 5 Ts on T24 cells;

[0047] Figure 9 To detect the recognition ability of the nucleic acid aptamer Sgc8 modified with a polynucleotide fragment of 5 Ts on sv-huc-1 cells;

[0048] Figure 10 Detection of orthotopic bladder cancer by perfusate for in vivo imaging in small animals;

[0049] Figure 11 To detect the effect of perfusion fluid on identifying orthotopic bladder cancer 12 hours after in vivo imaging in small animals;

[0050] Figure 12 To detect the recognition of human bladder tumor tissue by perfusate for small animal in vivo imaging;

[0051] Figure 13 Validation of the size recognition of human bladder tumor tissue by perfusate for in vivo imaging in small animals;

[0052] Figure 14 To detect the recognition of Sgc8-ICG on human bladder tumor tissue by fluorescence cystoscopy;

[0053] Figure 15 The incubation concentration required for fluorescence cystoscopic detection of bladder cancer in situ by irrigating fluid;

[0054] Figure 16The incubation time required for fluorescence cystoscopic detection of bladder cancer in situ by irrigating the solution;

[0055] Figure 17 Schematic diagram of the application of modified nucleic acid aptamer Sgc8 in cystoscopic detection. DETAILED DESCRIPTION

[0056] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0057] Example 1: Modified nucleic acid aptamer Sgc8

[0058] The structural formula of the modified nucleic acid aptamer Sgc8 provided in this embodiment is shown in Formula 1, and its structural schematic diagram is shown in Figure 1 As shown;

[0059] 5'-n(a)- n(b)-Y-3' formula 1;

[0060] In Formula 1, n(a) is a nucleic acid aptamer, and its nucleotide sequence is shown in SEQ ID NO: 1; n(b) is a polynucleotide fragment, and its nucleotide sequence is TTTTT; Y is a fluorescent group.

[0061] The modified nucleic acid aptamer Sgc8 is synthesized using conventional methods such as solid phase synthesis.

[0062] Example 2: Verification of the recognition ability of the modified nucleic acid aptamer Sgc8

[0063] In this example, the 3' end of the nucleic acid aptamer Sgc8 was designed to be extended by 0, 5, and 10 Ts, respectively, and the fluorescent molecule FAM was modified at the extended end, denoted as Sgc8-FAM. PTK7-positive cell line T24 cells were cultured. When the cells were cultured to about 80% of the culture flask, the culture medium was aspirated, and the cells were gently rinsed with an appropriate amount of PBS buffer, and the PBS buffer was discarded. 0.25% trypsin was added to cover the cell layer, and the culture flask was gently shaken to evenly distribute the trypsin. Incubate at 37°C for 3 min, and serum-containing culture medium was added to terminate the digestion reaction. Gently blow the cell layer with a pipette to completely detach the cells and disperse them into a single cell suspension. The cell suspension was transferred to a centrifuge tube and the cells were counted. To the suspension containing 200,000 cells, a 250 nM concentration of the nucleic acid aptamer Sgc8-FAM perfusion solution with 0, 5, and 10 T extensions was added and incubated at 4°C for 20 min. After incubation, the cells were washed with PBS buffer. After washing, cells were analyzed using a flow cytometer (BD FACSVerse TM ) to detect the fluorescence of FAM, and the detection results are as follows Figure 2 shown.

[0064] The results showed that the relative fluorescence intensity of the aptamer Sgc8 with 5 T extension was significantly enhanced compared with the relative fluorescence values of the aptamers Sgc8 with 0 and 10 T extensions, indicating that the recognition ability of the aptamer Sgc8 with 5 T extension was better than that of the aptamers Sgc8 with 0 and 10 T extensions.

[0065] To further evaluate the differences in tumor cell recognition abilities of the Sgc8 aptamer with 0, 5, and 10 T extensions, this example used flow cytometry to measure the dissociation constants (Kd) of these three Sgc8 aptamers. The specific experimental steps included: According to the aforementioned experimental method, a T24 cell suspension was obtained, and different concentrations of the FAM fluorescent molecule-labeled Sgc8 aptamer were prepared. The cells were then incubated with the T24 cell suspension at 4°C for 20 minutes. After washing, the cell surface fluorescence intensity was measured using flow cytometry, and binding curves were plotted and Kd values calculated.

[0066] The results are as follows Figure 3-5 As shown in the figure, the Kd value of the 5T extension is lower than the Kd values of the 0T and 10T extensions, indicating that the 5T extension of the nucleic acid aptamer has a higher affinity for binding to the tumor target and has a stronger tumor recognition ability.

[0067] In summary, extending 5 T bases at the end of the nucleic acid aptamer Sgc8 can effectively improve its targeting recognition performance, providing an important basis for the accurate identification of tumors.

[0068] Example 3: Binding of nucleic acid aptamers to bladder cancer cells

[0069] Bladder cancer cell lines 5637 (human bladder cancer cells), SCaBER cells (human bladder squamous cell carcinoma cells), and T24 cells (human bladder transitional cell carcinoma cells) were selected as PTK7-positive cell lines, and SV-HUC-1 cells (immortalized human ureteral epithelial cells) were selected as PTK7-negative cell lines. All four cell lines were cultured. When the cells reached approximately 80% confluence in the culture flask, the culture medium was aspirated and gently rinsed with an appropriate amount of PBS buffer, which was discarded. 0.25% trypsin was added to cover the cell layer, and the culture flask was gently shaken to evenly distribute the trypsin. Incubate at 37°C for 3 minutes, and then add serum-supplemented medium to terminate the digestion reaction. Gently pipette the cell layer to completely detach the cells and disperse them into a single-cell suspension. The cell suspension was transferred to a centrifuge tube and the cells were counted. A 250 nM perfusate (made by modifying the aptamer Sgc8 with a polynucleotide fragment containing five T residues and FAM and mixing it with saline) was added to a suspension containing 200,000 cells and incubated at 4°C for 20 minutes. After incubation, the cells were washed with PBS buffer. After washing, the cells were analyzed using a flow cytometer (BD FACSVerse TM ) to detect the fluorescence of Sgc8-FAM, and the detection results are as follows Figure 6-9 shown.

[0070] according to Figure 6-9 As can be seen, compared with the blank control, the relative fluorescence intensity of Sgc8 with a 5-T extension at the 3' end was significantly enhanced after incubation with 5637 cells, SCaBER cells, and T24 cells, indicating that the perfusate can effectively identify tumor cells. However, sv-huc-1 cells, a normal bladder cell line, did not show a significant increase in relative fluorescence intensity after incubation with Sgc8 with a 5-T extension at the 3' end compared to the blank control, indicating that they were unable to effectively bind to the perfusate. Therefore, it is shown that the Sgc8 modified according to this strategy has good recognition ability for various bladder cancer cells.

[0071] Example 4: Application of nucleic acid aptamers in bladder cancer recognition and fluorescence imaging

[0072] The 3' end of the nucleic acid aptamer Sgc8 was extended with a polynucleotide fragment containing five Ts and modified with the fluorescent molecule ICG at the 3' end to obtain the modified nucleic acid aptamer Sgc8. The modified nucleic acid aptamer Sgc8 was dissolved in physiological saline to obtain a perfusate solution, Sgc8-ICG, at a concentration of 250 nM. The perfusate Sgc8-ICG was used in the following experiments.

[0073] 4.1. Fluorescein-labeled T24 cells (purchased from Shanghai Fuheng Biotechnology Co., Ltd., catalog number FH0176) were inoculated into the bladders of Balb / c mice (purchased from Shanghai Experimental Animal Research Center) to establish an orthotopic mouse bladder cancer model. The specific steps for establishing an orthotopic mouse bladder cancer model are as follows: 5-6 week old female BALB / c nude mice were selected and anesthetized by intraperitoneal injection of 1.25% tribromoethanol (200 μL / 10 g). A 24G indwelling needle lubricated with paraffin oil was inserted through the urethra and pretreated with 100 μL of collagenase II (1 mg / mL) for 10 minutes. Subsequently, 100 μL of luciferase-labeled bladder cancer cell suspension (1×10 7 cells / mL) for 1 h to promote tumor cell colonization. On day 5 after model establishment, potassium luciferin (15 mg / mL, 100 μL / 10 g) was intraperitoneally injected. Under isoflurane anesthesia, tumor growth was monitored using a small animal in vivo imaging system 15 min later.

[0074] After the model was successfully established, the perfusate, Sgc8-ICG, was injected into the bladder of the model mouse and incubated for 1 hour. After the incubation period, the mouse bladder was gently pressed to stimulate excretion of the perfusate. Ten minutes later, the mouse was imaged using a small animal in vivo imaging system (IVISLUMINA II). Subsequently, the mouse bladder tissue was dissected, flushed with saline, and imaged using the small animal in vivo imaging system.

[0075] The results are as follows Figure 10 As shown, the LUC channel displays bioluminescent tumor tissue, confirming the successful establishment of the tumor model; the Sgc8-ICG channel displays the fluorescence signal of the ICG fluorescent molecule-modified nucleic acid aptamer Sgc8. The image on the left shows the results under 0T extension conditions. The tumor tissue region displayed by the LUC channel lacks the fluorescence signal of the nucleic acid aptamer Sgc8. In contrast, the image on the right under 5T extension conditions shows that the fluorescence signal of the Sgc8-ICG perfusate colocalizes with the tumor tissue region, indicating that the Sgc8-ICG perfusate successfully recognizes and targets the tumor tissue. This result demonstrates that under the same concentration of nucleic acid aptamer Sgc8 incubation conditions, the recognition ability of the 5T extension is significantly better than that of the 0T extension. Therefore, the 5T extension exhibits superior performance in targeted recognition applications.

[0076] 4.2. Using the same method as in 4.1, inject the perfusate Sgc8-ICG into the bladder of the model mouse and incubate for 1 hour. Gently press the mouse bladder to stimulate the model mouse to expel the perfusate. After feeding and drinking for 24 hours, the mouse was imaged using a small animal in vivo imaging system. The mouse bladder was dissected and flushed with saline. The mouse bladder was then imaged using a small animal in vivo imaging system. Simultaneously, ICG-containing saline was injected into the bladder of the model mouse as a control and bladder imaging was performed using the same method.

[0077] The results are as follows Figure 11 As shown in the figure, 24 hours after the perfusion fluid was injected into the tumor, the detection signal was still present in the tumor site, and the signal of the LUC channel overlapped with the signal of the Sgc8-ICG channel, indicating that the perfusion fluid Sgc8-ICG exhibited excellent retention in the tumor area, specifically enriching and retaining it in the tumor tissue for a long time. This efficient targeting and retention ability provides an excellent foundation for future applications in tumor diagnosis, targeted therapy, and intraoperative navigation, and has significant potential for clinical translation.

[0078] 4.3. During transurethral bladder cancer resection surgery, fresh cancer tissue from two patients and one fresh normal tissue were collected and immediately used for experiments. First, the tissue samples were rinsed with saline. The perfusion solution, Sgc8-ICG, was incubated with the tissue samples at 37°C for 20 minutes. After rinsing the incubated tissue samples with saline, the tissue samples were examined using a small animal in vivo imaging system. Simultaneously, a saline solution containing 250 nM ICG was incubated with the tissue samples as a control.

[0079] The results are as follows Figure 12 As shown, the perfusate Sgc8-ICG can accurately identify tumors, illuminating them while leaving normal tissue undetected and unilluminated. ICG alone, due to its lack of targeting, is unable to specifically identify and illuminate bladder cancer tissue. This comparison demonstrates the high specificity and targeting capabilities of the perfusate Sgc8-ICG, providing important evidence for precise tumor detection and treatment.

[0080] 4.4. Fresh cancer tissue was collected from two patients during transurethral bladder cancer resection surgery and immediately began the experiment. First, the tissue samples were rinsed with saline. The bladder cancer tumor tissue was divided into different sizes. The tissue samples were incubated with the perfusion solution Sgc8-ICG (containing 10% urine) at 37°C for 20 minutes. After rinsing the incubated tissue samples with saline, they were examined using a small animal in vivo imaging system.

[0081] The results are as follows Figure 13As shown in the figure, the left and right pictures show the results of dividing two tumors into small pieces of different sizes, indicating that the perfusion fluid can not only accurately identify tumor tissue, but also has the ability to identify small pieces of both left and right tumors, and the minimum size of identifiable tumor tissue can reach 2 mm.

[0082] 4.5. During transurethral bladder cancer resection, fresh cancer tissue from two patients and one normal tissue sample were collected and immediately used for the experiment. The tissue samples were rinsed with normal saline. The perfusion solution, Sgc8-ICG, was incubated with the tissue samples at 37°C for 20 minutes. After rinsing with normal saline, the tissue samples were examined using a fluorescence cystoscopy system (780 4K, Qingdao Aomeike Medical Technology Co., Ltd.).

[0083] The results are as follows Figure 14 As shown in the fluorescence cystoscopy system, the perfusate Sgc8-ICG can accurately identify and illuminate tumor tissue through the targeting effect of the nucleic acid aptamer Sgc8, while normal tissue has no such reaction. In contrast, pure ICG lacks targeting and cannot specifically identify and illuminate bladder cancer tissue. This comparison result significantly demonstrates the high specificity and targeting ability of the perfusate Sgc8-ICG, providing an important basis for the precise detection and treatment of tumors.

[0084] 4.6. Fluorescein-labeled T24 cells were inoculated into the mouse bladder to establish an orthotopic mouse bladder cancer model as previously described. 50 μL of different concentrations of Sgc8-ICG (100 nM, 250 nM, 400 nM, and 1000 nM) were injected into the bladder of the model mouse and incubated for 30 minutes. Gently press the mouse bladder to stimulate the model mouse to expel the perfusion solution. The mouse was imaged using a small animal in vivo imaging system. The mouse bladder was dissected, flushed with saline, and imaged using a fluorescent cystoscope.

[0085] The results are as follows Figure 15 As shown in the figure, when the Sgc8-ICG concentration in the perfusate reached 400 nM and 1000 nM, the bladder of the tumor model mouse was clearly illuminated, achieving accurate identification of the orthotopic tumor. This experimental result shows that when using cystoscopic detection, the effective illumination concentration of the perfusate must be higher than 400 nM.

[0086] 4.7. Fluorescein-labeled T24 cells were inoculated into the mouse bladder to establish an orthotopic mouse bladder cancer model as previously described. 50 μL of a 400 nM perfusate containing Sgc8-ICG was injected into the bladder of the model mouse and incubated for 10, 20, and 40 minutes, respectively. Gently press the mouse bladder to stimulate expulsion of the perfusate. The mouse was imaged using a small animal in vivo imaging system. The mouse bladder was dissected, flushed with saline, and imaged using a fluorescent cystoscope.

[0087] The results are as follows Figure 16 As shown, after incubation for 10-40 min, the perfusion solution Sgc8-ICG successfully illuminated and identified the in situ tumor.

[0088] In summary, the spatial distance between the fluorescent molecule and the aptamer recognition domain is increased by the polynucleotide fragment, which reduces the interference of the fluorescent molecule on the aptamer's recognition of the target molecule, thereby improving the recognition ability of the aptamer.

[0089] Example 5: Application of nucleic acid aptamers in cystoscopy

[0090] like Figure 17 As shown in Figure 2, the cystoscopy includes the following steps:

[0091] Step 1: Inject the perfusion solution containing the modified nucleic acid aptamer into the bladder. The injection volume of the perfusion solution is 50 mL, the concentration of the modified nucleic acid aptamer Sgc8 is 400 nM, and the solvent is normal saline.

[0092] Step 2: After the infusion solution remains in the bladder for 40 minutes, it is drained out and the bladder is flushed with 500 mL of normal saline.

[0093] Step 3: Infuse 150 mL of normal saline and insert a fluorescent cystoscope into the bladder to observe and detect the tumor area on the bladder wall by observing the fluorescent signal.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A modified nucleic acid aptamer, characterized in that The nucleic acid aptamer is nucleic acid aptamer Sgc8, and the nucleotide sequence of the nucleic acid aptamer Sgc8 is SEQ ID NO: 1; The 3' end of the nucleic acid aptamer is extended by a polynucleotide fragment, and one end of the polynucleotide fragment away from the nucleic acid aptamer is modified with a fluorescent molecule; The nucleotide sequence of the polynucleotide fragment is TTTTT; The fluorescent molecule is selected from ICG or FAM.

2. A method for modifying a nucleic acid aptamer, characterized in that: include: The nucleic acid aptamer is nucleic acid aptamer Sgc8, and the nucleotide sequence of the nucleic acid aptamer Sgc8 is SEQ ID NO: 1; a polynucleotide fragment is extended at the 3' end of the nucleic acid aptamer, and a fluorescent molecule is modified at the end of the polynucleotide fragment away from the nucleic acid aptamer to obtain a modified nucleic acid aptamer; The nucleotide sequence of the polynucleotide fragment is TTTTT; The fluorescent molecule is selected from ICG or FAM.

3. A composition comprising the modified nucleic acid aptamer according to claim 1.

4. The composition according to claim 3, characterized in that The composition further comprises at least one of physiological saline and PBS buffer.

5. The composition according to claim 3 or 4, characterized in that The concentration of the modified nucleic acid aptamer in the composition is 250 nM-1000 nM.

6. A product comprising the modified nucleic acid aptamer according to claim 1 or the composition according to any one of claims 3 to 5.

7. Use of the nucleic acid aptamer according to claim 1 or the composition according to any one of claims 3 to 5 in preparing a product for target molecule fluorescence imaging, wherein the nucleic acid aptamer specifically recognizes the target molecule.

8. The use according to claim 7, characterized in that The target molecule is tyrosine protein kinase 7 or cells and / or tissues that highly express tyrosine protein kinase 7.

Images