Development and application of intracellular tracing platform based on cavity hairpin tht optical nucleic acid switch

Abstract

The application provides development and application of an intracellular tracing platform based on a cavity hairpin ThT optical nucleic acid switch. A hairpin sequence carrying a CGG-AAA mismatch cavity is modified to a liposome, so that stable loading of low-concentration ThT (nM level) and efficient fluorescence excitation after the ThT enters the cell are realized without additional introduction of fluorescent markers or enzymes and other fluorescent developing substances and other delivery carriers assisting the ThT to enter the cell. The developed tracing platform has excellent imaging capability. The cavity hairpin ThT optical nucleic acid switch can delay passive ThT nuclear entry within 8 hours after the ThT enters the cell, so that the developed intracellular tracing platform further overcomes the problem of non-specific high background signal interference of ThT in the nuclear region after the ThT enters the cell, saves the amount of ThT, and is convenient for expanding the diversified application scenarios of ThT in the cell.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular biology, and relates to the application of a cavity hairpin capable of exciting ThT fluorescence and the development of a corresponding intracellular tracing platform. BACKGROUND

[0002] Thioflavin T (ThT) is a common fluorescent dye. In the middle of the last century, it was first reported to be used for quantitative analysis of amyloid in thyroid and kidney tissue sections, and in the following decade, its application was gradually expanded from histological analysis to structural and disease research, playing an important role in the field of biomedicine. In addition to the analysis of protein components, with the discovery of the advantages of ThT itself, such as photophysical stability, membrane permeability and low toxicity, more and more different conformational nucleic acid sequences have been reported to enhance ThT fluorescence, enabling ThT as a fluorescent molecule to not only indicate nucleic acid conformational changes to explore its biological functions, but also to a certain extent replace traditional fluorescent tags to achieve observation and imaging of special nucleic acid conformation distribution in cells, and even to be used for monitoring the expression and metabolic processes of functional genes with these structures.

[0003] However, while ThT has emerged as a key area in intracellular research, it also has some shortcomings. For example, in order to achieve similar imaging effects as commonly used nucleic acid dyes, it is necessary to increase the processing concentration (μM) or develop suitable nanocarriers to deliver ThT to address its limited intracellular fluorescence excitation or high background signals due to excessive dispersion, but high concentrations of ThT intracellular load are toxic to cells themselves.

[0004] Therefore, it is necessary to select nucleic acid conformations that can achieve ThT light excitation enhancement and stable loading in cells, and have few conformational formation condition constraints, and to reasonably build an intracellular imaging tracing platform to promote the application and development of ThT in the field of cell biology. SUMMARY

[0005] Based on the difficulties of low-concentration ThT intracellular imaging and the lack of specificity of ThT imaging due to complex intracellular environment, the technical problem to be solved by the present application is to apply a new double-stranded nucleic acid conformation (developed cavity hairpin ThT light nucleic acid switch) which has strong ThT fluorescence excitation and loading stability and few constraints on its own conformational formation, so as to help ThT as a fluorescent reporter molecule to establish a specific imaging tracing platform assisted by intracellular delivery carriers as the core element of intracellular tracing.

[0006] The present application provides a nucleic acid sequence having a base pair mismatch cavity structure capable of exciting ThT fluorescence potential, which is composed of CGG and AAA mismatches, wherein each base forms a mismatch cavity structure of A-C / A-G / A-G, and the sequence is modified at the 3' end with a cholsterol (chol) linker.

[0007] Preferably, the nucleic acid sequence is composed of ThT photoswitch hairpin sequence containing 1-2 CGG-AAA cavity structure, and then adding 3-8 nt polyA tail at 3' end and modifying cholesteryl linker.

[0008] The nucleic acid sequence is obtained by adding 5 nt polyA tail at 3' end and modifying cholesteryl linker.

[0009] The continuous nucleotides composed of CGG and the continuous nucleotides composed of AAA in the nucleic acid sequence are on the same nucleotide chain, forming a hairpin structure.

[0010] The nucleic acid sequence is selected from one of the following sequences:

[0011] A-C / A-G / A-G-3'chol: 5'-TTTTCGGTTTTAAAAAAAAAAAAAAAAAAAA-chol-3', SEQ ID NO. 1;

[0012] 6H3M1-3'chol: 5'-TTTTTTTTTTTCGGTTTTTTTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAchol-3', SEQ ID NO. 2;

[0013] 6H3M2-3'chol: 5'-TTTTTTCGGTTTTTTTCGGTTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-chol-3', SEQ ID NO. 3;

[0014] 6H3Mm-TET-3'chol: 5'-CGGTGGTGCGGTGGTGAAAAAACACCAAAA CACCACCGAAAAA-chol-3', SEQ ID NO. 4;

[0015] 6H3Mt-TET-3'chol: 5'-CGGTGGTGCGGTGGTGAAAAAACACCACCGC ACCAAAAAAAAA-chol-3', SEQ ID NO. 5;

[0016] 6H3Md-TET-3'chol: 5'-CGGTGGTGCGGTGGTGAAAAAACACCAAAA CACCAAAAAAAAA-chol-3', SEQ ID NO. 6;

[0017] H3Md-TET-3'chol: 5'-CGGTGGTGCGGTGGTGCACCAAAACACCAAAA AAA-chol-3', SEQ ID NO. 7;

[0018] 1H3Md-TET-3'chol: 5'-CGGTGGTGCGGTGGTGACACCAAAACACCA AAA AAAAAAAA-chol-3', SEQ ID NO. 8;

[0019] 2H3Md-TET-3'chol: 5'-CGGTGGTGCGGTGGTGAACACCAAAACACC AAAAAAAAA-chol-3', SEQ ID NO. 9;

[0020] 3H3Md-TET-3'chol: 5'-CGGTGGTGCGGTGGTGAAACACCAAAACAC CAAAAAAAAA-chol-3', SEQ ID NO. 10;

[0021] 4H3Md-TET-3'chol: 5'-CGGTGGTGCGGTGGTGAAAACACCAAAACA CCAAAAAAAAA-chol-3', SEQ ID NO. 11;

[0022] 8H3Md-TET-3'chol: 5'-CGGTGGTGCGGTGGTGAAAAAAAACACCAA AACACCAAAAAAAAA-chol-3', SEQ ID NO. 12;

[0023] 10H3Md-TET-3'chol: 5'-CGGTGGTGCGGTGGTGAAAAAAAAAACACCAAAACACCAAAAAAAAA-chol-3', SEQ ID NO. 13;

[0024] H3Mm-BB-3'chol: 5'-ACATAATTTAATACGGATGTTAACATAAATAT TAAATTATGTAAAAA-chol-3', SEQ ID NO. 14;

[0025] H3Mt-BB-3'chol: 5'-ACATAACGGAATATTTATGTTAACATAAATATT AAATTATGTTTTTT-chol-3', SEQ ID NO. 15;

[0026] H3Md-BB-3'chol: 5'-ACATAACGGAATACGGATGTTAACATAAATATTAAATTATGTTTTTT-chol-3', SEQ ID NO. 16.

[0027] In one aspect of the present application, an intracellular liposome non-enzyme free labeling fluorescent tracing imaging system is provided, comprising the following steps:

[0028] (1) The aforementioned nucleic acid sequence is coupled with liposomes at 25-37°C for 4-8h, the ratio of liposome phospholipid and sequence modification is controlled to be 100:1-1000:1, 5000g-8000g, and centrifuged for 3-10min; preferably, the coupling is performed at 37°C for 6h, the ratio of liposome phospholipid and sequence modification is controlled to be 500:1, 6000g, and centrifuged for 5min;

[0029] (2) ThT is added to the above system in a dark environment at 25-37°C, incubated for 30-120min, 5000g-8000g, centrifuged for 3-10min; preferably, ThT is added to the above system in a dark environment at 37°C, incubated for 60min, 6000g, and centrifuged for 5min;

[0030] (3) The above obtained liposome imaging system is co-incubated with cells, and then cultured for 2-16h, and then observed under a fluorescence microscope after being fixed by 4% paraformaldehyde; preferably, the co-incubation culture is 8h, and then observed under a fluorescence microscope after being fixed by 4% paraformaldehyde.

[0031] The present application also provides the use of the above nucleic acid sequence in constructing an intracellular phospholipid type delivery system. The phospholipid type delivery system is, for example, a liposome or an exosome.

[0032] In another aspect, the intracellular liposome non-enzyme free labeling fluorescent tracing imaging system provided by the present application is used in the fields of tracing imaging, drug delivery, targeted therapy, and pathway verification.

[0033] The present application has the following technical effects:

[0034] (1) The present application provides an intracellular delivery carrier tracing imaging platform based on a CGG-AAA mismatched cavity hairpin ThT optical nucleic acid switch. The platform has excellent high-loading stability of ThT and intracellular imaging capability, can realize stable loading of ThT, and effectively slows down the entry of ThT into the nucleus within 8h.

[0035] (2) The ThT intracellular imaging system based on the cavity hairpin of the present application can specifically track the intracellular carrier at the nM level without introducing other delivery carriers to assist the entry of ThT into the cell, effectively saving the processing concentration of ThT.

[0036] (3) The present application does not need to introduce additional fluorescent markers or enzymes and other fluorescent developing substances, which avoids complex operation and additional cost, and facilitates the diversified application of ThT in cells. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Preparation of liposomes, characterization and optimization. (A) DLS detection of particle size and PDI of liposomes. (B) Gel electrophoresis results of HC-Lip under different P / N ratios.

[0038] Figure 2 3T3-L cells were incubated with HC-Lip+ThT and H-Lip+ThT for 2h, and the CLSM images were obtained. The liposomes were stained with DID dye.

[0039] Figure 3 ThT UV-Vis absorption spectrum. Under the addition of 0-5 μM of (A) 3H3Md-TET, (B) 3H-TET, (D) H3Md-BB, (E) H-BB, (G) 6H3M1, (H) 6H3M0, the UV-Vis absorption spectrum of ThT, and the red shift degree of ThT caused by the cavity hairpin and the complementary hairpin in the non-functional hairpin group of (C) TET, (F) BB and (I) AT.

[0040] Figure 4 ThT fluorescence kinetic scanning. (A) Single hairpin addition group and (B) TET, (C) BB and (D) AT non-functional complementary and cavity hairpin group alternately added group of ThT real-time fluorescence change monitoring.

[0041] Figure 5 For confocal imaging, (A) HC-Lip+ThT and (B) H-Lip+ThT were incubated with 3T3-L1 cells for 4h, 6h, 8h, 12h and 16h, and the CLSM images were obtained. The liposomes were stained with DID dye, and the nuclei were stained with Hoechst 33342.

[0042] Figure 6 For colocalization Mander's correlation coefficient analysis. (A) ThT and liposomes. (B) ThT and nuclei. DETAILED DESCRIPTION

[0043] The application discloses a method for developing a cavity hairpin ThT light nucleic acid switch into an intracellular carrier-specific tracing imaging platform. Those skilled in the art can refer to the content of the application, and appropriately improve the process parameters to realize the method. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are regarded as being included in the application. The method and application of the application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described in the application without departing from the content, spirit and scope of the application, to realize and apply the technical solution of the application.

[0044] Example 1 Construction of intracellular liposome tracing imaging platform of ThT-CHTLNAS

[0045] The ThT light switch (Cavity hairpin ThT-light nucleic acid switches, CHTLNAS) containing CGG-AAA cavity hairpin: H3Md-BB sequence and the corresponding complementary H-BB sequence without cavity structure were modified with a chol linker at the 3' end (see Table 1), and H3Md-BB-3'chol or H-BB-3'chol was coupled with the synthesized liposome (DMPC) to obtain H3Md-BB-liposome and H-BB-liposome. Figure 1 A) The coupling was carried out at 37°C for 6h, and the best ratio of the nucleic acid sequence used in the synthesis process to the phospholipid (N / P) was determined to be 1:500 by 2% agarose gel electrophoresis to obtain the clearest and brightest band. Figure 1 B) and centrifuged at 5000g for 5min to obtain 5mM HC Lip / H-Lip (DMPC molar mass). Then, ThT was added to the above system at 37°C in the dark for 30min, and the final concentration was 2 times that of the nucleic acid sequence to obtain 1mM HC-Lip+ThT / H-Lip+ThT (DMPC molar mass), which was stored at 4°C.

[0046] Table 1 Sequence design of intracellular liposome tracing imaging of ThT-CHTLNAS

[0047]

[0048] Example 2 Pretreatment before cell experiment and confocal observation

[0049] The density of the cells was 2×10 53T3-L1 cells were seeded on μ-Slide 8 Well (ibidi GmbH, Germany). After 24 hours, the medium was replaced with DMEM + 10% FBS. Then, 100 μL of HC-LiP+-ThT / h-LiP+-ThT (ThT final concentration 200 nM, stained with DiD for 15 min) was added and the cells were co-incubated for another 4–16 hours. The medium was then carefully removed, and the cells were washed 3–5 times with DPBS. The cells were then fixed with 4% paraformaldehyde at room temperature for 15 min, followed by blocking with blocking solution (DPBS containing 0.1% Triton X-100) and washing 3–5 times with DPBS. The samples were observed using a CLSM (LSM900, Carl Zeiss, Germany).

[0050] like Figure 2 As shown, ThT fluorescence was clearly observable in the cytoplasm of the HC-Lip+ThT transfection group (experimental group). However, the ThT fluorescence in the H-Lip+ThT group (control group), which underwent the same treatment, was significantly lower than that in the experimental group. This indicates that H3Md-BB can still effectively excite ThT fluorescence in cells compared to H-BB, further confirming the value of cavity exploration. In particular, liposome signals and ThT signals showed good co-localization in the cytoplasm, which was not observed in the control group, demonstrating their excellent ability to assist ThT in intracellular liposome tracking and imaging.

[0051] Further observation Figure 2 The study found that in the control group, the dispersion trend of ThT into the nucleus was more obvious, suggesting that the cavity hairpin may be able to strongly excite ThT fluorescence while providing a more stable load compared to the complementary hairpin.

[0052] Example 3: Verification of the combined load capacity of CHTLNAS for ThT

[0053] First, the absorption spectra of 2.5 μM ThT and 0–5 μM sequences (containing CHTLNAS and corresponding complementary hairpins, sequences shown in Table 2) in 10 mM Tris-HCl buffer (pH 7.4) were collected using a UV2800 UV-Vis spectrometer (UNICO, USA). The background absorption of the buffer solution was then subtracted to obtain the changes in UV absorption spectra. It was found that ThT exhibited a characteristic absorption spectrum at 412 nm without DNA incorporation, while TET (…) showed a different absorption spectrum after sequence incorporation. Figure 3 AC), BB ( Figure 3 DF) and AT non-functional hairpin sequence ( Figure 3The spectral changes of GI were similar—that is, the absorption bands of ThT all showed a significant red shift. However, the red shift caused by the complementary hairpin sequences in each group was significantly smaller than that of the corresponding cavity hairpins, suggesting that the cavity hairpins formed a DNA-ThT complex more stably with ThT, and that the interaction between ThT and the CGG-AAA cavity was stronger.

[0054] Table 2. CHTLNAS sequence design for verifying ThT binding ability.

[0055]

[0056]

[0057] Simultaneously, the competitive binding effect of hairpins with and without cavities on ThT was evaluated based on real-time fluorescence changes. 1 μM ThT was added to 10 mM Tris-HCl buffer (pH 7.4), and the solution was allowed to stand at room temperature for 5 min to stabilize the fluorescence. The fluorescence emission wavelength was set to 490 nm, the excitation wavelength to 445 nm, and the reading interval to 0.02 s. Then, fluorescence kinetics were measured. 0.1 μM of complementary hairpins and cavity hairpins of equal concentration were alternately added to the system every 1 min until ThT fluorescence no longer increased with the addition of any sequence, at which point the monitoring process was terminated.

[0058] The results are as follows Figure 4 As shown, the fluorescence intensity did not change significantly after adding ThT to the initial measurement system. Later, by alternately adding complementary hairpins and cavity hairpins, the excessive ThT concentration caused its fluorescence to continuously increase with the addition of the sequence. However, in the three experimental groups ( Figure 4 (BD) The addition of each cavity hairpin sequence promoted a more significant fluorescence enhancement than the addition of the complementary hairpin one minute earlier, demonstrating the superior excitation effect of the cavity on ThT. However, as the total amount of nucleic acid in the system continued to increase, the excess ThT was gradually consumed, and the fluorescence enhancement became increasingly limited with increasing nucleic acid concentration, until the addition of the complementary hairpin resulted in no further increase in fluorescence intensity, indicating the first plateau phase, suggesting that ThT was completely consumed. Subsequently, further addition of cavity hairpins resulted in a small jump in fluorescence, reaching a second plateau phase. After this, it was found that further addition of either cavities or complementary hairpins did not change the fluorescence. However, the control group, which received the same sequence addition throughout, did not show any differential increase in ThT intensity or the second plateau phase during the gradient fluorescence increase period. Figure 4 A). This indicates that after the addition of nucleic acid hairpins reaches the ThT concentration saturation point, the cavity hairpins exhibit a stronger competitive binding ability for ThT than the complementary hairpins, enabling them to steal ThT from the complementary hairpins and further excite their fluorescence, thus leading to an increase in the fluorescence gradient.

[0059] Example 4 CHTLNAS retards ThT nuclear entry

[0060] Finally, the study evaluated the intracellular loading stability of ThT in the presence of CHTLNAS at different time points, and added Hoechst 33342 at the end of the staining step to label the nucleus for 30 min at room temperature to better evaluate the nuclear entry of ThT. It is particularly pointed out that Figure 5 The excitation voltage of the confocal control group ThT channel of B was adjusted, and the purpose was to ensure that the results were compared with Figure 5 The ThT channel of the experimental group A was compared with the same baseline.

[0061] It can be seen that the liposome signal is retained in the cytoplasm throughout the process, indicating that HC-Lip / H-Lip does not allow entry into the nucleus. With the uptake of HC-Lip+ThT by the cells, the ThT signal shows a clear co-localization signal with the liposome within 8h, confirming that the cavity hairpin can stably load ThT. With further time, the co-localization signal in the cytoplasm gradually decreases, indicating that the loading capacity of HC-Lip for ThT is weakened Figure 5 A). This phenomenon may be related to the degradation of the hairpin sequence caused by the presence of various nucleases in the cell. In contrast, the control group never showed a clear co-localization of ThT-liposome signal. It can be seen that after 4h of transfection, the ThT signal has a tendency to disperse throughout the cell, indicating that the complementary hairpin-modified liposome without a cavity does not significantly promote the loading of ThT.

[0062] It is particularly noted that with the extension of the cell uptake time, ThT in the control group has gradually distributed throughout the cell and appeared strong fluorescent signal in the nuclear region, especially in the nuclear spot region. After only 8h of transfection, the overlap of ThT and nuclear signal in the nuclear region can be clearly observed, while for the experimental group, the appearance of the blue co-localization in the nuclear region is much later than the control group (close to 16h). This phenomenon of ThT gradually concentrating towards the nucleus over time can be attributed to the presence of a large number of nucleic acid sequences in the nuclear genome, which have the potential to form G4 and other special nucleic acid structures to stimulate ThT fluorescence.

[0063] The quantitative analysis of the co-localization evaluation coefficient, Mander's coefficient (M value), further confirms the above changes. For the co-localization of ThT and liposome, the M value of the experimental group shows a general downward trend, but can remain above 0.6 within 4-8h. After 12h, the M value rapidly decreases to 0.3248 (12h) and 0.2756 (16h), gradually approaching the change value of the control group Figure 6 A). As Figure 6As shown in Figure B, the M values of both ThT and nuclei in the two groups increased. Before 8 h, the experimental group increased slowly from 0.3109 (4 h) to 0.4200 (8 h), and then began to rise rapidly, finally reaching 0.8302 (16 h). However, the M value of the control group exceeded 0.50 only after 4 h, and rapidly reached more than 0.8 at 12 h. These results show that the cavity hairpin not only can light up ThT, but also can stably load ThT after entering the cell for a period of time, thereby effectively delaying the process of ThT entering the nucleus and being excited by nucleic acid in the nuclear region. It is proved that the cavity hairpin can help ThT as an intracellular tracer of delivery carrier (such as liposome), which may solve the problem that ThT can only be passively located in the nuclear region for imaging due to its own characteristics.

[0064] The reagents or instruments used in the technical method provided by the present application can be purchased from the market. The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An intracellular liposome nonenzymatic label-free fluorescent carrier tracer system characterized in that, The system consists of ThT molecules and nucleic acid sequences that can excite the fluorescence of the liposome conjugate, the nucleic acid sequence has a base pair mismatch cavity structure that can excite the potential of ThT fluorescence, the base pair mismatch cavity structure consists of CGG and AAA mismatches, wherein each base forms a mismatch cavity structure of A-C / A-G / A-G, the 3' end of the sequence is modified with a chol linker, and the nucleic acid sequence is shown in one of the following sequences: 6H3M1 5'-TTTTTTTTTTTCGGTTTTTTTTTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3', SEQ ID NO. 19 3H3Md-TET 5'-CGGTGGTGCGGTGGTGAAACACCAAAACACCAAAA-3', SEQ ID NO. 21 H3Md-BB 5'-ACATAACGGAATACGGATGTTAACATAAATATTAAATTATGT-3', SEQ ID NO.

23.

2. A method for in vitro cell tracking using the system of claim 1, characterized by, Comprising the following steps: (1) The nucleic acid sequence of any one of claim 1 is coupled with liposome at 25~37℃ for 4~8h, the proportion of phospholipid of liposome and the proportion of sequence modification are controlled as 100:1~1000:1, centrifuged at 5000g~8000g for 3~10min; (2) ThT is added to the above system in a dark environment at 25~37℃, incubated for 30~120min; centrifuged at 5000g~8000g for 3~10min; (3) The above obtained liposome tracing system is co-incubated with cells, and then cultured for 2~16h, fixed with 4% polyformaldehyde, and observed under a fluorescence microscope.

3. The tracing system of claim 1 or the method of claim 2 is applied in in vitro intracellular tracing imaging.

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