Modified aptamer and application thereof

Abstract

The invention provides a modified nucleic acid aptamer and application thereof. On the first aspect, the invention provides a modified nucleic acid aptamer, the nucleic acid aptamer is a 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 through a polynucleotide fragment, and fluorescent molecular modification is carried out on the extended tail end of the polynucleotide fragment; the nucleotide sequence of the polynucleotide fragment is TTTTT. According to the invention, the base sequence with a certain length is added between the fluorescent molecule and the aptamer, so that the interference of the fluorescent molecule on an aptamer recognition structural domain can be effectively avoided, and the recognition and imaging efficiency is improved.

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 rapid aging of the population, the incidence of bladder cancer continues to rise. 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, and treatment is relatively convenient. 2 However, bladder cancer has a high recurrence rate and requires repeated examinations and treatments, which imposes a heavy economic burden on patients and society and also exacerbates the pain of patients. 2 Early bladder cancer is usually treated with electrosurgical resection under white light guidance. 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 bladder cancer has a high risk of recurrence. 3 Therefore, developing a highly specific and sensitive surgical auxiliary method to assist in the surgery to clean the tumor as much as possible 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), 5-aminolevulinic acid (5-ALA), etc., thereby improving the ability to identify cancerous areas. 4 These fluorescent imaging perfusion solutions will be selectively absorbed by 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, requiring only the addition of fluorescence imaging to the existing imaging system, without the need for complex additional equipment. 5 However, the fluorescent imaging perfusion fluid currently used only reflects the difference through the relative absorption of the tumor, and has no histological specificity. Therefore, it is still insufficient in specificity and sensitivity, and the efficiency of tumor recognition is still unsatisfactory, thus affecting the treatment effect.3 .

[0004] The nucleic acid aptamer Sgc8 was screened in 2006, and its target was identified as tyrosine protein kinase 7 (PTK7) in 2008. PTK7 is highly expressed in a variety of 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, which 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 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 by a fluorescent molecule;

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

[0018] As for the nucleic acid aptamer as described above, one end of the nucleic acid aptamer is the 5' end or the 3' end. The end of the polynucleotide fragment away from 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, that is, the nucleic acid aptamer and the fluorescent molecule are connected through the polynucleotide fragment.

[0019] As for 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] As described above, the fluorescent molecule is a substance that can re-emit light at a longer wavelength when absorbing light of a given wavelength, thereby enabling visualization and analysis. The fluorescent molecule can be an organic chemical molecule or a biological fluorescent molecule. The fluorescent molecule is covalently linked to the first nucleotide at the 5' end or 3' end of the polynucleotide fragment.

[0021] It is understood that, in some cases, the fluorescent molecule can be modified and then attached 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 a 3' end. When the 3' end of the nucleic acid aptamer is subjected to polynucleotide fragment 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] The 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; a polynucleotide fragment is extended at one 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;

[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 as 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. Further, 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] The fifth aspect of the present invention provides the 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 fluorescent imaging of a target molecule, wherein the nucleic acid aptamer specifically recognizes the target molecule.

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

[0036] The application as described above may include accurate identification and imaging of bladder tumors. Specifically, the perfusion solution containing the modified nucleic acid aptamer can be injected into the bladder, and the nucleic acid aptamer Sgc8 can accurately identify tumor cells. After the perfusion solution is incubated in the bladder for 10 min-40 min, accurate identification and imaging of bladder tumors are completed by detecting fluorescent signals within 10 min-12 h. In addition, the bladder can be flushed with solutions such as physiological saline, PBS buffer, and 5% glucose water to avoid interference of excessive perfusion solution on imaging.

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

[0038] The present invention provides a method for modifying a nucleic acid aptamer, wherein a polynucleotide fragment is used to extend one end of the nucleic acid aptamer, and a fluorescent molecule is modified at the extended end of the polynucleotide fragment. The polynucleotide fragment is used to increase the spatial distance between the fluorescent molecule and the nucleic acid aptamer recognition domain, thereby reducing the interference of the fluorescent molecule on the nucleic acid aptamer in recognizing the target molecule, thereby improving the recognition ability of the nucleic acid aptamer. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0040] Figure 2 is a comparison result diagram of the relative fluorescence intensity after extending nucleotides of different lengths 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 detect 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 The recognition ability of the nucleic acid aptamer Sgc8 modified with a polynucleotide fragment of 5 Ts on T24 cells was detected;

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

[0048] Fig.10 Identification of orthotopic bladder cancer using perfusate for in vivo imaging in small animals;

[0049] Fig.11 The effect of perfusion fluid on identifying orthotopic bladder cancer 12 hours after in vivo imaging of small animals was detected;

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

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

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

[0053] Fig.15 The incubation concentration required for fluorescent cystoscopic detection of irrigated fluid for identification of bladder cancer in situ;

[0054] Fig.16The incubation time required for fluorescent cystoscopic detection of bladder cancer in situ;

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

[0056] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination 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 creative work are 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; and Y is a fluorescent group.

[0061] The modified nucleic acid aptamer Sgc8 is synthesized by 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 embodiment, the 3' end of the nucleic acid aptamer Sgc8 is designed to extend 0, 5 and 10 Ts, respectively, and the fluorescent molecule FAM is modified at the extension end, which is expressed as Sgc8-FAM. The PTK7-positive cell line T24 cells were cultured. When the cells were cultured to about 80% of the culture bottle, the culture medium was aspirated, and the culture medium was gently rinsed with an appropriate amount of PBS buffer, and the PBS buffer was discarded. Add 0.25% trypsin to cover the cell layer, and shake the culture bottle gently to evenly distribute the trypsin. Incubate at 37°C for 3 min, add serum-containing culture medium, and 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. Transfer the cell suspension to a centrifuge tube and count the cells. Add 250 nM of nucleic acid aptamer Sgc8-FAM perfusion solution with extensions of 0, 5 and 10 Ts to the suspension containing 200,000 cells and incubate at 4°C for 20 min. After incubation, wash the cells with PBS buffer. After washing, the cells were analyzed by flow cytometry (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] In order to further evaluate the difference in the recognition ability of nucleic acid aptamers Sgc8 with extensions of 0, 5 and 10 T for tumor cells, this example uses a cell flow cytometer to detect the dissociation constants (Kd) of these three nucleic acid aptamers Sgc8 respectively. The specific experimental steps include: according to the above experimental method, obtain a T24 cell suspension, prepare different concentrations of nucleic acid aptamer Sgc8 labeled with FAM fluorescent molecules, and incubate with the T24 cell suspension at 4°C for 20 min. After washing, the cell surface fluorescence intensity is detected by flow cytometry, the binding curve is drawn and the Kd value is calculated.

[0066] The results are as follows Figure 3-5 As shown, the Kd value of extension 5T is lower than those of extension 0T and 10T, indicating that the nucleic acid aptamer extended by 5T has higher binding affinity with the tumor target and has 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 cells (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 (human ureteral epithelial immortalized cells) were selected as PTK7-negative cell lines. The above four cells were cultured. When the cells were cultured to about 80% of the culture flask, the culture medium was aspirated, and the appropriate amount of PBS buffer was used to gently rinse, 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, add serum-containing culture medium, and terminate the digestion reaction. Use a pipette to gently blow the cell layer to completely detach the cells and disperse them into a single cell suspension. Transfer the cell suspension to a centrifuge tube and count the cells. A perfusion solution with a concentration of 250 nM (the perfusion solution was obtained by modifying the nucleic acid aptamer Sgc8 with a polynucleotide fragment of 5 Ts and FAM and mixing it with physiological saline) was added to the suspension containing 200,000 cells and incubated at 4°C for 20 min. After the incubation, the cells were washed with PBS buffer. After washing, the cells were analyzed using a cell flow cytometer (BD FACSVerse TM ) to detect the fluorescence of Sgc8-FAM. The detection results are as follows Figure 6-9 shown.

[0070] according to Figure 6-9 It can be seen that compared with the blank control, the relative fluorescence intensity of Sgc8 with 5 Ts extended at the 3' end was significantly enhanced after incubation with 5637 cells, SCaBER cells and T24 cells, indicating that the perfusion solution can effectively identify tumor cells. However, sv-huc-1 cells, as a normal bladder cell line, did not show a significant increase in relative fluorescence intensity after incubation with Sgc8 with 5 Ts extended at the 3' end compared with the blank control, and could not effectively bind to the perfusion solution. Therefore, it is shown that the Sgc8 modified according to this strategy has a good recognition ability for a variety of 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 of 5 Ts, and the 3' end of the polynucleotide fragment was modified with a fluorescent molecule ICG to obtain a modified nucleic acid aptamer Sgc8; the modified nucleic acid aptamer Sgc8 was dissolved in physiological saline to obtain a perfusion solution Sgc8-ICG with a concentration of 250 nM. The perfusion solution Sgc8-ICG was used for the following experiment.

[0073] 4.1. Use fluorescein-labeled T24 cells (purchased from Shanghai Fuheng Biotechnology Co., Ltd., catalog number FH0176) to inoculate the bladder of Balb / c mice (purchased from Shanghai Experimental Animal Research Center) to construct an orthotopic mouse bladder cancer model. The specific steps for constructing 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 100 μL collagenase II (1 mg / mL) was instilled for pretreatment for 10 min, followed by injection of 100 μL of luciferase-labeled bladder cancer cell suspension (1×10 7 cells / mL) for 1 h to promote tumor cell colonization. On the 5th day after modeling, potassium luciferin (15 mg / mL, 100 μL / 10 g) was injected intraperitoneally, and under isoflurane gas anesthesia, the small animal in vivo imaging system was used to detect tumor growth 15 min later.

[0074] After the model was successfully constructed, the perfusion solution Sgc8-ICG was injected into the bladder of the model mouse and incubated for 1 hour. After the incubation, the mouse bladder was gently pressed to stimulate the model mouse to expel the perfusion solution. After 10 minutes, the mouse was imaged using the small animal in vivo imaging system (IVISLUMINA II). Subsequently, the mouse bladder tissue was dissected out, the bladder was flushed with saline, and the mouse bladder was imaged using the small animal in vivo imaging system.

[0075] The results are as follows Fig.10 As shown in the figure, the LUC channel shows the bioluminescent tumor tissue, confirming the successful construction of the tumor model; the Sgc8-ICG channel shows the fluorescence signal of the nucleic acid aptamer Sgc8 modified with the ICG fluorescent molecule. The left image shows the results under the 0T extension condition. The tumor tissue area shown by the LUC channel does not show the fluorescence signal of the nucleic acid aptamer Sgc8; in contrast, the image under the 5T extension condition on the right shows that the fluorescence signal of the Sgc8-ICG perfusion solution is co-localized with the tumor tissue area, indicating that the Sgc8-ICG perfusion solution successfully recognizes and targets the tumor tissue. This result shows that under the incubation conditions of the same concentration of the nucleic acid aptamer Sgc8, the recognition ability of the extension 5T is significantly better than that of the extension 0T. Therefore, the extension 5T shows better performance in targeted recognition applications.

[0076] 4.2. In the same way as in 4.1, inject the perfusion solution 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 perfusion solution. After feeding the mice for 24 hours without fasting or water deprivation, image the mice using the small animal in vivo imaging system. Dissect out the mouse bladder, flush the bladder with saline, and image the mouse bladder using the small animal in vivo imaging system. At the same time, inject ICG saline into the model mouse bladder as a control, and use the same method to image the bladder.

[0077] The results are as follows Fig.11 As shown in the figure, 24 hours after the perfusion fluid identified the tumor, the detection signal still existed 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 showed excellent retention in the tumor area and could be specifically enriched and retained in the tumor tissue for a long time. This efficient targeting and retention ability provides a good basic condition for future applications in tumor diagnosis, targeted therapy, and intraoperative navigation, and has important clinical translation potential.

[0078] 4.3. During the transurethral bladder cancer resection surgery, fresh cancer tissues from two patients and one fresh normal tissue were collected and the experiment was immediately carried out. First, the tissue samples were rinsed with saline. The perfusion solution Sgc8-ICG was incubated with the tissue samples at 37°C for 20 min. After the incubated tissue samples were rinsed with saline, the tissue samples were detected using a small animal in vivo imaging system. At the same time, a saline solution with a concentration of 250 nM ICG was incubated with the tissue samples as a control.

[0079] The results are as follows Fig.12 As shown in the figure, the perfusion fluid Sgc8-ICG can accurately identify tumors, thereby lighting up tumor tissues, while normal tissues will not be recognized and lit up. Pure ICG cannot specifically identify and light up bladder cancer tissues due to its lack of targeting. This comparison result fully demonstrates the high specificity and targeting ability of the perfusion fluid Sgc8-ICG, providing an important basis for the accurate detection and treatment of tumors.

[0080] 4.4. During the transurethral bladder cancer resection surgery, fresh cancer tissues from two patients were collected and the experiment was immediately carried out. First, the tissue samples were rinsed with saline. The bladder cancer tumor tissue was divided into different sizes. The perfusion fluid Sgc8-ICG (containing 10% urine) was incubated with the tissue samples at 37°C for 20 min. After the incubated tissue samples were rinsed with saline, the tissue samples were detected using the small animal live imaging system.

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

[0082] 4.5. During the transurethral bladder cancer resection, fresh cancer tissues from two patients and one fresh normal tissue were collected and the experiment was immediately carried out. The tissue samples were rinsed with normal saline. The perfusion fluid Sgc8-ICG was incubated with the tissue samples at 37°C for 20 min. After the tissue samples were rinsed with normal saline, the tissue samples were tested using a fluorescent cystoscope system (780 4K Qingdao Omek Medical Technology Co., Ltd.).

[0083] The results are as follows Fig.14 As shown in the figure, in the fluorescent cystoscope system, the perfusion fluid Sgc8-ICG can accurately identify and illuminate tumor tissues through the targeting effect of the nucleic acid aptamer Sgc8, while normal tissues have no such reaction; in contrast, pure ICG cannot specifically identify and illuminate bladder cancer tissues due to its lack of targeting. This comparison result significantly demonstrates the high specificity and targeting ability of the perfusion fluid Sgc8-ICG, providing an important basis for the accurate detection and treatment of tumors.

[0084] 4.6. Fluorescein-labeled T24 cells were inoculated into the mouse bladder, and an orthotopic mouse bladder cancer model was constructed according to the above method. 50 μL of different concentrations of perfusion solution Sgc8-ICG (100 nM, 250 nM, 400 nM, 1000 nM) was injected into the model mouse bladder and incubated for 30 min. The mouse bladder was gently pressed 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 out, the bladder was flushed with saline, and the mouse bladder was imaged using a fluorescent cystoscope.

[0085] The results are as follows Fig.15 As shown in the figure, when the concentration of Sgc8-ICG in the perfusion fluid reaches 400 nM and 1000 nM, it can be clearly observed that the bladder of the tumor model mouse is successfully illuminated, achieving accurate identification of the in situ tumor. The experimental results show that when using cystoscopy for detection, the effective illumination concentration of the perfusion fluid must be higher than 400 nM.

[0086] 4.7. Fluorescein-labeled T24 cells were inoculated into the mouse bladder, and an orthotopic mouse bladder cancer model was constructed according to the above method. 50 μL of 400 nM perfusion solution Sgc8-ICG was injected into the model mouse bladder and incubated for 10, 20, and 40 min, respectively. The mouse bladder was gently pressed 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 out, the bladder was flushed with saline, and the mouse bladder was imaged using a fluorescent cystoscope.

[0087] The results are as follows Fig.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 in recognizing the target molecule, thereby improving the recognition ability of the aptamer.

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

[0090] like Fig.17 As shown in the figure, during cystoscopy, the specific steps include the following:

[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 min, it is discharged and the bladder is flushed with 500 mL of normal saline.

[0093] Step 3: Infuse 150 mL of normal saline and insert the fluorescent cystoscope into the bladder to observe and detect the tumor area on the inner wall of the bladder 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned 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; 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 by a fluorescent molecule; The nucleotide sequence of the polynucleotide fragment is TTTTT.

2. The nucleic acid aptamer according to claim 1, characterized in that One end of the nucleic acid aptamer is the 5' end or the 3' end.

3. The nucleic acid aptamer according to claim 1 or 2, characterized in that The fluorescent molecule is selected from one or more of ICG or its derivatives, 5-ALA or its derivatives, FAM or its derivatives.

4. 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 one end of the nucleic acid aptamer, and a fluorescent molecule is modified at one 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.

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

6. The composition according to claim 5, characterized in that The composition further comprises at least one of physiological saline and PBS buffer.

7. The composition according to claim 5 or 6, characterized in that The concentration of the modified nucleic acid aptamer in the composition is 250 nM-1000 nM.

8. A product comprising the modified nucleic acid aptamer according to any one of claims 1 to 3 or the composition according to any one of claims 5 to 7.

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

10. The use according to claim 9, characterized in that: The target molecule is tyrosine protein kinase 7 or cells and / or tissues that highly express tyrosine protein kinase 7.

Images