An in situ detection method for ribosome-bound mRNA

Abstract

This invention discloses an in situ detection method for ribosome-bound mRNA, employing at least one probe set designed based on the target sequence on the ribosome-bound mRNA to be detected. Each probe set includes a target sequence recognition probe pair and a circular clip probe. This invention highly integrates nucleic acid probe technology, rolling circle amplification technology, and in situ detection technology, enabling quantitative and localized detection of mRNA expression in cells and tissues. It also allows for multiplex in situ detection by inserting gene-specific oligonucleotide tags and performing tag decoding via in situ sequencing of the amplified products. This method preserves the correlation between gene expression information and tissue morphology information without damaging cell structure. This provides crucial information for constructing gene expression regulatory networks, compensating for the lack of location information and data related to heterogeneous gene expression in traditional translatome techniques.

Description

Technical Field

[0001] This invention belongs to the field of spatial transcriptomics technology, specifically relating to an in situ detection method for ribosome-bound mRNA. Background Technology

[0002] A novel translatome technique based on adjacent-linked assays is an innovative detection method. This technique utilizes a special clip probe, the upper half of which contains a recognition sequence specifically complementary to the rRNA sequence, while the lower half contains a template sequence for probe circularization. During detection, the detection probe bound to the translating mRNA specifically hybridizes with the clip probe through base complementarity. Subsequently, the gap is closed using splintR ligase to form a circular structure. Detection probes not complementary to the target sequence are washed away. Finally, by detecting the detection probes bound to the target nucleic acid sequence, in-situ detection of the target nucleic acid—including qualitative, local, and quantitative analysis—is achieved. In-situ detection relies on the detection of the labeled signal of the detection probe. Currently, detection probe labeling mainly falls into two categories: fluorescent labeling and enzyme labeling. Fluorescent labeling typically involves covalently cross-linking fluorescent chemical molecules to the detection probe, thereby detecting the fluorescent signal. Enzyme labeling, on the other hand, involves covalently cross-linking catalytically active enzyme molecules to the detection probe, enabling detection through enzyme catalysis of the substrate to produce a colored product. For fluorescently labeled detection probes, the simultaneous labeling and detection of different target nucleic acid sequences can be achieved by utilizing fluorescence signals with different spectral characteristics. For enzyme-labeled detection probes, different enzymes catalyze the substrate to produce different colors, thereby enabling multiplex detection.

[0003] Novel translatomics technologies based on adjacent linkages can locate and quantify mRNAs undergoing translation in cell and tissue samples. Currently, the detection of mRNAs undergoing translation mainly relies on techniques such as polysome profiling, translation-associated protein capture (TRAP), ribosome affinity chromatography, and RIBOmap. Polysome profiling, TRAP, and RIBOmap have studied the translational status and regulatory mechanisms of mRNA from different perspectives. However, these techniques require cell disruption and purification of RNA molecules before use, leading to the loss of spatial location information of RNA molecules and making it impossible to link gene expression information with tissue morphology. Morphological information of gene expression is crucial for constructing gene expression regulatory networks; a high degree of consistency in the spatial distribution of different genes may indicate close connections between them. RIBOmap technology is a method that enables in situ detection of mRNA being translated. It relies on ribosomal RNA binding probes to circularize lock probes that specifically recognize mRNA, and then amplifies the detection signal by rolling circle amplification using primers that are adjacent to and partially complementary to the lock probes on the target mRNA. However, the design of its probe set is relatively complex, and there is still room for further improvement in efficiency. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an in situ detection method for ribosome-bound mRNA.

[0005] The technical solution of the present invention is as follows:

[0006] An in situ detection method for ribosome-bound mRNA, employing at least one probe set designed based on a target sequence on the target ribosome-bound mRNA to be detected, wherein each probe set includes:

[0007] A target sequence recognition probe pair consists of a first recognition probe and a second recognition probe. The first recognition probe, from 5' to 3', has a first target sequence recognition sequence, a first linker sequence, and a first circularization sequence, respectively. The second recognition probe, from 3' to 5', has a second target sequence recognition sequence, a second linker sequence, and a second circularization sequence, respectively.

[0008] A circularization clip probe has an rRNA sequence recognition sequence and a circularization template sequence connected in sequence. The circularization template sequence is complementary to the first circularization sequence and the second circularization sequence mentioned above, and serves as the circularization template for the first circularization sequence and the second circularization sequence.

[0009] Specifically, the steps include the following:

[0010] (1) At least one probe group is added to the sample to be tested for reaction, wherein:

[0011] Its target sequence recognition probes pair complementaryly with the target RNA via the first target sequence recognition sequence of the first recognition probe and the second target sequence recognition sequence of the second recognition probe, bringing the first and second target sequence recognition sequences close to each other.

[0012] Its circularization clip probes recognize hybridized ribosomal RNA through rRNA sequence recognition, and through complementary pairing of the circularization template sequence with both the first circularization sequence of the first recognition probe and the second circularization sequence of the second recognition probe, the first and second circularization sequences are brought close together.

[0013] Next, wash away the probes that do not have complementary pairing;

[0014] (2) Add ligase to react and link all the first target sequence recognition sequences and second target sequence recognition sequences that are close to each other in step (1), and link all the first circular sequences and second circular sequences that are close to each other, thereby forming at least one circular template.

[0015] (3) Perform rolling circle amplification on at least one circular template obtained in step (2) to obtain at least one rolling circle amplification product;

[0016] (4) Hybridize the detection probe corresponding to the target mRNA with at least one rolling circle amplification obtained in step (3) to perform in situ detection and obtain the corresponding detection signal.

[0017] In a preferred embodiment of the present invention, the sample to be tested is selected from cultured cells, cells in tissues, and cells in tissue sections.

[0018] In a preferred embodiment of the invention, the detection probe is modified with a reporter group.

[0019] More preferably, the reporter group is selected from fluorescent groups, radioactive isotopes, and enzyme molecules.

[0020] In a preferred embodiment of the present invention, at least one of the first recognition probe and the second recognition probe in the target sequence recognition probe pair has a gene-specific oligonucleotide tag.

[0021] In a preferred embodiment of the present invention, the ligase is splint R ligase.

[0022] The beneficial effects of this invention are:

[0023] 1. This invention highly integrates nucleic acid probe technology, rolling circle amplification technology, and in situ detection technology, enabling quantitative and localized detection of mRNA expression in cells and tissues. It can also achieve multiplex in situ detection by inserting gene-specific oligonucleotide tags and performing tag decoding through in situ sequencing of amplified products.

[0024] 2. Compared with traditional translatome techniques, this invention does not damage cell structure, thus preserving the correlation between gene expression information and tissue morphology information. This provides extremely important information for constructing gene expression regulatory networks, compensating for the lack of location information and data related to heterogeneous gene expression in traditional translatome techniques.

[0025] 3. Compared with RIBOmap technology, this invention not only simplifies the design of probe sets, but also improves detection efficiency. Attached Figure Description

[0026] Figure 1 The diagrams are schematic diagrams of Embodiments 1 and 2 of the present invention.

[0027] Figure 2 The figure shows the results of the feasibility verification experiment of Embodiment 1 of the present invention.

[0028] Figure 3 This is a diagram showing the results of a specificity verification experiment in Example 2 of the present invention.

[0029] Figure 4 The figure shows the experimental results of Embodiment 3 of the present invention. Detailed Implementation

[0030] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0031] Example 1: Comparative experiment on the technical feasibility of using MCF-7 cells on a smear (the principle is as follows). Figure 1 (As shown)

[0032] (I) Cell Culture and Fixation: Human breast adenocarcinoma cells MCF-7 were cultured in DMEM medium (containing 10% FBS) for 24-48 hours, then treated with trypsin to prepare a cell suspension, and seeded onto sterile glass slides coated with poly-L-lysine. The cells were cultured for another 12-24 hours. Subsequently, the cells were washed with DEPC-PBS and fixed with 4% PFA (prepared with DEPC-PBS) at room temperature for 15 minutes. After fixation, the cells were washed twice more with DEPC-PBS, and then dehydrated sequentially with a gradient of ethanol (70%, 85%, 100%) for 5 minutes each time. Finally, the glass slides were air-dried and stored at -80°C.

[0033] (II) Pretreatment of cell samples: The cell membranes of the cells were permeabilized and perforated. The samples were then placed in methanol solution and incubated at -20°C for 1 hour. Afterward, the samples were thawed in air and dried, and then washed three times with DEPC-PBS containing 0.1% (v / v) Tween 20.

[0034] (III) In-situ nucleic acid testing: The specific steps are as follows:

[0035] (1) Hybridization of probe with target gene: Add a hybridization reaction mixture containing 10% formamide, 4×SSC, 0.04 μM double-linked probe, and 0.1 μM clip probe to the sample. Incubate at 37°C for 10–12 h to allow the double-linked probe to specifically hybridize in situ with the mRNA molecule being translated. After the reaction, wash three times with DEPC-PBS-Tween 20. The ligation probe sequences are shown in Table 1 below (sequences are numbered from top to bottom as SEQ ID NO.01 to SEQ ID NO.29):

[0036] Table 1

[0037]

[0038]

[0039] (2) Ligation of the dual-linked probe pairs: After washing three times with DEPC-PBS-Tween 20, add 50 μL of ligation reaction mixture containing 0.2 μg / μL BSA, 1×SplintR buffer (NEB), 0.1 U / μL SplintR ligase (NEB), and 1 U / μL RiboLock RNase inhibitor (Thermo) to the sample. Incubate at 37°C for 30 min.

[0040] (3) Rolling circle amplification: After washing three times with DEPC-PBS-Tween 20, add 50 μL of rolling circle amplification reaction solution containing 5% glycerol, 0.2 μg / μL BSA, 1×Equiphi29 polymerase buffer (Thermo), 1 mM DTT, 1 mM dNTPs, and 1 U / μL Equiphi29 DNA polymerase (Thermo) to the sample. Incubate at 37°C for 10-12 h.

[0041] (IV) Detection Probe Hybridization: Add a mixture containing 4×SSC, 40% formamide, and 0.1 μM detection probe to the sample. Incubate at 37°C for 30 min to hybridize the rolling circle amplification product with the detection probe (the detection probes for ACTB and MALAT1 are 5'-tcatacactaaagataaaca-3' (SEQ ID NO. 30), with a fluorescent label at the 5' end). After the reaction, wash three times with DEPC-PBS-Tween 20. After the detection probe hybridization reaction, air-dry the cell samples. Finally, stain the cell nuclei with 0.5 μg / mL DAPI. The mounted samples can be examined and photographed under a fluorescence microscope. The detection results are as follows: Figure 2 As shown.

[0042] Example 2: Technically specific comparative experiment on MCF-7 cell smears (principle as follows) Figure 1 (As shown)

[0043] (I) Cell Culture and Fixation: Human breast adenocarcinoma cells MCF-7 were cultured in DMEM medium (containing 10% FBS) for 24-48 hours, then treated with trypsin to prepare a cell suspension, and seeded onto sterile glass slides coated with poly-L-lysine. The cells were cultured for another 12-24 hours. Subsequently, the cells were washed with DEPC-PBS and fixed with 4% PFA (prepared with DEPC-PBS) at room temperature for 15 minutes. After fixation, the cells were washed twice more with DEPC-PBS, and then dehydrated sequentially with a gradient of ethanol (70%, 85%, 100%) for 5 minutes each time. Finally, the glass slides were air-dried and stored at -80°C.

[0044] (II) Pretreatment of cell samples: The cell membranes of the cells were permeabilized and perforated. The samples were then placed in methanol solution and incubated at -20°C for 1 hour. Afterward, the samples were thawed in air and dried, and then washed three times with DEPC-PBS containing 0.1% (v / v) Tween 20.

[0045] (III) In-situ nucleic acid testing: The specific steps are as follows:

[0046] (1) Hybridization of probes with target genes: A hybridization reaction mixture containing 10% formamide, 4×SSC, 0.04 μM double-linked probes, and 0.1 μM clip probes was added to one group of samples. Hybridization reaction mixtures lacking clip probes complementary to the ribosomal sequence, DLP probes lacking the left end probe, and DLP probes lacking the right end probe, respectively, were added to the other three groups. The mixtures were incubated at 37°C for 10–12 h to allow the double-linked probes to specifically hybridize in situ with the translating mRNA molecules. After the reaction, the samples were washed three times with DEPC-PBS-Tween 20. The ligation probe sequences are shown in Table 2 below.

[0047] Table 2

[0048]

[0049] (2) Ligation of the dual-linked probe pairs: After washing three times with DEPC-PBS-Tween 20, add 50 μL of ligation reaction mixture containing 0.2 μg / μL BSA, 1×SplintR buffer (NEB), 0.1 U / μL SplintR ligase (NEB), and 1 U / μL RiboLock RNase inhibitor (Thermo) to the sample. Incubate at 37°C for 30 min.

[0050] (3) Rolling circle amplification: After washing three times with DEPC-PBS-Tween 20, add 50 μL of rolling circle amplification reaction solution containing 5% glycerol, 0.2 μg / μL BSA, 1×Equiphi29 polymerase buffer (Thermo), 1 mM DTT, 1 mM dNTPs, and 1 U / μL Equiphi29 DNA polymerase (Thermo) to the sample. Incubate at 37°C for 10-12 h.

[0051] (IV) Hybridization of Detection Probes: A mixture containing 4×SSC, 40% formamide, and 0.1 μM detection probe was added to the sample. The mixture was incubated at 37°C for 30 min to hybridize the rolling circle amplification product with the detection probe (5'-tcatacactaaagataaaca-3' (SEQ ID NO. 30), with a fluorescent label at the 5' end). After the reaction, the sample was washed three times with DEPC-PBS-Tween 20. After the detection probe hybridization reaction, the cell samples were air-dried. Finally, 0.5 μg / mL DAPI was added to stain the cell nuclei. The mounted samples could be examined and photographed under a fluorescence microscope. The detection results are as follows: Figure 3 As shown.

[0052] Example 3: Comparative experiment of the present invention and RIBOmap technology on MCF-7 cell smears.

[0053] (I) Cell Culture and Fixation: Human breast adenocarcinoma cells MCF-7 were cultured in DMEM medium (containing 10% FBS) for 24-48 hours, then treated with trypsin to prepare a cell suspension, and seeded onto sterile glass slides coated with poly-L-lysine. The cells were cultured for another 12-24 hours. Subsequently, the cells were washed with DEPC-PBS and fixed with 4% PFA (prepared with DEPC-PBS) at room temperature for 30 minutes. After fixation, the cells were washed twice more with DEPC-PBS, and then dehydrated sequentially with a gradient of ethanol (70%, 85%, 100%) for 5 minutes each time. Finally, the glass slides were air-dried and stored at -80°C.

[0054] (II) Pretreatment of cell samples: The cell membranes of the cells were permeabilized and perforated. The samples were then placed in methanol solution and incubated at -20°C for 1 hour. Afterward, the samples were thawed in air and dried, and then washed three times with DEPC-PBS containing 0.1% (v / v) Tween 20.

[0055] (III) In-situ nucleic acid detection of the present invention:

[0056] The recognition hybridization of the probe and the target gene, the ligation of the double-linked probe pairs, the rolling circle amplification reaction steps, and the probe and primer sequences are consistent with those in Example 1.

[0057] (iv) In-situ nucleic acid detection using RIBOmap technology:

[0058] The specific experimental procedures, reaction system, and probe sequences strictly followed the published literature on RIBOmap technology, as detailed below:

[0059] (1) Oligonucleotide hybridization

[0060] Hybridization reaction mixture containing 10% formamide, 2×SSC, 0.1 mg / mL yeast tRNA (Thermo Scientific), 0.4 U / μL SUPERase·In RNase inhibitor (Thermo Scientific), 0.1% (v / v) Tween 20, 1 nM lock probe, 1 nM primer probe, and 100 nM clip probe was added to the sample and incubated at 40 °C for 12 h. The lock probe hybridized directly and specifically with the target mRNA molecule in situ, the primer probe hybridized specifically with the target mRNA molecule and its lock probe in situ, and the clip probe hybridized simultaneously with ribosomal RNA and the lock probe. After the reaction was complete, the sample was washed twice with PBS (PBSTR) containing 0.1% (v / v) Tween 20 and 0.1 U / μL SUPERase·In RNase inhibitor, 2 min each time. Then, it was incubated with PBSTR solution containing 4×SSC at 37°C for 20 min, and finally rinsed once with PBSTR. The specific sequences of the lock probe, primer probe, and clip probe used to detect ACTB and MALAT1 RNA molecules are shown in Table 3 below.

[0061] Table 3

[0062]

[0063]

[0064] Note: * indicates thiophosphate modification.

[0065] (2) Locking probe connection

[0066] Add a reaction mixture containing 1×T4 DNA reaction buffer (Thermo Scientific), 0.4 U / μL SUPERase·In RNase inhibitor, 0.5 mg / mL BSA, and 0.25 U / μL T4 DNA ligase (Thermo Scientific) to the sample and incubate at room temperature for 2 h. After the reaction is complete, wash three times with PBSTR solution.

[0067] (3) Rolling circle amplification

[0068] A rolling circle amplification reaction mixture containing 1×phi29 DNA polymerase reaction buffer (Thermo Scientific), 0.25 mM dNTPs (Thermo Scientific), 0.5 U / μL phi29 DNA polymerase (Thermo Scientific), 0.4 U / μL SUPERase·In RNase inhibitor, and 0.5 mg / mL BSA was added to the sample. The mixture was incubated at 4°C for 30 min, followed by incubation at 30°C for 2 h to obtain the rolling circle amplification product. After the reaction was complete, the sample was washed three times with PBSTR solution.

[0069] (v) Detection of rolling circle amplification products

[0070] The rolling circle amplification products generated by this invention and RIBOmap technology were detected according to the following steps: A mixture containing 2×SSC, 20% formamide, and 0.1 μM detection probe was added to the sample. The mixture was incubated at 30°C for 30 min to allow the rolling circle amplification products to hybridize with the detection probe (the detection probe is 5'-tcatacactaaagataaaca-3' (SEQ ID NO. 30), with a fluorescent group labeled at the 5' end). After the reaction, the sample was washed three times with DEPC-PBS-Tween 20. After the detection probe hybridization reaction was complete, the cell samples were air-dried. Finally, 0.5 μg / mL DAPI was added to stain the cell nuclei. The mounted samples could be examined and photographed under a fluorescence microscope. The detection results are as follows: Figure 4 As shown: In this embodiment, the present invention has higher detection efficiency and better specificity than RIBOmap, so it has more advantages in the detection of ACTB.

[0071] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for in situ detection of ribosome-bound mRNA, characterized in that: The test is performed using at least one probe set designed based on the target sequence on the target mRNA that binds to the ribosome, wherein each probe set includes: A target sequence recognition probe pair consists of a first recognition probe and a second recognition probe. The first recognition probe, from 5' to 3', has a first target sequence recognition sequence, a first linker sequence, and a first circularization sequence, respectively. The second recognition probe, from 3' to 5', has a second target sequence recognition sequence, a second linker sequence, and a second circularization sequence, respectively. A circularization clip probe has an rRNA sequence recognition sequence and a circularization template sequence connected in sequence. The circularization template sequence is complementary to the first and second circularization sequences mentioned above, serving as the circularization mechanism for the first and second circularization sequences. The specific steps include: (1) At least one probe group is added to the sample to be tested for reaction, wherein: Its target sequence recognition probes pair complementaryly with the target RNA via the first target sequence recognition sequence of the first recognition probe and the second target sequence recognition sequence of the second recognition probe, bringing the first and second target sequence recognition sequences close to each other. Its circularization clip probes recognize hybridized ribosomal RNA through rRNA sequence recognition, and through complementary pairing of the circularization template sequence with both the first circularization sequence of the first recognition probe and the second circularization sequence of the second recognition probe, the first and second circularization sequences are brought close together. Next, wash away the probes that do not have complementary pairing; (2) Add ligase to react and link all the first target sequence recognition sequences and second target sequence recognition sequences that are close to each other in step (1), and link all the first circular sequences and second circular sequences that are close to each other, thereby forming at least one circular template. (3) Perform rolling circle amplification on at least one circular template obtained in step (2) to obtain at least one rolling circle amplification product; (4) Hybridize the detection probe corresponding to the target mRNA with at least one rolling circle amplification obtained in step (3) to perform in situ detection and obtain the corresponding detection signal.

2. The in-situ detection method as described in claim 1, characterized in that: The test samples are selected from cultured cells, cells in tissues, and cells in tissue sections.

3. The in-situ detection method as described in claim 1, characterized in that: The detection probe is modified with a reporter group.

4. The in-situ detection method as described in claim 3, characterized in that: The reporter group is selected from fluorescent groups, radioactive isotopes, and enzyme molecules.

5. The in-situ detection method as described in claim 1, characterized in that: At least one of the first and second recognition probes in the target sequence recognition probe pair has a gene-specific oligonucleotide tag.

6. The in-situ detection method as described in claim 1, characterized in that: The ligase is splint R ligase.

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