Fluorescent probe based on nucleic acid amplification technology, construction method of fluorescent probe and application of fluorescent probe in in-situ visualization of glycoRNA on cell
By designing a fluorescent probe based on nucleic acid amplification technology, combining G-quadrilateral and CSTS dye, the problem of difficulty in detection of glycoRNA at the cell level is solved, and high sensitivity and accurate quantities are achieved in situ visualization, ensuring cell integrity and Low cost testing.
Patent Information
- Application Number
- CN202411371006.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-29
- Publication Date
- 2025-01-14
AI Technical Summary
The prior art is difficult to detect and visualize the spatial distribution and expression levels of glycosylated RNA (glycoRNA) directly and effectively at the cellular level, especially due to its low expression levels and detection difficulties caused by protein protection.
A fluorescent probe based on nucleic acid amplification technology was designed, including three DNA strands: DNA sequence AK that is complementary to glycoRNA, strand H1 that is complementary to AK part, and strand H2 that achieves signal amplification, combining G-quadrilateral and CSTS Dyes, which realizes specific amplification detection and visualization of glycoRNA at the cellular level.
Improve the sensitivity and accurate quantification of glycoRNA detection, real-time, dynamic, and damage-free in situ visualization in living cells, reduce background noise, reduce detection costs, and avoid steric hindrance caused by antibodies, ensuring cells Integrity.
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Figure CN119307591A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biochemical nanomaterials; in particular, it relates to a fluorescent probe based on nucleic acid amplification technology and its construction and application in directly visualizing glycoRNA in situ on cells. Background Art
[0002] In the past, it was generally believed that cell membranes were mainly composed of lipids, proteins and sugars, but did not contain RNA. However, scientists have discovered that a class of RNA molecules that were little known in the past, maxRNA, is stably attached to the outer surface of human cells. More interestingly, scientists have recently identified a surface molecule, glycosylated RNA (glycoRNA), on multiple cell lineages (including cancer cells), which is modified by sialic acid-containing N-glycans to form a new glycoconjugate. They have great potential for understanding cancer biology, because similar RNA modifications have been shown to affect cancer-related processes such as cancer cell migration, invasion and drug resistance. Prior to this, although there were potential signs that glycoRNA was related to glycoproteins, there was a lack of direct evidence to support this claim. At this time, the identification and localization of glycosylated RNA (glycoRNA) on the cell surface connects different aspects of glycobiology and RNA biology, which were often separated in the past. Because glycoRNA is located on the outside of the cell membrane, it is easier to access than targets inside the cell and can become a more convenient drug target. Characterization of the spatial distribution and expression level of glycoRNA will help further reveal its biological functions and its role in disease. However, the characterization of glycoRNA is highly complex. Compared with common RNA, glycoRNA not only has glycosylation modification, but may also have other types of modifications, such as phosphorylation, methylation, etc. The presence of these modifications makes the detection and characterization of glycoRNA more difficult. In addition, the expression level of glycoRNA is low, and glycoRNA is protected by proteins, etc., and traditional methods are prone to off-target effects. Therefore, there is an urgent need for a method to directly visualize the spatial distribution of glycoRNA in situ. Fluorescent probes have high sensitivity: glycoRNA can be accurately quantified; fluorescent probes are non-destructive: fluorescent detection can perform non-destructive detection of glycoRNA while maintaining cell integrity; real-time dynamic monitoring: fluorescent probes have the ability to monitor in real time, in situ, and dynamically. Therefore, fluorescent probes may become an effective means to study glycoRNA.
[0003] From the perspective of the structure of glycoRNA, it contains two parts: sialic acid ligand and RNA. The research on polysaccharide clusters is very mature. Although the existing high-throughput sequencing technology has identified the sequence of the RNA, no further description is given for its content and distribution. The present invention designs a fluorescent probe based on nucleic acid amplification technology of hybridization chain reaction for the RNA part of glycoRNA, and introduces G-quadruplex to improve the sensitivity, hoping to achieve specific amplification detection of glycoRNA at the cellular level. Summary of the invention
[0004] The primary purpose of the present invention is to provide a new design of a fluorescent probe (hereinafter referred to as FHCR) based on nucleic acid amplification technology, aiming to achieve in situ specific amplification detection of glycoRNA.
[0005] Fluorescent probe based on nucleic acid amplification technology, including three DNA chains:
[0006] (1) DNA sequence AK complementary to glycoRNA;
[0007] (2) strand H1 that is partially complementary to AK;
[0008] (3) Chain H2 achieves signal amplification.
[0009] Further,
[0010] The DNA sequence AK complementary to the glycoRNA includes, from the 5' end, an HCR initiating sequence (18-24 nt), a T spacer sequence (6-12 nt), and a sequence complementary to U1 (50-59 nt);
[0011] The chain H1 complementary to the AK portion includes a sequence complementary to the HCR initiation portion of AK (19-24nt), a sequence complementary to H2 (19-25nt), and a G-quadruplex portion (13-18nt);
[0012] The signal amplifying chain H2 includes a sequence complementary to the 5' end of H1 (19-25 nt), a sequence complementary to the 3' end of H1 (19-25 nt), and a G-quadruplex portion (13-18 nt).
[0013] Furthermore,
[0014] The T spacer sequence in the DNA sequence AK complementary to the glycoRNA is used to reduce the effect of steric hindrance on the recognition ability;
[0015] The G-quadruplex part is used to bind CSTS dye to achieve visualization effect.
[0016] Specifically, the fluorescent probe based on nucleic acid amplification technology preferably has the following sequence:
[0017] The sequence of AK is AGT TGT AGT CAG ACT ATT CGA TTTTTTTT CTG GGA AAA CCA CCTTCG TGA TCA TGG TAT CTC CCC TGC CAG GTA AGT AT;
[0018] The sequence of H1 is ATC GAA TAG TCT GAC TAC AAC T TG AAA CAG TTG TAG TCA GAC TAGGG TTG GGC GGG ATG GGG;
[0019] The H2 sequence is AGT TGT AGT CAG ACT ATT CGA T TA GTC TGA CTA CAA CTG TTT CAGGG TTG GGC GGG ATG GGG.
[0020] The second aspect of the present invention aims to provide a method for constructing the fluorescent probe based on nucleic acid amplification technology, which is synthesized and constructed according to the design requirements of the fluorescent probe based on nucleic acid amplification technology.
[0021] The third object of the present invention is to provide an application method of the fluorescent probe based on nucleic acid amplification technology for in situ visual detection of glycoRNA on cells.
[0022] Especially for testing for non-diagnostic and therapeutic purposes.
[0023] The present invention mainly includes three DNA chains
[0024] (1) The DNA sequence AK complementary to glycoRNA is designed to include, from the 5' end, the HCR initiation sequence (the horizontal line portion, 21 nt); the T spacer sequence (8 nt) to reduce the effect of steric hindrance on recognition ability; and the complementary sequence to U1 to recognize U1 glycoRNA (the bold portion, 50 nt).
[0025]
[0026] (2) strand H1, which is partially complementary to AK, includes a sequence complementary to the HCR initiation portion of AK (the horizontal line portion, 22 nt);
[0027] Complementary sequence to H2 (italic part 22 nt); G-quadruplex part, used to bind CSTS dye to achieve visualization effect (bold part 18 nt).
[0028]
[0029] (3) Chain H2, which achieves signal amplification, includes a complementary sequence to the 5' end of H1 (the underlined part, 22 nt) and a complementary sequence to the 3' end of H1 (the italic part, 22 nt); and a G-quadruplex part for binding to the CSTS dye (the bold part, 18 nt).
[0030]
[0031] Furthermore, AK opens the hairpin structure of H1, and then the exposed part of H1 continues to open the hairpin structure of H2, finally forming a long-chain nanocluster complex of the three cross-complementary structures.
[0032] The present invention mainly includes the following two parts:
[0033] (1) Signal amplification based on hybridization chain reaction
[0034] (2) G-quadruplex-enriched fluorescent aptamer
[0035] Hybridization chain reaction can help us amplify and increase the signal strength of glycoRNA, thereby improving its detection sensitivity. This is a nucleic acid amplification technology that does not require enzyme participation, is easy to prepare, and can reduce detection costs.
[0036] FHCR nanoassemblies enrich multiple G-quadruplexes, and the specific binding of CSTS dye to G-quadruplexes can reduce the background, thereby improving its detection sensitivity.
[0037] The use of fluorescent aptamers enables us to observe and quantify the content and distribution of glycoRNA in real time in living cells. CSTS dye is a near-infrared dye that contains negatively charged groups that are repelled by the cell membrane. It is a membrane-impermeable dye that not only has extremely low biological toxicity, but also can overcome the disadvantage of the high fluorescence background of the cell itself.
[0038] The present invention combines nucleic acid amplification technology and fluorescent aptamer means to achieve direct in situ visualization of glycoRNA at the living cell level while avoiding steric hindrance caused by antibodies, etc., so as to analyze its content and spatial distribution.
[0039] The present invention first verifies the recognition ability of the hybridization sequence R-Cy5 through the following experiments.
[0040] R-Cy5 is complementary to U1 glycoRNA and modified with fluorophore Cy5. The specific sequence is CTG GGAAAA CCA CCT TCG TGA TCA TGG TAT CTC CCC TGC CAG GTA AGT AT-Cy5
[0041] (1) Preparation and in vitro characterization of nanoassemblies
[0042] (2) Imaging analysis of glycoRNA by FHCR on the cell membrane
[0043] (3) Flow cytometry verification
[0044] (4) Cell co-localization analysis
[0045] (5) Generality analysis
[0046] The present invention has found that the concentration and time of incubation of the hybridization sequence R-Cy5 with cells have a significant impact on the recognition ability and need to be optimized.
[0047] Furthermore, the concentration of R-Cy5 was designed to be 0 μM, 0.5 μM, 1 μM, 1.5 μM, and 2 μM, and the incubation time was set to 15 min, 30 min, 45 min, and 60 min.
[0048] Preferably, the R-Cy5 concentration is selected to be 1.5 μM.
[0049] Preferably, the incubation time is 30 min.
[0050] Furthermore, the incubation conditions of FHCR-treated cells with the dye CSTS have a significant impact on the imaging results, and several factors need to be optimized: dye concentration, CSTS incubation time.
[0051] Preferably, the dye concentration is selected to be 4 μM and the incubation time is 15 min.
[0052] Furthermore, when performing flow cytometry, using trypsin to digest cells will damage the structure of glycoRNA, so a milder EDTA digestion solution is needed to disperse the cells.
[0053] Beneficial effects of the present invention
[0054] 1. RNA localization can be performed without pretreatment.
[0055] 2. HCR nanoassemblies can amplify the output signal of glycoRNA by enriching multiple G-quadruplexes, thereby improving the sensitivity of detection.
[0056] 3. The specific binding of G4 dye to G-quadruplex can reduce the background, allowing us to observe and quantify the content and distribution of glycoRNA in real time in living cells.
[0057] 4. The present invention is an enzyme-free and modification-free technology, which reduces the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS
[0058] Figure 1 This is a schematic diagram of the FHCR provided by the present invention to realize glycoRNA imaging on the cell membrane surface.
[0059] Figure 2 Provided in Example 1 of the present invention: A) confocal imaging of hybridization sequences and cells incubated; B) flow cytometry data of hybridization sequences and cells incubated.
[0060] Figure 3 Preparation and in vitro characterization of the nanoassembly provided in Example 2 of the present invention: A) gel electrophoresis results; B) fluorescence results of each part.
[0061] Figure 4 Provided for Examples 3 and 4 of the present invention: A) Imaging analysis of glycoRNA by FHCR on the cell membrane; B) Flow cytometry verification of glycoRNA by FHCR on the cell membrane.
[0062] Figure 5 This is the confocal imaging result of cell co-localization analysis provided in Example 5 of the present invention.
[0063] Figure 6 The universal analysis of FHCR on glycoRNA in the cell membrane provided in Example 6 of the present invention: A) the content of different glycoRNAs on Hela cells; B) the content of different glycoRNAs on L02 cells; C) the content of different glycoRNAs on A549 cells. DETAILED DESCRIPTION
[0064] Unless otherwise specified, the cells, drugs and reagents used in the examples of the present invention are all from regular and easily available channels. Among them, all nucleic acid chains were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. and modified accordingly. The specific sequences are as follows.
[0065]
[0066]
[0067] Example 1. Verification of hybridization sequence recognition ability
[0068] HeLa cells were seeded in 15 mm confocal dishes and cultured overnight. 2+and Ca 2+ The cells were washed three times with DPBS and then blocked with DPBS containing 10% goat serum at 4°C for 30 minutes. The R-Cy5 probe (1.5 μM) was incubated with FISH buffer (50 mM Tris-HCl, 10 mM MgCl, pH 7.5) at 37°C for 30 minutes, and the cells were washed three times with DPBS to remove the unfixed probe, and then the cell membrane fluorescence was analyzed using the CLSM confocal imaging system. To verify the effect of RNase A digestion or Competition chain (complementary to U1 glycoRNA for 59 bases), the cells were first blocked with DPBS and washed three times, incubated with RNase A (50 U / mL) or Competition chain (4.5 μM) at 37°C for 30 minutes, and finally incubated with R-Cy5 probe.
[0069] The results are as follows Figure 2 A), after incubation of cells with R-Cy5 probe, fluorescence enhancement was observed around the cells. Due to the extremely low content of glycoRNA, this fluorescence was weak. When cells pre-treated with RNase A were incubated with R-Cy5 probe, negligible fluorescence was observed (compared with the positive group). The same result was observed in the group treated with Competition chain.
[0070] Furthermore, if Figure 2 B), further verified by flow cytometry.
[0071] Example 2. Preparation and in vitro characterization of nanoassemblies
[0072] All probes were annealed before use. First, probes H1 (2 μM), H2 (2 μM) and AK (1.5 μM) or KT (KT does not contain a complementary sequence to U1 glycoRNA and can only trigger HCR reaction) were mixed in FISH buffer and incubated at 37 ° C for 2 hours to form nanoassemblies. Subsequently, the synthesized nanoassemblies were characterized by 2.5% agarose gel electrophoresis (120V, 90min). Then, the CSTS dye was dissolved in G4 buffer (20mM Tris-HCl, 100mM KCl, pH 7.5), incubated with the formed nanoassemblies at 25 ° C for 15min, and then measured with a fluorescence spectrophotometer (Ex: 680nm, Em: 700-750nm).
[0073] The results are as follows Figure 3A), comparing lanes 1-5, 11, and 12, it shows that H1 can open AK, and H2 alone does not react with AK. In lane 11, H1 and H2 do not react. Comparing lanes 11 and 12, it shows that long-chain nanoassemblies can only be generated in the presence of AK. Lanes 6 and 7 show that KT can also trigger HCR reactions. Figure 3 As shown in B), the activation effect of AK alone on CSTS is negligible, while after HCR amplification, the fluorescence increases by nearly 60 times. Compared with the AK+H1 group, the complete HCR group also produces nearly 13-fold enhancement. Further optimization of the conditions can achieve a 16-fold fluorescence amplification effect.
[0074] Example 3. Imaging analysis of glycoRNA by FHCR on the cell membrane
[0075] 3.1. HeLa cells (1×10 6 ) were seeded in a 15 mm confocal dish and cultured overnight. All cell samples were rinsed three times with DPBS before the experiment, and no fixative was used to fix the cells during imaging. (1) The cells were first blocked with DPBS containing 10% sheep serum at 4°C for 30 minutes, and then the residual blocking solution was washed away with DPBS. (2) The cells were incubated with AK (1.5 μM) or KT (1.5 μM) in FISH buffer at 37°C for 30 minutes, H1 (2 μM) and H2 (2 μM) were added as premixes, and the hybridization chain reaction was performed at 37°C for 1 hour. Unbound probes were removed by washing with DPBS. (3) The CSTS solution was incubated with the cells at 25°C for 15 minutes. The U1 glycoRNA was then directly visualized using a CLSM imaging system.
[0076] 3.2. RNase A or the competitive chain was incubated with the cells for 30 minutes, and then the steps of Example 3.1 were performed and analyzed using a CLSM imaging system.
[0077] The results are as follows Figure 4 As shown in Figure A), bright fluorescence enhancement is produced around the cells treated with FHCR, and the fluorescence of the cells treated with RNase A and the competitive chain is significantly weakened, which is consistent with the results in the previous example 1, indicating that the present invention can achieve amplified imaging of glycoRNA at the cellular level.
[0078] Example 4. Flow cytometry validation
[0079] 4.1 HeLa cells (1×10 6 ) were inoculated in a 24-well plate and cultured overnight. Example 1 was repeated, and the cells were incubated with R-Cy5 probes and digested with 0.04% EDTA solution for 5 minutes, and then the cells were dispersed and analyzed by flow cytometry using DPBS.
[0080] 4.2 Repeat the steps of "Example 3". After the FHCR step, digest the cells with 0.04% EDTA solution for 5 minutes, then incubate the cells with CSTS solution at 25°C for 15 minutes. Finally, disperse the cells with DPBS solution for flow cytometry analysis.
[0081] The results are as follows Figure 4 As shown in Figure B), the peaks of cells treated with RNase A and competition chain shifted significantly, and the effect of the competition chain group was slightly weaker than that of the RNase A treatment group, which was consistent with the confocal imaging results, further demonstrating that the present invention can directly visualize the distribution of glycoRNA on the cell membrane.
[0082] Example 5. Cell co-localization analysis
[0083] To verify the distribution of glycoRNA, we incubated FHCR-treated cells with DiO (2×) cell membrane dye at 37°C for 15 minutes, then added Hoechst 33342 (1×) live cell dye and incubated at room temperature for 10 minutes. Finally, CSTS solution was added and incubated at room temperature for 15 minutes, and CLSM imaging was used to analyze the cell co-localization results.
[0084] The results are as follows Figure 5 As shown, cells treated with CSTS alone have no fluorescence, while cells treated with FHCR show bright fluorescence on the cell membrane after incubation with CSTS dye, and the fluorescence of CSTS overlaps with the fluorescence of DIO cell membrane dye to a large extent, further demonstrating that our invention can achieve visualization imaging of specific RNA on the cell membrane. Combined with the fluorescence of the living cell dye Hoechst, the application of the present invention at the living cell level is confirmed.
[0085] Example 6. Generality analysis
[0086] The hybridization sequence of other glycoRNA (Y5 / U3 / U35a) was used to replace the U1 glycoRNA hybridization sequence on AK, and the steps of Example 3 were followed. The results were as follows: Figure 6 A), indicating that FHCR can also be used to visualize different glycoRNAs on Hela cells. Figure 6 B) and C) were also performed on A549 cells and L02 cells according to Example 3, further demonstrating the versatility of this method.
Claims
1. A fluorescent probe based on nucleic acid amplification technology, characterized in that: It consists of three DNA strands: (1) DNA sequence AK complementary to glycoRNA; (2) strand H1 that is partially complementary to AK; (3) Chain H2 achieves signal amplification.
2. The fluorescent probe based on nucleic acid amplification technology according to claim 1, characterized in that: The DNA sequence AK complementary to the glycoRNA includes, from the 5' end, an HCR initiating sequence, a T spacer sequence, and a sequence complementary to U1; The chain H1 complementary to the AK portion includes a sequence complementary to the HCR initiation portion of AK, a sequence complementary to H2, and a G-quadruplex portion; The chain H2 that achieves the signal amplification function comprises a sequence complementary to the 5' end of H1, a sequence complementary to the 3' end of H1, and a G-quadruplex portion.
3. The fluorescent probe based on nucleic acid amplification technology according to claim 1 or 2, characterized in that: The T spacer sequence in the DNA sequence AK complementary to the glycoRNA is used to reduce the effect of steric hindrance on the recognition ability; The G-quadruplex part is used to bind CSTS dye to achieve visualization effect.
4. The fluorescent probe based on nucleic acid amplification technology according to claim 1, 2 or 3, characterized in that: The sequence of AK is AGT TGT AGT CAG ACT ATT CGA TTTTTTTT CTG GGA AAA CCA CCT TCGTGA TCA TGG TAT CTC CCC TGC CAG GTA AGT AT; The sequence of H1 is ATC GAA TAG TCT GAC TAC AAC T TG AAA CAG TTG TAG TCA GAC TA GGGTTG GGC GGG ATG GGG; The H2 sequence is AGT TGT AGT CAG ACT ATT CGA T TA GTC TGA CTA CAA CTG TTT CA GGGTTG GGC GGG ATG GGG.
5. The method for constructing a fluorescent probe based on nucleic acid amplification technology according to any one of claims 1 to 4, characterized in that: It is synthesized and constructed according to the design requirements of fluorescent probes based on nucleic acid amplification technology.
6. The method for applying the fluorescent probe based on nucleic acid amplification technology according to any one of claims 1 to 4, characterized in that: Used for in situ visualization and detection of glycoRNA in cells.
7. The application method according to claim 6, characterized in that: Testing for non-diagnostic and therapeutic purposes.