A live-cell in situ imaging method for CRISPR / Cas12a system activated by adjacent dual recognition strand substitution probes.

Abstract

This invention discloses a method for in situ imaging of glycosylated RNA in live cells based on a CRISPR / Cas12a system activated by a neighboring dual-recognition strand substitution probe. The method includes: embedding a cholesterol-modified tetrahedral DNA reporter molecule into the live cell membrane; adding a neighboring dual-recognition strand substitution probe, which is formed by partial complementary pairing of a first DNA strand (containing glycosyl recognition elements, spacer sequences, and a Cas12a / crRNA trigger sequence) and a second DNA strand; and adding a Cas12a / crRNA complex. Only when the probe simultaneously recognizes and binds to the glycosyl and RNA sequences of the same glycosylated RNA molecule will a strand substitution reaction be triggered, releasing the trigger sequence and activating the trans-cleavage activity of Cas12a. This cleaves the reporter molecule, generating a fluorescent signal, thus achieving in situ imaging. The entire process is performed under physiological conditions. This invention eliminates the need for nucleic acid amplification, is simple to operate, has high specificity, significantly reduces background signal, and is suitable for rapid, high-resolution visualization of glycosylated RNA in live cells, providing an effective tool for tumor cell classification and progression assessment.

Description

Technical Field

[0001] This invention relates to the field of glycosylated RNA technology, specifically to a live-cell glycosylated RNA in situ imaging method based on a CRISPR / Cas12a system activated by a neighboring dual recognition strand substitution probe. Background Technology

[0002] Glycosylation is a ubiquitous and important post-translational modification process, widely involved in various biological processes such as cell recognition, signal transduction, immune regulation, and tumorigenesis. For a long time, glycosylation modification was thought to primarily occur on proteins and lipids. However, a recent groundbreaking study discovered that non-coding RNA can also undergo glycosylation modification, forming a novel class of biomolecules called "glycosylated RNA (glycoRNA)." Research shows that glycosylated RNA is widely present on the surface of various mammalian cells and on extracellular vesicles, playing a crucial regulatory role in key physiological and pathological processes such as immune recognition, cell migration, inflammatory responses, and angiogenesis. Therefore, developing efficient detection and imaging technologies for glycosylated RNA is of great significance for further elucidating its biological functions and clinical application potential.

[0003] Currently, methods for detecting glycosylated RNA mainly include metabolic labeling combined with click chemistry, proximity oxidation labeling (such as rPAL), and sequencing-based analysis techniques. For example, metabolic labeling utilizes azide-modified non-natural sugars, integrating them into nascent glycans through the cell's own sugar metabolism pathways. Subsequently, click chemistry is used to link reporter groups such as biotin, enabling the extraction and in vitro analysis of glycosylated RNA. The rPAL method utilizes periodate to selectively oxidize the vicinal diol of sialic acid to generate an aldehyde group, which is then linked to aminooxybiotin, achieving direct chemical labeling without metabolic labeling. However, most of these methods require cell lysis or RNA extraction, belonging to in vitro population analysis. They not only cannot achieve in situ detection in a living cell environment but also struggle to reflect the spatial distribution, single-cell level differences, and dynamic changes of glycosylated RNA under real physiological conditions, severely limiting in-depth research on its biological functions.

[0004] To overcome these limitations, researchers have developed several in situ imaging methods for intracellular glycosylated RNA. For example, the ARPLA technique, based on ligation reactions, utilizes sialic acid aptamers and RNA in situ hybridization probes to recognize the glycosylation and RNA portions of glycosylated RNA, respectively. A proximity effect triggers a ligation reaction to form a circular DNA template, followed by rolling circle amplification (RCA) and fluorescent probe hybridization, enabling imaging of glycosylated RNA fixed on the cell surface. The HieCo2 technique introduces sialic acid-encoding probes through metabolic labeling and click chemistry, which, combined with RNA recognition probes, exposes primers for the hybridization chain reaction (HCR) after multi-stage decoding. In situ imaging of specific RNA sialylation on the living cell membrane is achieved through HCR signal amplification. Despite these significant advancements in imaging, they still suffer from considerable technical limitations: First, all of them rely on nucleic acid amplification processes (such as RCA or HCR) to amplify the signal. This not only requires complex primer design and multi-step cascade reactions, resulting in complicated experimental steps, high operational difficulty, and long time consumption (usually more than 2.5 to 4 hours), but also introduces the risk of non-specific background signals caused by amplification. Secondly, the application scenarios are limited. ARPLA requires cell fixation and permeabilization, making it unsuitable for live-cell dynamic studies. While HieCo2 can be used in live cells, it still relies on metabolic labeling, resulting in complex procedures and labeling efficiency easily affected by cell state. In summary, existing technologies generally lack a glycosylated RNA in situ imaging method that combines high signal-to-noise ratio, high specificity, ease of operation, no need for nucleic acid amplification, and is truly applicable to the live-cell environment.

[0005] In recent years, the CRISPR / Cas system has demonstrated great potential in gene editing and biosensing due to its unique targeting and collateral cleavage activities. In particular, the complex formed by the Cas12a protein after binding to specific crRNAs, once activated by its targeted single-stranded or double-stranded DNA trigger sequence, exhibits strong, non-specific trans-cleavage activity; this "collateral cleavage" effect can serve as an effective signal amplification mechanism. This characteristic provides a novel approach for developing biosensing methods that achieve high-sensitivity detection without traditional nucleic acid amplification. Meanwhile, nucleic acid aptamers targeting specific small molecules or glycosyl structures (such as sialic acid aptamers) possess advantages such as high affinity, ease of chemical synthesis and modification, and can serve as ideal molecular recognition elements. Summary of the Invention

[0006] The technical problem this invention aims to solve is to overcome the above-mentioned technical difficulties and provide a live-cell glycosylated RNA in situ imaging method based on a proximity dual-recognition strand substitution probe-activated CRISPR / Cas12a system. This method designs dual-recognition probes that target both the glycosyl group and the RNA of the glycosylated RNA. Only when both recognition elements bind to the same glycosylated RNA molecule does the spatial proximity effect lead to the release of the Cas12a trigger sequence, activating the trans-cleavage activity of Cas12a, cleaving the pre-embedded fluorescent reporter molecule in the cell membrane, and generating a detectable fluorescent signal. This mechanism not only effectively suppresses false-positive signals caused by a single glycosyl group or RNA through proximity dual recognition, but also utilizes the signal amplification function of Cas12a to achieve highly specific and sensitive in situ imaging of live-cell glycosylated RNA under conditions without nucleic acid amplification. This provides a powerful technical platform for studying the biological functions of glycosylated RNA and its role in tumorigenesis and immune regulation.

[0007] To solve the above-mentioned technical problems, the technical solution provided by the present invention is: a method for in situ imaging of live-cell glycosylated RNA based on a neighboring dual-recognition strand substitution probe to activate a CRISPR / Cas12a system, comprising the following steps: (1) Inserting cholesterol-modified tetrahedral DNA reporter molecules into living cell membranes; (2) Add a neighboring double recognition strand replacement probe to a living cell, wherein the neighboring double recognition strand replacement probe binds to glycosylated RNA on the cell membrane; (3) Add the complex formed by Cas12a protein and crRNA to activate the trans-cleavage activity of Cas12a, cut the tetrahedral DNA reporter molecule to generate a fluorescent signal, and realize in situ imaging of glycosylated RNA in living cells. The adjacent dual recognition strand replacement probe comprises a partially complementary first DNA strand and a second DNA strand; the first DNA strand contains a glycosyl recognition element, a spacer sequence, and a trigger sequence for Cas12a / crRNA; the second DNA strand contains an RNA recognition sequence complementary to the target glycosylated RNA sequence. When the adjacent dual recognition strand displacement probes simultaneously recognize and bind to the glycosylation portion and RNA sequence of the same glycosylated RNA molecule, the second DNA strand hybridizes with the target glycosylated RNA to trigger a strand displacement reaction, releasing the trigger sequence in the first DNA strand. The trigger sequence is recognized by the Cas12a / crRNA complex and activates the trans-cleavage activity of Cas12a.

[0008] As an improvement, the trigger sequence of the Cas12a / crRNA in the first DNA strand contains a mismatched base, which is used to prevent non-specific activation of the Cas12a / crRNA complex.

[0009] As an improvement, there are two base mismatches in the hybridization portion of the T1 and T2 chains.

[0010] As an improvement, the glycosyl recognition element is an aptamer that specifically recognizes the glycosyl portion of glycosylated RNA; the RNA recognition sequence is a nucleic acid sequence that specifically recognizes a specific RNA sequence on glycosylated RNA.

[0011] As an improvement, steps (1) to (3) are all performed under physiological conditions, and the total incubation time does not exceed 1 hour.

[0012] As an improvement, the glycosyl recognition element is a sialic acid aptamer; the trigger sequence of the Cas12a / crRNA contains four mismatched bases.

[0013] As an improvement, the second DNA strand has a single-stranded region for recognizing the target glycosylated RNA and triggering a strand displacement reaction.

[0014] As an improvement, the glycosylated RNA is glycosylated U1 RNA or glycosylated U3 RNA.

[0015] A proximity double-strand recognition probe for use in the method of any one of claims 1 to 7; the probe is formed by a first DNA strand and a second DNA strand through complementary base pairing to form a partially double-stranded structure. The first DNA strand contains, from the 5' end to the 3' end, seven T, Cas12a / crRNA trigger sequences, spacer sequences, and glycosyl recognition elements. The second DNA strand contains an RNA recognition sequence complementary to the target glycosylated RNA sequence, and the second DNA strand partially complements the first DNA strand, such that the trigger sequence in the first DNA strand is blocked when it does not bind to the target glycosylated RNA.

[0016] It also includes the application of the live-cell glycosylated RNA in situ imaging method in tumor cell classification or tumor progression assessment. By detecting the difference in expression levels of glycosylated U1 RNA and / or glycosylated U3 RNA on the surface of tumor cells, combined with principal component analysis, it is possible to distinguish between different tumor cell lines or tumor cells of different malignant degrees.

[0017] The advantages of this invention compared to existing technologies are as follows: This invention creatively utilizes the trans-cleavage activity activated by the CRISPR / Cas12a complex as a signal amplification mechanism. As described in claim 1, this method replaces traditional rolling circle amplification or hybridization chain reaction by "activating the trans-cleavage activity of Cas12a to cleave the tetrahedral DNA reporter molecule and generate a fluorescent signal." This mechanism eliminates the need for complex amplification primers, circular templates, or multi-level hairpin probes; detection can be completed with only a carefully designed double-stranded probe and the Cas12a / crRNA complex. This not only significantly simplifies the experimental procedure and reduces operational difficulty but also fundamentally avoids the accumulation of non-specific products and background signal amplification that may occur during nucleic acid amplification, significantly improving the reliability of detection. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the principle of the imaging method of the present invention.

[0019] Figure 2 This is a diagram showing the in vitro and cellular validation results of the probe's feasibility using the method of this invention.

[0020] Figure 3 This is a diagram showing the verification results of the feasibility and specificity of the method of the present invention for cell imaging.

[0021] Figure 4 This is an imaging result of glycosylated U1 and U3 in cells at different stages of breast cancer progression using the method of this invention. Figure 5 This is an in situ imaging and classification result of glycosylated U1 and U3 in six different tumor cell lines using the method of this invention. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] Example 1: Design of a Neighboring Dual Recognition Chain Displacement Probe like Figure 1 As shown, this invention discloses a live-cell glycosylated RNA in situ imaging method based on a neighboring double-stranded DNA probe activating the CRISPR / Cas12a system. The core of this method lies in the design of a double-stranded DNA probe comprising: T1 chain: contains glycosyl recognition elements (such as sialic acid aptamers), spacer sequences, and trigger sequences for Cas12a / crRNA (which contain 4 mismatched bases). T2 chain: Contains an RNA recognition sequence complementary to the target glycosylated RNA sequence.

[0024] T1 and T2 form a partially double-stranded structure through complementary base pairing, with T2 containing a single-stranded region for recognizing the target RNA. When the probe simultaneously recognizes and binds to both the glycosylation region and the RNA sequence of the same glycosylated RNA molecule, T2 completely hybridizes with the target RNA, triggering a strand displacement reaction and releasing the trigger sequence from the T1 strand. This trigger sequence is recognized by the Cas12a / crRNA complex, activating the trans-cleavage activity of Cas12a. Simultaneously, a cholesterol-modified tetrahedral DNA reporter molecule pre-embedded in the cell membrane is cleaved by the activated Cas12a, generating a fluorescent signal. The entire imaging process is completed sequentially under physiological conditions: first, the reporter molecule is embedded; then, it binds to the probe; finally, the Cas12a / crRNA complex is added. The total incubation time does not exceed one hour, achieving amplification-free, highly specific, and high signal-to-noise ratio in situ imaging of live-cell glycosylated RNA.

[0025] Example 2: Cholesterol-modified tetrahedral DNA reporter molecule This example illustrates reporter molecules used for cell membrane anchoring. For example... Figure 1 As shown, the reporter molecule is a cholesterol-modified tetrahedral DNA reporter molecule. This reporter molecule is pre-embedded in the cell membrane and is cleaved by activated Cas12a, generating a fluorescent signal.

[0026] Example 3: In situ imaging method for live-cell glycosylated RNA This embodiment uses human U1 glycosylated RNA in HeLa cells as the detection target to illustrate the complete imaging operation steps of the present invention. The entire imaging process is completed sequentially under physiological conditions, with a total incubation time of no more than 1 hour.

[0027] Step 1: Reporter molecules embed into the cell membrane Cholesterol-modified tetrahedral DNA reporter molecules were embedded into the membranes of living cells. Specifically, the reporter molecules prepared in Example 2 were added to HeLa cells and incubated at 37°C for 15 minutes to anchor the reporter molecules to the cell membrane surface. After incubation, the cells were washed with phosphate-buffered saline.

[0028] Step 2: Adjacent dual recognition chain replacement probe binds to target Add the adjacent dual recognition strand substitution probe designed in Example 1 to the live cells treated in Step 1. Incubate the cells at 37°C for 15 minutes after adding the probe. Figure 1As shown in the "Probe Recognition and Binding" and "Strand Displacement Triggering" sections, when the probe simultaneously recognizes and binds to both the glycosylation region and the RNA sequence of the same glycosylated RNA molecule, T2 completely hybridizes with the target RNA, triggering a strand displacement reaction and releasing the trigger sequence from the T1 strand. After incubation, the cells are washed with phosphate-buffered saline.

[0029] Step 3: Activation and fluorescence imaging of the Cas12a / crRNA complex Add the complex formed by Cas12a protein and crRNA to the cells treated in step two, and incubate at 37°C for 30 minutes. Figure 1 As shown in the "Cas12a Activation and Signal Output" section, the released trigger sequence is recognized by the Cas12a / crRNA complex, activating the trans-cleavage activity of Cas12a. The activated Cas12a cleaves cholesterol-modified tetrahedral DNA reporter molecules pre-embedded in the cell membrane, generating a fluorescent signal. After incubation, live cells are observed and imaged using a laser scanning confocal microscope, achieving in-situ imaging of glycosylated RNA in live cells.

[0030] Step 4: Feasibility verification of probe design To verify the feasibility of the adjacent dual recognition probe design in the method of this invention, it was first validated in vitro and at the cellular level, and the results are as follows: Figure 2 As shown.

[0031] (1) In vitro fluorescence verification: After co-incubating the adjacent dual recognition strand replacement probe with the target RNA, the Cas12a / crRNA complex and ssDNA reporter molecule were added. The results are as follows: Figure 2 As shown in a-2b, a significant enhancement of fluorescence signal was observed in the presence of the target U1 RNA, while the background of the control group without the target was extremely low, demonstrating that the probe can effectively activate the trans-cleavage activity of Cas12a in vitro through RNA recognition.

[0032] (2) Probe integrity control: The above operations were performed using a T2 probe with a random Cas12a / crRNA trigger sequence, a random aptamer sequence, or a single-stranded probe lacking RNA binding. Results are as follows: Figure 2 As shown in c-2d, the fluorescence signals of these control groups almost disappeared, and only the probe group with the complete structure (PDAC) showed obvious fluorescence signals, indicating that the signal is strictly dependent on the integrity of the probe structure.

[0033] Step 5: Setting up a control experiment for specificity verification To verify the specificity of the method of the present invention, the following control group was set up, and the results are as follows: Figure 3 As shown.

[0034] (1) RNA-dependent control: Cells were treated with RNase I and RNase A before probe treatment. The results are as follows: Figure 3 As shown in a-3b, the fluorescence signal decreased by approximately 88% and 93%, respectively, demonstrating that the signal depends on the presence of the RNA portion.

[0035] (2) RNA sequence specificity verification: A competition experiment was conducted using blockers targeting different glycosylated RNAs. The results are as follows: Figure 3 As shown in c-3f, the fluorescence signal was completely eliminated only when an inhibitor complementary to the target U1 RNA sequence was added, while inhibitors targeting U3 or U8 glycosylated RNA had no significant effect, confirming the specificity of RNA sequence recognition.

[0036] (3) Glycosylation-dependent control: Before probe treatment, cells were treated with PNGase F, α2-3,6,8,9-neuraminidase, or O-glycosidase, respectively. Results are as follows: Figure 3 As shown in g-3h, the fluorescence signal decreased by 82% and 84% after treatment with PNGaseF and neuraminidase, respectively, while the effect of O-glycosidase treatment was minimal, indicating that the signal mainly originated from N-glycosylated RNA.

[0037] Example 4: Application of the method of the present invention in tumor cell classification and malignancy assessment This embodiment demonstrates the application of the method of the present invention in detecting differences in glycosylated RNA expression in different tumor cells, and in performing cell classification and disease progression assessment.

[0038] (1) Analysis of breast cancer progression models Cell models representing different stages of breast cancer progression were selected: non-tumorigenic breast epithelial cells MCF-10A, malignant breast cancer cells MCF-7, and highly metastatic breast cancer cells MDA-MB-231. Following the steps described in Example 3, in situ imaging of these three cell types was performed using probes targeting glycosylated U1 and glycosylated U3.

[0039] The results are as follows Figure 4 As shown in c-4d, the fluorescence signal intensity of glycosylated U1 and U3 decreased sequentially in MCF-10A, MCF-7, and MDA-MB-231 cells, indicating a negative correlation between glycosylated RNA expression levels and tumor malignancy. This result demonstrates that the method of this invention can be used to distinguish tumor cells at different stages of progression.

[0040] (2) Classification of various tumor cell lines Six different tumor cell lines were selected: A375, PC-3, MCF-7, HeLa, MDA-MB-231, and A549. In situ imaging of these cells was performed using probes targeting glycosylated U1 and glycosylated U3.

[0041] The results are as follows Figure 5 As shown in a-5b, obvious glycosylated U1 and U3 fluorescence signals were observed in all six cell lines. Figure 5 As shown in Figure c, there are significant differences in fluorescence intensity among the cell lines. Figure 5 As shown in d, principal component analysis based on glycosylated U1 and U3 signals can clearly distinguish six cell lines, demonstrating the potential of this method in tumor cell classification.

[0042] Sequence List The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A live-cell glycosylated RNA in situ imaging method based on a neighboring dual-recognition strand substitution probe activating a CRISPR / Cas12a system, characterized in that: Includes the following steps: (1) Inserting cholesterol-modified tetrahedral DNA reporter molecules into living cell membranes; (2) Add a neighboring double recognition strand replacement probe to a living cell, wherein the neighboring double recognition strand replacement probe binds to glycosylated RNA on the cell membrane; (3) Add the complex formed by Cas12a protein and crRNA to activate the trans-cleavage activity of Cas12a, cut the tetrahedral DNA reporter molecule to generate a fluorescent signal, and realize in situ imaging of glycosylated RNA in living cells. The adjacent dual recognition strand replacement probe comprises a partially complementary first DNA strand and a second DNA strand; the first DNA strand contains a glycosyl recognition element, a spacer sequence, and a trigger sequence for Cas12a / crRNA; the second DNA strand contains an RNA recognition sequence complementary to the target glycosylated RNA sequence. When the adjacent dual recognition strand displacement probes simultaneously recognize and bind to the glycosylation portion and RNA sequence of the same glycosylated RNA molecule, the second DNA strand hybridizes with the target glycosylated RNA to trigger a strand displacement reaction, releasing the trigger sequence in the first DNA strand. The trigger sequence is recognized by the Cas12a / crRNA complex and activates the trans-cleavage activity of Cas12a.

2. The method according to claim 1, characterized in that, The trigger sequence of the Cas12a / crRNA in the first DNA strand contains mismatched bases, which are used to prevent non-specific activation of the Cas12a / crRNA complex.

3. The method according to claim 1, characterized in that, The glycosyl recognition element is an aptamer that specifically recognizes the glycosyl portion of glycosylated RNA; the RNA recognition sequence is a nucleic acid sequence that specifically recognizes a specific RNA sequence on glycosylated RNA.

4. The method according to claim 1, characterized in that, All steps (1) to (3) are performed under physiological conditions, and the total incubation time does not exceed 1 hour.

5. The method according to claim 1, characterized in that, The glycosyl recognition element is a sialic acid aptamer; the trigger sequence of the Cas12a / crRNA contains 4 mismatched bases, and there are two base mismatches in the hybridization part of the T1 and T2 chains.

6. The method according to claim 1, characterized in that, The second DNA strand contains a single-stranded region that is used to recognize the target glycosylated RNA and trigger a strand displacement reaction.

7. The method according to claim 1, characterized in that, The glycosylated RNA is either glycosylated U1 RNA or glycosylated U3 RNA.

8. A neighboring dual recognition chain displacement probe, characterized in that, Used in the method according to any one of claims 1 to 7; the probe is a partially double-stranded structure formed by complementary base pairing of a first DNA strand and a second DNA strand; The first DNA strand contains, from the 5' end to the 3' end, seven T, Cas12a / crRNA trigger sequences, spacer sequences, and glycosyl recognition elements. The second DNA strand contains an RNA recognition sequence complementary to the target glycosylated RNA sequence, and the second DNA strand partially complements the first DNA strand, such that the trigger sequence in the first DNA strand is blocked when it does not bind to the target glycosylated RNA.

9. The application of the live-cell glycosylated RNA in situ imaging method according to any one of claims 1 to 7 in tumor cell classification or tumor progression assessment.

10. The application according to claim 9, characterized in that, By detecting the differences in expression levels of glycosylated U1 RNA and / or glycosylated U3 RNA on the surface of tumor cells, and combining this with principal component analysis, it is possible to distinguish between different tumor cell lines or tumor cells of different malignant degrees.

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