In situ multi-flux glycosylation RNA imaging method

Abstract

This invention relates to an in situ multi-throughput glycosylated RNA imaging method, comprising the following steps: constructing different proxHCR systems for different glycoRNAs, thereby performing multi-throughput in situ imaging of multiple glycoRNAs with the same sialic acid composition but different RNA sequences; the proxHCR system includes a sialic acid recognition module, an RNA hybridization module, and a fluorescently labeled hairpin probe (AR) acting as an amplification reporter. The sialic acid recognition module and the proxHCR module are used for dual recognition of sialic acid and RNA units of glycoRNAs on the cell membrane surface. A complete activation foothold switch is formed only when the sialic acid and RNA units of the glycoRNAs on the cell membrane surface are spatially adjacent, triggering HCR of the fluorescently labeled hairpin probe (AR), thereby visualizing the glycoRNAs on the cell surface through amplified fluorescence signals. This invention solves the problem of the lack of prior art for simultaneous multi-throughput analysis of multiple different glycosylated RNAs.

Description

Technical Field

[0001] This invention belongs to the field of in situ imaging technology, and in particular relates to an in situ multi-throughput glycosylated RNA imaging method. Background Technology

[0002] The cell membrane surface contains various two-component biomolecules, such as glycoproteins, glycolipids, and glycosylated RNA. Sialidized glycosylated RNA (glycoRNA) is a two-component biomolecule composed of sialylated glycans and small non-coding RNA. Depending on the type of glycan and the RNA sequence, various glycoRNA molecules have been found to be widely distributed on the cell membrane surface, closely related to RNA maturation, protein translation, chromatin structure regulation, and RNA epigenetics. Therefore, in situ multi-throughput imaging of glycoRNAs on the cell membrane is of great significance for a deeper understanding of the distribution characteristics and biological functions of different glycosylated RNAs.

[0003] Methods based on proximity labeling and imaging techniques have been developed for the detection of glycosylated RNAs on the cell surface. However, these methods only enable the detection and imaging of single glycosylated RNAs, and methods for in situ multi-throughput visualization of multiple different glycosylated RNAs are still lacking. Summary of the Invention

[0004] The existing technology lacks a method for in situ multi-throughput visualization of multiple different glycosylated RNAs. Based on this, the present invention provides an in situ multi-throughput glycosylated RNA imaging method.

[0005] The method provided by this invention is a proxHCR-based method that enables in situ multi-throughput imaging of glycosylated RNA on the cell membrane surface, thereby solving the problem that existing technologies lack the ability to perform multi-throughput analysis of multiple different glycosylated RNAs simultaneously.

[0006] Ortho-induced hybridization chain reaction (proxHCR) has been developed and applied to two-component target detection and protein interaction analysis, with significant advantages such as isothermal, enzyme-free, rapid, simple operation and low cost.

[0007] This invention constructs a sialic acid recognition module (SRM), an RNA hybridization module (RHM), and an amplifying reporter (fluorescently labeled hairpin probe, AR) using DNA sequences. The SRM and RHM enable dual recognition of sialic acid and RNA units of glycoRNAs on the cell membrane surface. Only when these two units are spatially adjacent can they form a complete activation foothold switch, triggering a hybridization chain reaction (HCR) at the AR. This allows visualization of glycoRNAs on the cell surface through amplified fluorescence signals. Multiple orthogonal proxHCR systems enable multi-throughput in situ imaging of various structurally similar glycoRNAs with different glycan components or RNA sequences.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] This invention provides an in situ multi-throughput imaging method for glycosylated RNA, which is a method for in situ multi-throughput imaging of glycosylated RNA on the cell membrane surface based on proxHCR, comprising the following steps:

[0010] Different proxHCR systems were constructed for different glycoRNAs to enable multi-throughput in situ imaging of multiple glycoRNAs with the same sialic acid composition but different RNA sequences.

[0011] The proxHCR system includes a sialic acid recognition module (SRM), an RNA hybridization module (RHM), and a fluorescently labeled hairpin probe (AR) serving as an amplification reporter. The sialic acid recognition module (SRM) is used for dual recognition of sialic acid and RNA units of glycoRNAs on the cell membrane surface.

[0012] A complete activation base switch is formed and triggers HCR of AR (fluorophore-labeled hairpin probe) only when the sialic acid and RNA units of glycoRNA on the cell membrane surface are spatially adjacent, thereby visualizing the glycoRNA on the cell surface through amplified fluorescence signals.

[0013] The foothold is a continuous single-stranded DNA segment that can trigger the HCR reaction. In the scheme provided in this application, both SRM and RHM have foothold fragments. When glycoRNA is absent, SRM and RHM cannot specifically bind, and the foothold is not continuous, thus failing to trigger HCR. However, when glycoRNA is present, SRM and RHM can specifically bind, and the foothold is continuous, enabling the activation of HCR.

[0014] The proxHCR system undergoes a specific DNA hybridization reaction in the presence of glycoRNA.

[0015] In one embodiment of the present invention, the proxHCR system is a single-throughput glycoRNA imaging system, wherein the sequence of the sialic acid recognition module (SRM) is shown in SEQ ID NO.1. The sialic acid recognition module (SRM) includes a nucleic acid aptamer domain that specifically binds to N-acetylneuraminic acid (Neu5Ac) (bold portion of the sialic acid recognition module sequence in Table 1), a spacer domain for reducing steric hindrance (normal portion of the sialic acid recognition module sequence in Table 1), a segmented foothold region (underlined portion of the sialic acid recognition module sequence in Table 1), and a connecting domain (italicized portion of the sialic acid recognition module sequence in Table 1).

[0016] In one embodiment of the present invention, the proxHCR system is a single-throughput glycoRNA imaging system, wherein the sequence of the RNA hybridization module (RHM) is shown in SEQ ID NO.2. The RNA hybridization module, also abbreviated as GlycoRNA-U1, includes an RNA-binding domain that can hybridize in situ with RNA units of glycoRNA (bold portion of the RNA hybridization module sequence in Table 1), a spacer domain for reducing steric hindrance (normal portion of the RNA hybridization module sequence in Table 1), a segmented foothold region (underlined portion of the RNA hybridization module sequence in Table 1), and a linker domain that hybridizes complementaryly with the linker domain of SRM to connect and fix GRM and RHM (italicized portion of the RNA hybridization module sequence in Table 1).

[0017] In one embodiment of the present invention, the proxHCR system is a single-throughput glycoRNA imaging proxHCR system, wherein the RNA binding domain is designed with a corresponding DNA sequence based on the RNA unit composition of the target glycoRNA.

[0018] In one embodiment of the present invention, the proxHCR system is a single-throughput glycoRNA imaging proxHCR system, wherein the amplified reporter (AR) comprises two DNA hairpins H1a and H2a with partially complementary sequences, which achieve signal quenching by modifying fluorescence resonance energy transfer; wherein H1a and H2a are shown in Table 1. The sequence of H1a is: ATG AAGGAC GA / dT-Alexa Fluor 488 / TGT ATG CTT AGG GTC GAC TTC CAT AGACCCTAAGCATACAT / BHQ1 /

[0019] The H2 a sequence is: GAC CCT AAG CAT ACA TCG TCC TTC ATA TGT ATG CTT AGG GTCTAT GGA AGTC.

[0020] H1a and H2a are the footpoint switch and DNA hairpin sequence, respectively, which have been verified to successfully induce HCR.

[0021] In one embodiment of the invention, the maximum spatial distance between sialic acid units and RNA units sufficient to activate the foothold switch and trigger a hybridization chain reaction in AR is 18.08 nm; beyond this distance, a hybridization chain reaction cannot be triggered.

[0022] When the proxHCR system is a single-throughput glycoRNA imaging proxHCR system, the linker domain is a complementary sequence with a length of 7-9 nt. The length of the linker domain is adjusted according to the spatial scale of cellular glycoRNAs.

[0023] Table 1. DNA sequences in the proxHCR system used for single-throughput glycoRNA imaging.

[0024]

[0025]

[0026] GlycoRNA has two units: a sugar unit and an RNA unit. The RNA hybridization module pairs with the RNA unit in GlycoRNA (in bold). The RNA hybridization module for GlycoRNA-U1 is the U1-RNA binding region, for GlycoRNA-U35a it is the U35a-RNA binding region, and for GlycoRNA-Y5 it is the U5-RNA binding region. H1a and H2a form the AR (Arithmetic Hybridization) region.

[0027] In one embodiment of the present invention, the proxHCR system is a dual-throughput glycoRNA imaging system, wherein the sequence of the sialic acid recognition module (SRM) is shown in SEQ ID NO.3. The sialic acid recognition module (SRM) includes a nucleic acid aptamer domain that specifically binds to N-acetylneuraminic acid (Neu5Ac) (bold portion of the sialic acid recognition module sequence in Table 2), a spacer domain for reducing steric hindrance (normal portion of the sialic acid recognition module sequence in Table 1), a segmented anchorage region (underlined portion of the sialic acid recognition module sequence in Table 2), and a connecting domain (italicized portion of the sialic acid recognition module sequence in Table 2).

[0028] In one embodiment of the present invention, the proxHCR system is a dual-throughput glycoRNA imaging system, wherein the RNA hybridization module (RHM) sequence includes RHM-U1 and RHM-U35a, RHM-U1 as shown in SEQ ID NO.4, which is the RHM used for imaging GlycoRNA-U1, and RHM-U35a as shown in SEQ ID NO.5, which is the RHM used for imaging GlycoRNA-U35a. Both RHM-U1 and RHM-U35a contain an RNA-binding domain capable of in situ hybridization with RNA units of glycoRNA (bold portion of the RNA hybridization module sequence in Table 2), a spacer domain for reducing steric hindrance (normal body portion of the RNA hybridization module sequence in Table 2), a segmented foothold region (underlined portion of the RNA hybridization module sequence in Table 2), and a linker domain that complementarily hybridizes with the linker domain of SRM to connect and fix GRM and RHM (italicized portion of the RNA hybridization module sequence in Table 2).

[0029] In one embodiment of the present invention, the proxHCR system is a dual-throughput glycoRNA imaging proxHCR system, wherein the RNA binding domain is designed with a corresponding DNA sequence based on the RNA unit composition of the target glycoRNA.

[0030] In one embodiment of the present invention, the proxHCR system is a dual-throughput glycoRNA imaging proxHCR system, wherein the amplified reporter (AR) comprises DNA hairpins H1a, H2a, H1b, H2b, and H2c with partially complementary sequences, achieving signal quenching through modification of fluorescence resonance energy transfer; wherein H1a, H2a, H1b, H2b, and H2c are shown in Table 2.

[0031] The H1 a sequence is: ATG AAG GAC GA / dT-Alexa Fluor 488 / TGT ATGCTTAGGGTC GAC TTCCAT AGA CCC TAA GCA TAC AT / BHQ1 / ;

[0032] The H2 a sequence is: GAC CCT AAG CAT ACA TCG TCC TTC ATA TGT ATG CTT AGG GTCTAT GGA AGTC;

[0033] The H1 b sequence is: TCT AGT CGT T / dT-Alexa Fluor 594 / GATG ATGCTTAGGGTC CGA CAGATA AGACCCT AAG CAT CAT C / BHQ2 / ;

[0034] The H2 b sequence is: GAC CCT AAG CAT CATC AACGA CTA GAG ATG ATG CTT AGG GTC TTATCT GTCG;

[0035] H1a and H2a are hairpin probes used for GlycoRNA-U1 imaging, and H1b and H2b are hairpin probes used for GlycoRNA-U35a imaging.

[0036] Table 2. DNA sequences in the proxHCR system used for dual-throughput glycoRNA imaging.

[0037]

[0038]

[0039] In one embodiment of the present invention, the proxHCR system is a three-throughput glycoRNA imaging system, wherein the sequence of the sialic acid recognition module (SRM) is shown in SEQ ID NO.6, and the sialic acid recognition module (SRM) includes a nucleic acid aptamer domain that specifically binds to N-acetylneuraminic acid (Neu5Ac) (the bold portion of the sialic acid recognition module sequence in Table 3), a spacer domain for reducing steric hindrance (the normal portion of the sialic acid recognition module sequence in Table 1), a segmented foothold region (the underlined portion of the sialic acid recognition module sequence in Table 3), and a connecting domain (the italicized portion of the sialic acid recognition module sequence in Table 3).

[0040] In one embodiment of the present invention, the proxHCR system is a three-throughput glycoRNA imaging system, wherein the RNA hybridization module (RHM) sequence includes RHM-U1, RHM-U35a and RHM-Y5, RHM-U1 as shown in SEQ ID NO.7, is an RHM for imaging GlycoRNA-U1, RHM-U35a as shown in SEQ ID NO.8, is an RHM for imaging GlycoRNA-U35a, and RHM-Y5 as shown in SEQ ID NO.9, is an RHM for imaging GlycoRNA-Y5. RHM-U1, RHM-U35a, and RHM-Y5 all contain an RNA-binding domain that can hybridize in situ with RNA units of glycoRNA (bold portion of the RNA hybridization module sequence in Table 3), a spacer domain for reducing steric hindrance (normal portion of the RNA hybridization module sequence in Table 3), a segmented foothold region (underlined portion of the RNA hybridization module sequence in Table 3), and a linker domain that hybridizes complementaryly with the linker domain of SRM to connect and fix GRM and RHM (italicized portion of the RNA hybridization module sequence in Table 3).

[0041] In one embodiment of the present invention, the proxHCR system is a three-throughput proxHCR system for glycoRNA imaging, wherein the RNA binding domain is designed with a corresponding DNA sequence based on the RNA unit composition of the target glycoRNA.

[0042] In one embodiment of the present invention, the proxHCR system is a three-throughput glycoRNA imaging proxHCR system, wherein the amplified reporter (AR) comprises six DNA hairpins H1a, H2a, H1b, H2b, H1c, and H2c with partially complementary sequences, and signal quenching is achieved by modifying fluorescence resonance energy transfer; wherein,

[0043] The H1 a sequence is: ATG AAG GAC GA / dT-Alexa Fluor 488 / TGT ATGCTTAGGGTC GAC TTCCAT AGA CCC TAAGCATAC AT / BHQ1 /

[0044] The H2 a sequence is: GAC CCT AAG CAT ACATCG TCC TTC ATATGT ATG CTT AGG GTC TATGGAAGTC

[0045] The H1 b sequence is: GAC CCT AAG CAT ACA TCG TCC TTC ATA TGT ATG CTT AGG GTCTAT GGAAGTC

[0046] The H2 b sequence is: TCT AGT CGT T / dT-Alexa Fluor 594 / GATG ATGCTTAGGGTC CGACAGATAAGACCCT AAG CAT CAT C / BHQ2 /

[0047] The H1 c sequence is: CAT AGG GTTC / dT-Alexa Fluor 647 / GGA TATG CTT AGG GTCGCAGCA TCAAGACCCT AAG CAT ATC C / BHQ3 /

[0048] The H2 c sequence is: GAC CCT AAG CAT ATCC GAACC CTATG GGAT ATG CTT AGG GTC TTGATG CTG C;

[0049] H1a, H2a, H1b, H2b, H1c, and H2c are shown in Table 3. H1a and H2a are hairpin probes used for GlycoRNA-U1 imaging, H1b and H2b are hairpin probes used for GlycoRNA-U35a imaging, and H1c and H2c are hairpin probes used for GlycoRNA-Y5 imaging.

[0050] Table 3. DNA sequences in the proxHCR system used for three-throughput glycoRNA imaging.

[0051]

[0052]

[0053] Referring to Tables 1, 2, and 3, proxHCR has three components: SRM, RHM, and SR. The SRM sequence information is fixed, while the RHM and SR sequences vary depending on the specific glycoRNA sequence.

[0054] Furthermore, the present invention also provides a method for in situ amplification and visualization of glycoRNAs on the cell membrane surface based on a neighbor-induced hybridization chain reaction, comprising the following steps:

[0055] 1) Synthesis of the proxHCR system:

[0056] The proxHCR for visualization of in situ glycosylated RNA consists of three functional modules:

[0057] The sialic acid recognition module (SRM) contains a sialic acid aptamer domain that can specifically bind to N-acetylneuraminic acid (Neu5Ac);

[0058] RNA hybridization module (RHM) contains an RNA binding domain that is complementary to the target RNA sequence;

[0059] Amplified reporter (AR), a pair of DNA hairpins with partially complementary sequences, achieves signal quenching by modifying fluorescence resonance energy transfer (FRET) pairs;

[0060] Reference to the three functional modules of proxHCR Figure 1 A. When SRM and RHM recognize and bind to the sialic acid and RNA units of glycoRNA, respectively, they are spatially adjacent and form a complete activation base switch, thereby triggering the opening of the AR hairpin and HCR, producing an amplified fluorescence signal (see reference). Figure 1 B). Multiple orthogonal proxHCR systems were designed and synthesized for different glycoRNAs to achieve multi-throughput detection.

[0061] 2) In situ multi-throughput imaging of cell surface glycoRNAs using proxHCR

[0062] Using normal human hepatocytes (QSG-7701), human hepatocellular carcinoma cells (HepG2), and human hepatocellular carcinoma cells (MHCC97-H) as model cells, and small nuclear RNA U1 (glycoRNA-U1), SNORD35a (glycoRNA-U35a), and Ro60-associated Y5 RNA (glycoRNA-Y5) as representative targets, the developed proxHCR strategy was used to perform monochrome, dual-color, and tri-color in situ imaging of the three glycoRNAs on the cell membrane surface.

[0063] This invention designs a proxHCR probe based on DNA, which can be used for in situ multi-throughput imaging of glycosylated RNA on the cell membrane surface.

[0064] This application constructs a dual recognition module (SRM) and an RNA hybridization module (RHM) to recognize the sialic acid and RNA units of glycoRNAs, thereby activating a foothold switch to trigger hemoglobin-mediated glycogenic regeneration (HCR). Using multiple sets of orthogonal proxHCR systems, in situ imaging and abundance analysis of various glycoRNAs with similar structures were successfully performed in normal human hepatocytes and human hepatocellular carcinoma cells.

[0065] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0066] 1. By dual recognition of sialylated glycosyl groups and RNA units, the signal is ensured to be generated only when the two are spatially adjacent, avoiding interference from a single target or a target far away.

[0067] 2) Multi-throughput imaging capability: The proxHCR system can simultaneously identify and amplify multiple target signals, making it suitable for the parallel detection of various two-component biomolecules with similar structures. This helps to reveal the distribution and abundance of complex two-component biomolecules (such as glycoRNA) on the cell surface.

[0068] 3) Automatic background suppression: Based on the fluorescence signal activation mode, it can effectively suppress background noise, improve the signal-to-background ratio, and improve imaging accuracy.

[0069] 4) No enzymatic reaction required: It does not depend on the quality of the enzyme or the reaction conditions, which significantly improves the stability and consistency of the experiment and reduces the cost.

[0070] 5) Fast and efficient: The entire process is completed within 6 hours, making it suitable for high-throughput detection of large-scale cell samples. Attached Figure Description

[0071] Figure 1 The functional module structure of the glycoRNA imaging probe, and the schematic diagram of activating the fulcrum switch to trigger HCR;

[0072] Figure 2 The proxHCR experimental procedure and results;

[0073] Figure 3 In situ two-color imaging experiment of sialoglycoRNA and experimental results;

[0074] Figure 4 In situ three-color imaging experiment of sialoglycoRNA and experimental results. Detailed Implementation

[0075] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0076] Example 1

[0077] This embodiment provides a method for in situ amplification and visualization of glycoRNAs on the cell membrane surface based on a neighbor-induced hybridization chain reaction, comprising the following steps:

[0078] 1) Synthesis of the proxHCR system:

[0079] The proxHCR for visualization of in situ glycosylated RNA consists of three functional modules:

[0080] The sialic acid recognition module (SRM) contains a sialic acid aptamer domain that can specifically bind to N-acetylneuraminic acid (Neu5Ac);

[0081] RNA hybridization module (RHM) contains an RNA binding domain that is complementary to the target RNA sequence;

[0082] Amplified reporter (AR), a pair of DNA hairpins with partially complementary sequences, achieves signal quenching by modifying a fluorescence resonance energy transfer (FRET) pair.

[0083] Specifically, refer to Figure 1 The glycoRNA imaging system consists of three functional modules: a sialic acid recognition module (SRM), an RNA hybridization module (RHM), and an amplifiable reporter (AR) module. Figure 1 A). The SRM contains four domains: the nucleic acid aptamer region, the spacer region to avoid steric hindrance during hybridization, the discontinuous anchorage region, and the linker region (Tables 1-3). The RNA hybridization module (RHM) also contains four domains: the region for in situ orthogonal hybridization with glycoRNA, the spacer region, the discontinuous anchorage region, and the linker region (Tables 1-3). Figure 1 A, Supporting Information). AR contains two DNA hairpins with partially complementary sequences, one of which consists of a pair of... Resonance energy transfer (FRET) dye labeling. Proximal binding of the two recognition modules allows for stable hybridization of the two junctional regions and further activates the fulcrum switch to trigger HCR ( Figure 1 B). This proximity-triggered HCR signal amplification strategy ensures that signal generation is based solely on the dual recognition of two nearby targets (i.e., glycan and RNA), avoiding false positive signals that may arise from a single target or two more distant targets. When SRM and RHM recognize and bind to the sialic acid and RNA units of glycoRNA, respectively, they are spatially adjacent and form a complete activation foothold switch, thereby triggering the opening of the AR hairpin and HCR, producing an amplified fluorescence signal (see reference). Figure 1 B). Multiple orthogonal proxHCR systems were designed and synthesized for different glycoRNAs to achieve multi-throughput detection.

[0084] Experimental procedure:

[0085] Cells were distributed at 3 × 10⁶ cells per well. 5Cells were seeded at a density of 100 nM in 29 mm glass-bottom imaging dishes and cultured for 24 hours. Cells were washed three times with DEPC-treated PBS and then fixed with 4% paraformaldehyde (PFA) at 37°C for 15 minutes. To block nonspecific interactions, cells were incubated at 37°C for 30 minutes with 100 nM polyT oligonucleotides and 0.25 μg / μl BSA in 1× (50 mM Tris-HCl buffer and 10 mM MgCl2, pH 7.4) hybridization buffer. Subsequently, cells were incubated at 37°C for 30 minutes with 500 nM MSRM, 0.25 μg / μl BSA, and 250 mM NaCl in 1× (50 mM Tris-HCl buffer and 10 mM MgCl2, pH 7.4) hybridization buffer. Then, cells were washed sequentially at 37°C for 10 minutes each time with 500 μL of 1× PBS. Cells were then incubated for 2 h at 37°C with 100 μl of 100 nM glycan recognition module, 100 μl of 50 nM RHM, 0.25 μg / μl BSA, and 100 nM polyT oligonucleotide buffer (50 mM Tris-HCl, 5 mM KCl, 100 mM NaCl, and 1 mM MgCl2, pH 7.4). Next, cells were washed for 10 min at room temperature with 500 μl of (a) 75% 2×SSC / 25% PBS, (b) 50% 2×SSC / 50% PBS, (c) 25% 2×SSC / 75% PBS, and (d) 100% PBS, respectively. All wash buffers were preheated to 37°C before use. All samples were stored in the dark and examined using a Zeiss confocal microscope (×63 oil immersion objective; laser line excitation / emission wavelengths of 488 nm / 520 nm). Images were processed using Zen 3.6 (blue version). (Composed of 2×SSC: 6M NaCl and 0.6M sodium citrate)

[0086] 2) In situ multi-throughput imaging of cell surface glycoRNAs using proxHCR

[0087] Using normal human hepatocytes (QSG-7701), human hepatocellular carcinoma cells (HepG2), and human hepatocellular carcinoma cells (MHCC97-H) as model cells, and small nuclear RNA U1 (glycoRNA-U1), SNORD35a (glycoRNA-U35a), and Ro60-associated Y5 RNA (glycoRNA-Y5) as representative targets, the developed proxHCR strategy was used to perform monochrome, dual-color, and tri-color in situ imaging of the three glycoRNAs on the cell membrane surface.

[0088] Monochrome: All samples were preserved in darkness using a Zeiss confocal microscope (×63 oil immersion objective; laser line excitation / emission wavelengths of 488nm / 520nm). Images were processed using Zen 3.6 (blue edition).

[0089] Two-color: All samples were preserved in darkness and examined using a Zeiss confocal microscope (×63 oil immersion objective; laser line excitation wavelengths of 488nm and 560nm, and emission wavelengths of 520nm and 590nm). Images were processed using Zen 3.6 (blue edition).

[0090] Three-color: All samples were preserved in darkness and examined using a Zeiss confocal microscope (×63 oil immersion objective; laser line excitation wavelengths of 488nm, 560nm, and 650nm; emission wavelengths of 520nm, 590nm, and 690nm). Images were processed using Zen 3.6 (blueedition).

[0091] Specifically, firstly, the normal human hepatocyte line QSG-7701 was selected as the model cell, and nuclear small RNA U1 (glycoRNA-U1) was selected as the representative target. For example... Figure 2 As shown in Figure A, a bright fluorescent signal was observed on the cell membrane using a confocal laser scanning microscope (CLSM), indicating that glycoRNA-U1 can be imaged by proxHCR.

[0092] To further evaluate the applicability of the glycoRNA imaging method, three GlycoRNA-U1s were visualized and compared in malignant (HepG2) and metastatic (MHCC97-H) hepatocellular carcinoma cell lines. Figure 2 As shown in CD, using proxHCR visualization, glycoRNA-U1 specifically exhibited the strongest fluorescence signal in the non-tumorigenic cell line QSG-7701, while the fluorescence signal in the malignant cell line HepG2 and the metastatic cell line MHCC97-H decreased by 38.81% and 74.58%, respectively. This suggests that the abundance of glycoRNA-U1 on the cell surface may decrease during tumor malignancy and metastasis.

[0093] Two orthogonal proxHCR systems were designed to specifically target the corresponding glycoRNA-U1 and glycoRNA-U35a. Figure 3 A).

[0094] The experimental procedure is as follows:

[0095] Cells were distributed at 3 × 10⁶ cells per well. 5Cells were seeded at a density of 100 nM in 29 mm glass-bottom imaging dishes and cultured for 24 hours. Cells were washed three times with DEPC-treated PBS and then fixed with 4% paraformaldehyde (PFA) at 37°C for 15 minutes. To block nonspecific interactions, cells were incubated at 37°C for 30 minutes with 100 nM polyT oligonucleotides and 0.25 μg / μl BSA in 1× (50 mM Tris-HCl buffer and 10 mM MgCl2, pH 7.4) hybridization buffer. Subsequently, cells were incubated at 37°C for 30 minutes with 500 nM MSRM, 0.25 μg / μl BSA, and 250 mM NaCl in 1× (50 mM Tris-HCl buffer and 10 mM MgCl2, pH 7.4) hybridization buffer. Then, cells were washed sequentially at 37°C for 10 minutes each time with 500 μL of 1× PBS. Cells were then incubated for 2 h at 37°C with 100 μl of 100 nM glycan recognition module, 100 μl of 50 nM RHM, 0.25 μg / μl BSA, and 100 nM polyT oligonucleotide buffer (50 mM Tris-HCl, 5 mM KCl, 100 mM NaCl, and 1 mM MgCl2, pH 7.4). Next, cells were washed for 10 min at room temperature with 500 μl of (a) 75% 2×SSC / 25% PBS, (b) 50% 2×SSC / 50% PBS, (c) 25% 2×SSC / 75% PBS, and (d) 100% PBS, respectively. All wash buffers were preheated to 37°C before use. All samples were stored in the dark and examined using a Zeiss confocal microscope (×63 oil immersion objective; laser line excitation / emission wavelengths of 488 nm / 520 nm). Images were processed using Zen 3.6 (blue version). (Composed of 2×SSC: 6M NaCl and 0.6M sodium citrate).

[0096] like Figure 3 As shown in Figure B, after a 355-minute dual-throughput glycoRNA imaging workflow, signals of both glycoRNA-U1 and glycoRNA-U35a could be simultaneously observed on the QSG-7701 cell membrane. Furthermore, this proxHCR for 2-plex imaging demonstrated excellent applicability in different cell lines. Next, the spatial distribution of the two glycoRNAs on the cell membrane was explored using proxHCR-based dual-throughput imaging technology. Figure 3 As shown in Figure B, glycoRNA-U1 and glycoRNA-U35a exhibit significant co-localization on the cell membrane in QSG-7701 cells, with a Pearson correlation coefficient (PCC) of 0.72. This co-localization pattern has also been observed in different cell lines.

[0097] To further illustrate the parallel imaging capability of proxHCR, three orthogonal proxHCR systems were designed (as shown in Table 3) to simultaneously visualize glycoRNA-U1, glycoRNA-U35a, and glycoRNA-Y5. Figure 4 A).

[0098] The experimental procedure is as follows:

[0099] Cells were distributed at 3 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 nM in 29 mm glass-bottom imaging dishes and cultured for 24 hours. Cells were washed three times with DEPC-treated PBS and then fixed with 4% paraformaldehyde (PFA) at 37°C for 15 minutes. To block nonspecific interactions, cells were incubated at 37°C for 30 minutes with 100 nM polyT oligonucleotides and 0.25 μg / μl BSA in 1× (50 mM Tris-HCl buffer and 10 mM MgCl2, pH 7.4) hybridization buffer. Subsequently, cells were incubated at 37°C for 30 minutes with 500 nM MSRM, 0.25 μg / μl BSA, and 250 mM NaCl in 1× (50 mM Tris-HCl buffer and 10 mM MgCl2, pH 7.4) hybridization buffer. Then, cells were washed sequentially at 37°C for 10 minutes each time with 500 μL of 1× PBS. Cells were then incubated for 2 h at 37°C with 100 μl of 100 nM glycan recognition module, 100 μl of 50 nM RHM, 0.25 μg / μl BSA, and 100 nM polyT oligonucleotide buffer (50 mM Tris-HCl, 5 mM KCl, 100 mM NaCl, and 1 mM MgCl2, pH 7.4). Next, cells were washed for 10 min at room temperature with 500 μl of (a) 75% 2×SSC / 25% PBS, (b) 50% 2×SSC / 50% PBS, (c) 25% 2×SSC / 75% PBS, and (d) 100% PBS, respectively. All wash buffers were preheated to 37°C before use. All samples were stored in the dark and examined using a Zeiss confocal microscope (×63 oil immersion objective; laser line excitation / emission wavelengths of 488 nm / 520 nm). Images were processed using Zen 3.6 (blue version). (Composed of 2×SSC: 6M NaCl and 0.6M sodium citrate)

[0100] Following the established proxHCR workflow, signals from these three cell surface glycoRNAs were successfully detected in QSG-7701, HepG2, and MHCC97-H cell lines. Figure 4B). Notably, the significant co-localization of these three glycoRNAs on the cell membrane observed in three-throughput imaging further demonstrates the aggregation of multiple glycoRNAs on the cell membrane.

[0101] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An in situ multi-throughput glycosylated RNA imaging method, characterized in that, A method for in situ multi-throughput imaging of glycosylated RNA on the cell membrane surface based on a neighbor-induced hybridization chain reaction includes the following steps: Different proxHCR systems were constructed for different glycoRNAs to enable multi-throughput in situ imaging of multiple glycoRNAs with the same sialic acid composition but different RNA sequences. The proxHCR system includes a sialic acid recognition module, an RNA hybridization module, and a fluorescently labeled hairpin probe (AR) serving as an amplification reporter. The sialic acid recognition module is used for dual recognition of sialic acid and RNA units of glycoRNA on the cell membrane surface. A complete activation foothold switch is formed only when the sialic acid and RNA units of glycoRNA on the cell membrane surface are spatially adjacent, triggering HCR of the fluorescently labeled hairpin probe AR, thereby visualizing the glycoRNA on the cell surface through an amplified fluorescence signal.

2. The in situ multi-throughput glycosylated RNA imaging method according to claim 1, characterized in that, The basis is a continuous single-stranded DNA that can trigger an HCR reaction; SRM and RHM each have a base segment. When glycoRNA is absent, SRM and RHM cannot bind stably. At this time, the base is not continuous and HCR cannot be triggered. However, when glycoRNA is present, SRM and RHM can bind specifically. At this time, the base is continuous and HCR can be activated.

3. The in situ multi-throughput glycosylated RNA imaging method according to claim 1, characterized in that, The sialic acid recognition module includes a nucleic acid aptamer domain that can specifically bind to N-acetylneuraminic acid (Neu5Ac), a spacer domain for reducing steric hindrance, a segmented foothold region, and a connection domain.

4. The in situ multi-throughput glycosylated RNA imaging method according to claim 3, characterized in that, The linker domain is a complementary sequence of 7-9 nt in length; the length of the linker domain is regulated according to the spatial scale of cellular glycoRNAs.

5. The in situ multi-throughput glycosylated RNA imaging method according to claim 1, characterized in that, The RNA hybridization module includes an RNA-binding domain capable of in situ hybridization with RNA units of glycoRNA, a spacer domain for reducing steric hindrance, a segmented foothold region, and a linker domain that hybridizes complementaryly with the linker domain of SRM to connect and fix GRM and RHM.

6. The in situ multi-throughput glycosylated RNA imaging method according to claim 1, characterized in that, The maximum spatial distance between sialic acid units and RNA units sufficient to activate the foothold switch and trigger the hybridization chain reaction of AR is 18.08 nm; beyond this distance, the hybridization chain reaction cannot be triggered.

7. The in situ multi-throughput glycosylated RNA imaging method according to claim 1, characterized in that, The proxHCR system is a single-throughput glycoRNA imaging system. The sequence of the sialic acid recognition module is shown in SEQ ID NO.1; The sequence of the RNA hybridization module is shown in SEQ ID NO.2; The amplified reporter comprises two DNA hairpins, H1a and H2a, whose sequences are partially complementary. The H1 a sequence is: ATG AAG GAC GA / dT-Alexa Fluor 488 / TGT ATG CTT AGG GTC GACTTC CAT AGACCCTAAGCATACAT / BHQ1 / The H2 a sequence is: GAC CCT AAG CAT ACA TCG TCC TTC ATA TGT ATG CTT AGG GTC TATGGAAGTC.

8. The in situ multi-throughput glycosylated RNA imaging method according to claim 1, characterized in that, The proxHCR system is a dual-throughput glycoRNA imaging system, wherein... The sequence of the sialic acid recognition module is shown in SEQ ID NO.3; The RNA hybridization module includes sequences of RHM-U1 and RHM-U35a, RHM-U1 as shown in SEQ ID NO.4, which is an RHM used for imaging GlycoRNA-U1, and RHM-U35a as shown in SEQ ID NO.5, which is an RHM used for imaging GlycoRNA-U35a. The amplifying reporter includes DNA hairpins H1a, H2a, H1b, H2b, and H2c with partially complementary sequences, which achieve signal quenching through modification of fluorescence resonance energy transfer; wherein, The H1 a sequence is: ATG AAG GAC GA / dT-Alexa Fluor 488 / TGT ATGCTTAGGGTC GAC TTC CATAGACCC TAA GCA TAC AT / BHQ1 / ; The H2 a sequence is: GAC CCT AAG CAT ACA TCG TCC TTC ATA TGT ATG CTT AGG GTC TATGGAAGTC; The H1 b sequence is: TCT AGT CGT T / dT-Alexa Fluor 594 / GATG ATGCTTAGGGTC CGACAG ATAAGACCCT AAG CAT CAT C / BHQ2 / ; The H2 b sequence is: GAC CCT AAG CAT CATC AACGA CTA GAG ATG ATG CTT AGG GTC TTA TCTGTCG; H1a and H2a are hairpin probes used for GlycoRNA-U1 imaging, and H1b and H2b are hairpin probes used for GlycoRNA-U35a imaging.

9. The in situ multi-throughput glycosylated RNA imaging method according to claim 1, characterized in that, The proxHCR system is a three-throughput glycoRNA imaging system, wherein... The sequence of the sialic acid recognition module is shown in SEQ ID NO.6; The RNA hybridization module includes sequences of RHM-U1, RHM-U35a, and RHM-Y5, where RHM-U1 is shown in SEQ ID NO.7 and is the RHM used for imaging GlycoRNA-U1; RHM-U35a is shown in SEQ ID NO.8 and is the RHM used for imaging GlycoRNA-U35a; and RHM-Y5 is shown in SEQ ID NO.9 and is the RHM used for imaging GlycoRNA-Y5. The amplifying reporter comprises six DNA hairpins with partially complementary sequences H1a, H2a, H1b, H2b, H1c, and H2c, which achieve signal quenching through modification of fluorescence resonance energy transfer; wherein, The H1 a sequence is: ATG AAG GAC GA / dT-Alexa Fluor 488 / TGT ATGCTTAGGGTC GAC TTC CATAGA CCC TAA GCA TAC AT / BHQ1 / The H2 a sequence is: GAC CCT AAG CAT ACA TCG TCC TTC ATA TGT ATG CTT AGG GTC TATGGA AGTC The H1 b sequence is: GAC CCT AAG CAT ACA TCG TCC TTC ATA TGT ATG CTT AGG GTC TATGGA AGTC The H2 b sequence is: TCT AGT CGT T / dT-Alexa Fluor 594 / GATG ATGCTTAGGGTC CGA CAG ATAAGACCCT AAG CAT CAT C / BHQ2 / The H1 c sequence is: CAT AGG GTTC / dT-Alexa Fluor 647 / GGA TATG CTT AGG GTCGCAGCATCAAGACCCT AAG CAT ATC C / BHQ3 / The H2 c sequence is: GAC CCT AAG CAT ATCC GAACC CTATG GGAT ATG CTT AGG GTC TTG ATGCTG C; H1a and H2a are hairpin probes used for imaging GlycoRNA-U1, H1b and H2b are hairpin probes used for imaging GlycoRNA-U35a, and H1c and H2c are hairpin probes used for imaging GlycoRNA-Y5.

10. A method for in-situ amplification and visualization of glycoRNAs on the cell membrane surface based on a neighbor-induced hybridization chain reaction, characterized in that, Includes the following steps: 1) Synthesis of the proxHCR system: proxHCR for visualization of in situ glycosylated RNA consists of three functional modules composition: The sialic acid recognition module includes a sialic acid aptamer domain that can specifically bind to N-acetylneuraminic acid; RNA hybridization module, containing an RNA binding domain complementary to the target RNA sequence; Amplifying reporter, namely a DNA hairpin with complementary sequence, achieves signal quenching by modifying fluorescence resonance energy transfer pairs; 2) In situ multi-throughput imaging of cell surface glycoRNAs using proxHCR Using normal human hepatocytes, human hepatocellular carcinoma cells, and human hepatocellular carcinoma cells as model cells, and small nuclear RNA U1, SNORD35a, and Ro60-related Y5 RNA as representative targets, the proxHCR system was used to perform monochrome, two-color, and three-color in situ imaging of the three glycoRNAs on the cell membrane surface.

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