Haplotypes d-amino acid oxidase and its application in proximity labeling multi-omics technology

Abstract

This invention first constructs a monomeric D-amino acid oxidase and, based on this, proposes for the first time a non-toxic, multifunctional proximity labeling method (PRADA). By linking the monomeric D-amino acid oxidase to APEX2, or constructing the two in adjacent spatial locations, an enzymatic cascade reaction can be achieved to generate H2O2 in situ in vivo, avoiding the use of exogenous H2O2, significantly reducing toxicity, while retaining the chemical multifunctionality of peroxidase. PRADA can label not only proteins and RNA but also support the assembly of functional polymers, meeting the needs of multidimensional analysis and broadening its application range. This technology can be directly applied to various in vivo models under physiological conditions without relying on mutants or ex vivo tissues. Furthermore, the PRADA-MaP seq technology based on PRADA has for the first time achieved the resolution of subcellular compartment-specific RNA structures in vivo, overcoming the spatial resolution limitations of traditional methods.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a monomeric D-amino acid oxidase and its application in proximity labeling multi-omics technology. Background Technology

[0002] Proximity labeling is a method that uses highly reactive enzymes to label biomolecules near specific subcellular regions in living cells. By positioning the enzyme to the target region, spatially specific labeling of proteins or RNA is achieved, thereby revealing complex spatial organization and molecular functions within cells. This technique is of great value in studying the localization of biomolecules and their biological functions.

[0003] Currently, commonly used proximity labeling technologies include BioID and APEX2. BioID uses biotin ligase to label neighboring proteins, while APEX2 uses peroxidase to label proteins and RNA. However, these technologies have significant limitations in in vivo applications. APEX2 relies on exogenous hydrogen peroxide (H2O2), a toxic substance that interferes with cellular redox signaling pathways, inducing cellular stress and even death, thus limiting its use in in vivo models. Although BioID uses a non-toxic biotin substrate, its function is limited to protein labeling and cannot label RNA or achieve other functional applications.

[0004] Furthermore, existing technologies often require mutants or ex vivo tissues in in vivo models (such as nematodes, zebrafish, fruit flies, and mice), making them difficult to operate under physiological conditions. For RNA structure resolution, traditional chemical probe-based techniques (such as DMS-MaPseq and SHAPE-MaP) can only provide structural information at the whole-cell level, lacking subcellular compartment specificity, resulting in an insufficient understanding of RNA folding states.

[0005] Therefore, there is an urgent need in this field for a non-toxic proximity labeling technology that can label proteins and RNA in living organisms of various model organisms, thereby enabling multi-omics research. Summary of the Invention

[0006] The purpose of this invention is to provide a monomeric D-amino acid oxidase and its application in proximity labeling multi-omics technology.

[0007] In a first aspect, the present invention provides a fusion protein having a structure as shown in formula (I), formula (II), formula (III) or formula (IV):

[0008] (Z1-Z2)-Z3-(Z4-Z5)(I);

[0009] (Z4-Z5)-Z3-(Z1-Z2)(II);

[0010] Z1-Z2-Z5(III); or

[0011] Z1-Z4-Z5(IV);

[0012] In the formula,

[0013] Z1 is none, or is selected from the following group: location sequence, connection sequence, label sequence, or a combination thereof;

[0014] Z2 is the hydrogen peroxide utilization module;

[0015] Z3 is a connection sequence;

[0016] Z4 is the hydrogen peroxide generation module;

[0017] Z5 is absent, or selected from the following group: positioning sequence, connection sequence, or a combination thereof;

[0018] "-" indicates a peptide bond that connects the above sequences;

[0019] (Z1-Z2) is selected from the following group: Z1-Z2 or Z2-Z1;

[0020] (Z4-Z5) is selected from the following group: Z4-Z5 or Z5-Z4.

[0021] In another preferred embodiment, the fusion protein has the structure shown in formulas (V) to (XII):

[0022] Z1-Z2-Z3-Z4-Z5(V);

[0023] Z1-Z2-Z3-Z5-Z4(VI);

[0024] Z2-Z1-Z3-Z4-Z5(VII);

[0025] Z2-Z1-Z3-Z5-Z4(VIII);

[0026] Z4-Z5-Z3-Z1-Z2(IX);

[0027] Z4-Z5-Z3-Z2-Z1(X);

[0028] Z5-Z4-Z3-Z1-Z2(XI);

[0029] Z5-Z4-Z3-Z2-Z1(XII);

[0030] In the formula,

[0031] Z1 is none, or is selected from the following group: location sequence, connection sequence, label sequence, or a combination thereof;

[0032] Z2 is the hydrogen peroxide utilization module;

[0033] Z3 is a connection sequence;

[0034] Z4 is the hydrogen peroxide generation module;

[0035] Z5 is absent, or selected from the following group: positioning sequence, connection sequence, or a combination thereof;

[0036] "-" indicates a peptide bond connecting the above sequences.

[0037] In another preferred embodiment, the hydrogen peroxide utilization module is a peroxidase.

[0038] In another preferred embodiment, the peroxidase includes: APEX2, APEX, and HRP.

[0039] In another preferred embodiment, the peroxidase is APEX2.

[0040] In another preferred embodiment, the APEX2 has an amino acid sequence as shown in SEQ ID NO:1.

[0041] In another preferred embodiment, the hydrogen peroxide generating module is a D-amino acid oxidase.

[0042] In another preferred embodiment, the D-amino acid oxidase is derived from fungi.

[0043] In another preferred embodiment, the D-amino acid oxidase is derived from Rhodotorula gracilis.

[0044] In another preferred embodiment, the D-amino acid oxidase is selected from the group consisting of: dimerized D-amino acid oxidase, monomeric D-amino acid oxidase, or combinations thereof.

[0045] In another preferred embodiment, the D-amino acid oxidase is a monomeric D-amino acid oxidase.

[0046] In another preferred embodiment, the monomeric D-amino acid oxidase has an amino acid sequence as shown in SEQ ID NO:4.

[0047] In another preferred embodiment, the localization sequence is selected from the group consisting of: cytoplasmic matrix localization sequences, nuclear localization sequences, nucleolar localization sequences, mitochondrial matrix localization sequences, endoplasmic reticulum outer membrane localization sequences, cell membrane localization sequences, or combinations thereof.

[0048] In another preferred embodiment, the cytoplasmic matrix localization sequence has an amino acid sequence as shown in SEQ ID NO:23.

[0049] In another preferred embodiment, the nuclear localization sequence has an amino acid sequence as shown in SEQ ID NO:24.

[0050] In another preferred embodiment, the nucleolar positioning sequence is selected from the group consisting of the amino acid sequence shown in SEQ ID NO:24, the amino acid sequence shown in SEQ ID NO:25, or a combination thereof.

[0051] In another preferred embodiment, the mitochondrial matrix localization sequence has an amino acid sequence as shown in SEQ ID NO:26.

[0052] In another preferred embodiment, the endoplasmic reticulum outer membrane localization sequence has an amino acid sequence as shown in SEQ ID NO:27.

[0053] In another preferred embodiment, the cell membrane localization sequence has an amino acid sequence as shown in SEQ ID NO:28.

[0054] In another preferred embodiment, the connection sequence is selected from the group consisting of flexible connection sequences, rigid connection sequences, or combinations thereof.

[0055] In another preferred embodiment, the connection sequence is a flexible connection sequence.

[0056] In another preferred embodiment, the linker sequence consists of 1-30 glycine (G), and / or serine (S), and / or arginine (R), and / or asparagine (N).

[0057] In another preferred embodiment, the linker sequence is selected from the group consisting of the amino acid sequence shown in SEQ ID NO:17, the amino acid sequence shown in SEQ ID NO:18, the amino acid sequence shown in SEQ ID NO:19, the amino acid sequence shown in SEQ ID NO:20, the amino acid sequence shown in SEQ ID NO:21, the amino acid sequence shown in SEQ ID NO:22, or combinations thereof.

[0058] In another preferred embodiment, the tag sequence is selected from the group consisting of amino acid sequences such as those shown in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or combinations thereof.

[0059] In another preferred embodiment, the Z3 has an amino acid sequence as shown in SEQ ID NO:17.

[0060] In another preferred embodiment, the N-terminus of formula (I) or formula (II) also contains methionine (M).

[0061] In another preferred embodiment, the fusion protein has a structure as shown in formula (XIII) or formula (XIV):

[0062] Z2-Z3-Z4(XIII); or

[0063] Z4-Z3-Z2(XIV);

[0064] In the formula,

[0065] Z2 is APEX2; APEX2 has the amino acid sequence shown in SEQ ID NO:1;

[0066] Z3 is a linker sequence; the linker sequence has the amino acid sequence shown in SEQ ID NO:17;

[0067] Z4 is a monomeric D-amino acid oxidase; the monomeric D-amino acid oxidase has the amino acid sequence shown in SEQ ID NO:4;

[0068] "-" indicates a peptide bond connecting the above sequences.

[0069] In another preferred embodiment, the fusion protein has a structure as shown in formula (XV):

[0070] Z1-Z2-Z3-Z4(XV);

[0071] In the formula,

[0072] Z1 is a mitochondrial matrix localization sequence and a tag sequence; the mitochondrial matrix localization sequence has the amino acid sequence shown in SEQ ID NO:26, and the tag sequence has the amino acid sequence shown in SEQ ID NO:14;

[0073] Z2 is APEX2, which has the amino acid sequence shown in SEQ ID NO:1;

[0074] Z3 is a linker sequence having an amino acid sequence as shown in SEQ ID NO:17;

[0075] Z4 is a monomeric D-amino acid oxidase having the amino acid sequence shown in SEQ ID NO:4;

[0076] "-" indicates a peptide bond connecting the above sequences.

[0077] In another preferred embodiment, the fusion protein has a structure as shown in formula (XV):

[0078] Z1-Z2-Z3-Z4(XV);

[0079] In the formula,

[0080] Z1 is the location sequence, the connection sequence, and the label sequence;

[0081] Z2 is APEX2, which has the amino acid sequence shown in SEQ ID NO:1;

[0082] Z3 is a linker sequence having an amino acid sequence as shown in SEQ ID NO:17;

[0083] Z4 is a monomeric D-amino acid oxidase having the amino acid sequence shown in SEQ ID NO:4;

[0084] "-" indicates a peptide bond that connects the above sequences;

[0085] In Z1, the positioning sequence is selected from the group consisting of the amino acid sequence shown in SEQ ID NO:27 and the amino acid sequence shown in SEQ ID NO:28.

[0086] The linker sequence is selected from the group consisting of the amino acid sequence shown in SEQ ID NO:18 and the amino acid sequence shown in SEQ ID NO:19;

[0087] The tag sequence has an amino acid sequence as shown in SEQ ID NO:14.

[0088] In another preferred embodiment, the fusion protein has a structure as shown in formula (XV):

[0089] Z1-Z2-Z3-Z4(XV);

[0090] In the formula,

[0091] Z1 is an endoplasmic reticulum outer membrane localization sequence, a linker sequence, and a tag sequence; the endoplasmic reticulum outer membrane localization sequence has an amino acid sequence as shown in SEQ ID NO:27, the linker sequence has an amino acid sequence as shown in SEQ ID NO:19, and the tag sequence has an amino acid sequence as shown in SEQ ID NO:14.

[0092] Z2 is APEX2, which has the amino acid sequence shown in SEQ ID NO:1;

[0093] Z3 is a linker sequence having an amino acid sequence as shown in SEQ ID NO:17;

[0094] Z4 is a monomeric D-amino acid oxidase having the amino acid sequence shown in SEQ ID NO:4;

[0095] "-" indicates a peptide bond that connects the sequences mentioned above.

[0096] In another preferred embodiment, the fusion protein has a structure as shown in formula (XV):

[0097] Z1-Z2-Z3-Z4(XV);

[0098] In the formula,

[0099] Z1 is a cell membrane localization sequence, a linker sequence, and a tag sequence; the cell membrane localization sequence has an amino acid sequence as shown in SEQ ID NO:28, the linker sequence has an amino acid sequence as shown in SEQ ID NO:18, and the tag sequence has an amino acid sequence as shown in SEQ ID NO:14.

[0100] Z2 is APEX2, which has the amino acid sequence shown in SEQ ID NO:1;

[0101] Z3 is a linker sequence having an amino acid sequence as shown in SEQ ID NO:17;

[0102] Z4 is a monomeric D-amino acid oxidase having the amino acid sequence shown in SEQ ID NO:4;

[0103] "-" indicates a peptide bond that connects the sequences mentioned above.

[0104] In another preferred embodiment, the fusion protein has a structure as shown in formula (V):

[0105] Z1-Z2-Z3-Z4-Z5(V);

[0106] In the formula,

[0107] Z1 is a tag sequence; the tag sequence has an amino acid sequence as shown in SEQ ID NO:14;

[0108] Z2 is APEX2; APEX2 has the amino acid sequence shown in SEQ ID NO:1;

[0109] Z3 is a linker sequence; the linker sequence has the amino acid sequence shown in SEQ ID NO:17;

[0110] Z4 is a monomeric D-amino acid oxidase; the monomeric D-amino acid oxidase has the amino acid sequence shown in SEQ ID NO:4;

[0111] Z5 is a cytoplasmic matrix localization sequence; the cytoplasmic matrix localization sequence has the amino acid sequence shown in SEQ ID NO:23;

[0112] "-" indicates a peptide bond that connects the sequences mentioned above.

[0113] In another preferred embodiment, the fusion protein has a structure as shown in formula (V):

[0114] Z1-Z2-Z3-Z4-Z5(V);

[0115] In the formula,

[0116] Z1 is the location sequence, the connection sequence, and the label sequence;

[0117] Z2 is APEX2; APEX2 has the amino acid sequence shown in SEQ ID NO:1;

[0118] Z3 is a linker sequence; the linker sequence has the amino acid sequence shown in SEQ ID NO:17;

[0119] Z4 is a monomeric D-amino acid oxidase; the monomeric D-amino acid oxidase has the amino acid sequence shown in SEQ ID NO:4;

[0120] Z5 consists of the connection sequence and the positioning sequence;

[0121] "-" indicates a peptide bond that connects the above sequences;

[0122] In Z1, the positioning sequence has an amino acid sequence as shown in SEQ ID NO:24, the linking sequence has an amino acid sequence as shown in SEQ ID NO:18, and the tag sequence has an amino acid sequence as shown in SEQ ID NO:14.

[0123] In Z5, the linker sequence is selected from the group consisting of amino acid sequences as shown in SEQ ID NO:18 and amino acid sequences as shown in SEQ ID NO:20; the positioning sequence is selected from the group consisting of amino acid sequences as shown in SEQ ID NO:24 and amino acid sequences as shown in SEQ ID NO:25.

[0124] In another preferred embodiment, the fusion protein has a structure as shown in formula (V):

[0125] Z1-Z2-Z3-Z4-Z5(V);

[0126] In the formula,

[0127] Z1 is a positioning sequence, a linking sequence, and a tag sequence; the positioning sequence has an amino acid sequence as shown in SEQ ID NO:24, the linking sequence has an amino acid sequence as shown in SEQ ID NO:18, and the tag sequence has an amino acid sequence as shown in SEQ ID NO:14.

[0128] Z2 is APEX2; APEX2 has the amino acid sequence shown in SEQ ID NO:1;

[0129] Z3 is a linker sequence; the linker sequence has the amino acid sequence shown in SEQ ID NO:17;

[0130] Z4 is a monomeric D-amino acid oxidase; the monomeric D-amino acid oxidase has the amino acid sequence shown in SEQ ID NO:4;

[0131] Z5 is a linker sequence and a nuclear localization sequence; the linker sequence has an amino acid sequence as shown in SEQ ID NO:18, and the nuclear localization sequence has an amino acid sequence as shown in SEQ ID NO:24;

[0132] "-" indicates a peptide bond that connects the sequences mentioned above.

[0133] In another preferred embodiment, the fusion protein has a structure as shown in formula (V):

[0134] Z1-Z2-Z3-Z4-Z5(V);

[0135] In the formula,

[0136] Z1 is a positioning sequence, a linking sequence, and a tag sequence; the positioning sequence has an amino acid sequence as shown in SEQ ID NO:24, the linking sequence has an amino acid sequence as shown in SEQ ID NO:18, and the tag sequence has an amino acid sequence as shown in SEQ ID NO:14.

[0137] Z2 is APEX2; APEX2 has the amino acid sequence shown in SEQ ID NO:1;

[0138] Z3 is a linker sequence; the linker sequence has the amino acid sequence shown in SEQ ID NO:17;

[0139] Z4 is a monomeric D-amino acid oxidase; the monomeric D-amino acid oxidase has the amino acid sequence shown in SEQ ID NO:4;

[0140] Z5 is a linker sequence and a nucleolar positioning sequence; the linker sequence has an amino acid sequence as shown in SEQ ID NO:20, and the nucleolar positioning sequence has an amino acid sequence as shown in SEQ ID NO:25;

[0141] "-" indicates a peptide bond that connects the sequences mentioned above.

[0142] In another preferred embodiment, the fusion protein is selected from the group consisting of: PRADA-cytosol (NES), nucleus (NLS)-PRADA, nucleolus (NIK)-PRADA, mitochondria (MITO)-PRADA, outer endoplasmic reticulum membrane (EMR)-PRADA, and cell membrane (MEM)-PRADA.

[0143] In another preferred embodiment, the fusion protein is selected from the group consisting of:

[0144] (1) It has the amino acid sequence shown in SEQ ID NO:5 (PRADA-NES);

[0145] (2) It has the amino acid sequence shown in SEQ ID NO:6 (NLS-PRADA);

[0146] (3) It has the amino acid sequence shown in SEQ ID NO:7 (NIK-PRADA);

[0147] (4) It has the amino acid sequence shown in SEQ ID NO:8 (MITO-PRADA);

[0148] (5) It has the amino acid sequence shown in SEQ ID NO:9 (EMR-PRADA);

[0149] (6) It has the amino acid sequence shown in SEQ ID NO:10 (MEM-PRADA);

[0150] Or a combination thereof.

[0151] In another preferred embodiment, the fusion protein has a structure as shown in formula (III) or (IV):

[0152] Z1-Z2-Z5(III); or

[0153] Z1-Z4-Z5(IV);

[0154] In the formula,

[0155] Z1 is selected from the following group: location sequence, connection sequence, tag sequence, or a combination thereof;

[0156] Z2 is APEX2; APEX2 has the amino acid sequence shown in SEQ ID NO:1;

[0157] Z4 is a monomeric D-amino acid oxidase; the monomeric D-amino acid oxidase has the amino acid sequence shown in SEQ ID NO:4;

[0158] Z5 is selected from the following group: positioning sequences, connection sequences, or combinations thereof;

[0159] "-" indicates a peptide bond that connects the sequences mentioned above.

[0160] In another preferred embodiment, in the structure shown in formula (III) or (IV), the positioning sequence is selected from the group consisting of amino acid sequences such as those shown in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, or combinations thereof.

[0161] In another preferred embodiment, in the structure shown in formula (III) or (IV), the linker sequence is selected from the group consisting of the amino acid sequence shown in SEQ ID NO:21, the amino acid sequence shown in SEQ ID NO:22, or a combination thereof.

[0162] In another preferred embodiment, in the structure shown in formula (III) or (IV), the tag sequence is selected from the group consisting of amino acid sequences such as those shown in SEQ ID NO:15, amino acid sequences such as those shown in SEQ ID NO:16, or combinations thereof.

[0163] In another preferred embodiment, the fusion protein has a structure as shown in formula (III):

[0164] Z1-Z2-Z5(III);

[0165] In the formula,

[0166] Z1 consists of the location sequence, label sequence, and connection sequence;

[0167] Z2 is APEX2; APEX2 has the amino acid sequence shown in SEQ ID NO:1;

[0168] Z5 consists of the connection sequence and the positioning sequence;

[0169] "-" indicates a peptide bond that connects the above sequences;

[0170] In Z1, the positioning sequence has an amino acid sequence as shown in SEQ ID NO:29;

[0171] The tag sequence has an amino acid sequence as shown in SEQ ID NO:15;

[0172] The linker sequence has an amino acid sequence as shown in SEQ ID NO:21;

[0173] In Z5, the linker sequence has an amino acid sequence as shown in SEQ ID NO:22;

[0174] The positioning sequence has an amino acid sequence as shown in SEQ ID NO:30.

[0175] In another preferred embodiment, the fusion protein has a structure as shown in formula (III):

[0176] Z1-Z2-Z5(III);

[0177] In the formula,

[0178] Z1 consists of the location sequence, label sequence, and connection sequence;

[0179] Z2 is APEX2; APEX2 has the amino acid sequence shown in SEQ ID NO:1;

[0180] Z5 consists of the connection sequence and the positioning sequence;

[0181] "-" indicates a peptide bond that connects the above sequences;

[0182] In Z1, the positioning sequence has an amino acid sequence as shown in SEQ ID NO:29;

[0183] The tag sequence has an amino acid sequence as shown in SEQ ID NO:15;

[0184] The linker sequence has an amino acid sequence as shown in SEQ ID NO:21;

[0185] In Z5, the linker sequence has an amino acid sequence as shown in SEQ ID NO:18;

[0186] The positioning sequence has an amino acid sequence as shown in SEQ ID NO:31.

[0187] In another preferred embodiment, the fusion protein has a structure as shown in formula (IV):

[0188] Z1-Z4-Z5(IV);

[0189] In the formula,

[0190] Z1 is the label sequence;

[0191] Z4 is a monomeric D-amino acid oxidase; the monomeric D-amino acid oxidase has the amino acid sequence shown in SEQ ID NO:4;

[0192] Z5 is the positioning sequence;

[0193] "-" indicates a peptide bond that connects the above sequences;

[0194] In Z1, the tag sequence has an amino acid sequence as shown in SEQ ID NO:16;

[0195] In Z5, the positioning sequence has an amino acid sequence as shown in SEQ ID NO:32.

[0196] In another preferred embodiment, the fusion protein is selected from the group consisting of:

[0197] (1) The amino acid sequence as shown in SEQ ID NO:11;

[0198] (2) The amino acid sequence as shown in SEQ ID NO:12;

[0199] (3) The amino acid sequence shown in SEQ ID NO:13.

[0200] In another preferred embodiment, a fusion protein having the structure shown in Formula (III) is used in combination with a fusion protein having the structure shown in Formula (IV).

[0201] In another preferred embodiment, the fusion protein having the structure shown in Formula (III) and the fusion protein having the structure shown in Formula (IV) are located on the same cell.

[0202] In another preferred embodiment, the fusion protein having the structure shown in Formula (III) and the fusion protein having the structure shown in Formula (IV) are located on different cells.

[0203] In a second aspect, the present invention provides a monomeric D-amino acid oxidase for constructing the fusion protein described in the first aspect of the present invention, the monomeric D-amino acid oxidase comprising or consisting of an amino acid sequence as shown in SEQ ID NO:4.

[0204] In another preferred embodiment, the D-amino acid oxidase comprises or consists of an amino acid sequence having at least 80%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4.

[0205] In another preferred embodiment, the monomeric D-amino acid oxidase has an amino acid sequence as shown in SEQ ID NO:4.

[0206] In another preferred embodiment, the monomeric D-amino acid oxidase is derived from wild-type D-amino acid oxidase or dimerized D-amino acid oxidase.

[0207] In another preferred embodiment, the wild-type D-amino acid oxidase or dimerized D-amino acid oxidase is derived from fungi.

[0208] In another preferred embodiment, the wild-type D-amino acid oxidase or dimerized D-amino acid oxidase is derived from Rhodotorula gracilis.

[0209] In another preferred embodiment, the activity of the fusion protein composed of the monomeric D-amino acid oxidase is significantly enhanced.

[0210] In another preferred embodiment, "significantly improved" means that, compared with the activity A0 of the fusion protein composed of the dimerized D-amino acid oxidase, the activity A1 of the fusion protein composed of the monomeric D-amino acid oxidase satisfies A1 / A0≥120%, such as ≥125%.

[0211] A third aspect of the present invention provides a polynucleotide encoding the fusion protein described in the first aspect of the present invention.

[0212] In another preferred embodiment, the polynucleotide comprises or consists of a nucleotide sequence having at least 80%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:37.

[0213] In another preferred embodiment, the polynucleotide comprises or consists of a nucleotide sequence having at least 80%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:38.

[0214] In another preferred embodiment, the polynucleotide comprises or consists of a nucleotide sequence having at least 80%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:39.

[0215] In another preferred embodiment, the polynucleotide comprises or consists of a nucleotide sequence having at least 80%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:40.

[0216] In another preferred embodiment, the polynucleotide comprises or consists of a nucleotide sequence having at least 80%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:41.

[0217] In another preferred embodiment, the polynucleotide comprises or consists of a nucleotide sequence having at least 80%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:42.

[0218] In another preferred embodiment, the polynucleotide comprises or consists of a nucleotide sequence having at least 80%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:43.

[0219] In another preferred embodiment, the polynucleotide comprises or consists of a nucleotide sequence having at least 80%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:44.

[0220] In another preferred embodiment, the polynucleotide comprises or consists of a nucleotide sequence having at least 80%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:45.

[0221] In a fourth aspect, the present invention provides a carrier containing the polynucleotide described in the third aspect of the present invention.

[0222] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0223] In another preferred embodiment, the carrier is an expression carrier.

[0224] In another preferred embodiment, the vector is a eukaryotic expression vector.

[0225] In a fifth aspect, the present invention provides a cell line expressing the protein described in the first aspect of the present invention, having integrated the polynucleotide described in the third aspect of the present invention into its genome, or containing the vector described in the fourth aspect of the present invention.

[0226] In another preferred embodiment, the cell line is derived from: humans, non-human mammals, fish, nematodes, or insects.

[0227] In another preferred embodiment, the non-human mammal includes rodents.

[0228] In another preferred embodiment, the rodents include mice and rats.

[0229] In another preferred embodiment, the insect includes: the black-bellied fruit fly.

[0230] In another preferred embodiment, the nematode includes: Caenorhabditis elegans.

[0231] In another preferred embodiment, the fish includes zebrafish.

[0232] In another preferred embodiment, the cell line includes: embryonic kidney cells, tumor cells, nerve cells, and muscle cells.

[0233] A sixth aspect of the present invention provides a method for performing proximity labeling in vivo, the method comprising the steps of:

[0234] (S1) Provide an organism containing the fusion protein described in the first aspect of the present invention;

[0235] (S2) Delivering a D-amino acid substrate and a labeled probe substrate carrying a reactive group into the organism for proximity labeling;

[0236] Specifically, the hydrogen peroxide generation module in the fusion protein converts the D-amino acid substrate into hydrogen peroxide. The hydrogen peroxide activates the hydrogen peroxide utilization module in the fusion protein. Under the catalysis of the activated hydrogen peroxide utilization module, the labeled probe substrate is covalently linked to the target through its carried reactive groups, thereby labeling the labeled probe on the target and obtaining a target labeled with the labeled probe.

[0237] In another preferred embodiment, the reactive group is selected from the group consisting of phenolic group, aniline group, phenol derivatives, aniline derivatives, or combinations thereof.

[0238] In another preferred embodiment, the organism includes: a human, a non-human mammal, a fish, a nematode, or an insect.

[0239] In another preferred embodiment, the non-human mammal includes rodents.

[0240] In another preferred embodiment, the rodents include mice and rats.

[0241] In another preferred embodiment, the insect includes: the black-bellied fruit fly.

[0242] In another preferred embodiment, the nematode includes: Caenorhabditis elegans.

[0243] In another preferred embodiment, the fish includes zebrafish.

[0244] In another preferred embodiment, the fusion protein is expressed in the cells / tissues / organs of the organism.

[0245] In another preferred embodiment, the fusion protein is expressed in cells / tissues / organs including: muscle, tumor cells, neurons, and tumor cells.

[0246] In another preferred embodiment, the fusion protein is expressed in a cellular structure.

[0247] In another preferred embodiment, the fusion protein is expressed in a cellular structure selected from the group consisting of: the cell nucleus, cytoplasm, mitochondrial matrix, cell membrane, or a combination thereof.

[0248] In another preferred embodiment, the fusion protein is selected from the group consisting of:

[0249] (1) It has the amino acid sequence shown in SEQ ID NO:5;

[0250] (2) It has the amino acid sequence shown in SEQ ID NO:6;

[0251] (3) It has the amino acid sequence shown in SEQ ID NO:7;

[0252] (4) It has the amino acid sequence shown in SEQ ID NO:8;

[0253] (5) Has an amino acid sequence as shown in SEQ ID NO:9;

[0254] (6) Has an amino acid sequence as shown in SEQ ID NO:10;

[0255] (7) Has the amino acid sequence shown in SEQ ID NO:11;

[0256] (8) Has an amino acid sequence as shown in SEQ ID NO:12;

[0257] (9) Has the amino acid sequence shown in SEQ ID NO:13;

[0258] Or a combination thereof.

[0259] In another preferred embodiment, the organism comprises: (i) having an amino acid sequence as shown in SEQ ID NO:11, and / or having an amino acid sequence as shown in SEQ ID NO:12; and

[0260] (ii) Having an amino acid sequence as shown in SEQ ID NO:13.

[0261] In another preferred embodiment, the D-amino acid substrate is selected from the group consisting of D-methionine (D-Met), D-phenylalanine (D-Phe), D-alanine (D-Alanine), D-norleucine (D-Norvaline), D-proline (D-Proline), D-serine (D-Serine), D-valine (D-Valine), D-leucine (D-Leucine), D-cysteine ​​(D-Cysteine), D-tryptophan (D-Tryptophan), or combinations thereof.

[0262] In another preferred embodiment, the D-amino acid substrate is selected from the group consisting of D-Met, D-Phe, or combinations thereof.

[0263] In another preferred embodiment, the D-amino acid substrate is D-Met.

[0264] In another preferred embodiment, the concentration of the D-amino acid substrate is ≥50mM, such as 70mM or 100mM.

[0265] In another preferred embodiment, the labeled probe substrate carrying a reactive group includes: a labeled probe substrate carrying a phenol group, a labeled probe substrate carrying an aniline group, or a combination thereof.

[0266] In another preferred embodiment, the labeled probe substrate carrying a phenol group is selected from the group consisting of: fluorescein (FITC)-phenol, biotin-phenol (BP), biotin-xx-phenol (BxxP), alkynyl-phenol (AP), azide-phenol (NP), biomolecules containing a phenol group, or combinations thereof.

[0267] In another preferred embodiment, the substrate of the labeled probe carrying a phenol group is alkynyl-phenol.

[0268] In another preferred embodiment, the labeled probe substrate carrying an aniline group includes: biotin-aniline (BA) and a biomacromolecule containing an aniline group.

[0269] In another preferred embodiment, the hydrogen peroxide generating module is a D-amino acid oxidase.

[0270] In another preferred embodiment, the hydrogen peroxide generating module is a monomeric D-amino acid oxidase.

[0271] In another preferred embodiment, the hydrogen peroxide utilization module is a peroxidase.

[0272] In another preferred embodiment, the hydrogen peroxide utilization module is APEX2.

[0273] In another preferred embodiment, the delivery method includes: local, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal delivery.

[0274] In another preferred embodiment, the delivery method includes injection or infusion.

[0275] In another preferred embodiment, the delivery method is selected from the group consisting of: intratumoral injection, intravenous injection, intradermal injection, intraperitoneal injection, transdermal delivery, or combinations thereof.

[0276] In another preferred embodiment, the time for performing the proximity marking is ≥40 seconds, such as ≥10 minutes, ≥30 minutes, ≥60 minutes, or ≥180 minutes.

[0277] In another preferred embodiment, the time for performing the proximity marking is ≥30 minutes.

[0278] In another preferred embodiment, the target includes: cells, proteins, and RNA.

[0279] In another preferred embodiment, the cells are tumor cells.

[0280] In another preferred embodiment, the method further includes the step of: (S3) incubating a reactive probe targeting the labeled probe with the labeled probe to perform a click chemical reaction, thereby performing click chemical modification on the labeled probe target;

[0281] In this process, the reaction probe undergoes a click chemical reaction with the labeling probe, thereby connecting the reaction probe to the target labeled with the labeling probe, resulting in a target containing click chemical modification.

[0282] In another preferred embodiment, the reaction probe comprises a biomacromolecule containing azide (azide).

[0283] In another preferred embodiment, the azide-containing biomacromolecules include: azide-biotin, azide-Cy3, and azide-fluorescein.

[0284] In another preferred embodiment, the method further includes the step of: (S4) incubating the substrate for enriching the click-chemically modified target with the click-chemically modified target to perform immunoblotting analysis and / or enrichment.

[0285] In another preferred embodiment, the substrate for enriching the target of click chemical modification includes: a biomacromolecule containing streptavidin, a solid matrix containing streptavidin, and a probe containing fluorescein.

[0286] In another preferred embodiment, the solid matrix containing streptavidin is streptavidin magnetic beads.

[0287] In another preferred embodiment, the biomacromolecule containing streptavidin or the solid matrix containing streptavidin is used to enrich a biotin-modified target.

[0288] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0289] A seventh aspect of the present invention provides a method for controlling the electrical activity of nerve cells, the method comprising the steps of:

[0290] (Z1) Provides a nerve cell, wherein the cell membrane of the nerve cell expresses the fusion protein described in the first aspect of the present invention;

[0291] (Z2) The D-amino acid substrate and the electrofunctional substrate are incubated with the nerve cells to cause the electrofunctional substrate to undergo a polymerization reaction to form an electrofunctional polymer, thereby controlling the electroactivity of the nerve cells;

[0292] The hydrogen peroxide generation module in the fusion protein converts the D-amino acid substrate into hydrogen peroxide, which activates the hydrogen peroxide utilization module in the fusion protein. The activated hydrogen peroxide utilization module catalyzes the polymerization reaction of the electrofunctional substrate to obtain an electrofunctional polymer.

[0293] If the electrofunctional polymer is a conductive polymer, the cell membrane capacitance of the nerve cell decreases and the electrical activity of the nerve cell increases; if the electrofunctional polymer is an insulating polymer, the cell membrane capacitance of the nerve cell increases and the electrical activity of the nerve cell decreases.

[0294] In another preferred embodiment, the nerve cells are derived from human or non-human mammals.

[0295] In another preferred embodiment, the non-human mammal includes rodents.

[0296] In another preferred embodiment, the rodents include mice and rats.

[0297] In another preferred embodiment, the fusion protein has an amino acid sequence as shown in SEQ ID NO:10.

[0298] In another preferred embodiment, the D-amino acid substrate includes: D-alanine, D-methionine, D-phenylalanine, D-leucine, D-valine, D-proline, D-serine, D-valine, D-leucine, D-cysteine, D-tryptophan, or combinations thereof.

[0299] In another preferred embodiment, the electrofunctional substrate includes aniline and diaminobenzidine (DAB).

[0300] In another preferred embodiment, the conductive polymer comprises polyaniline (PANI).

[0301] In another preferred embodiment, the insulating polymer comprises polydiaminobenzidine (PDAB).

[0302] In another preferred embodiment, the hydrogen peroxide generating module is a D-amino acid oxidase.

[0303] In another preferred embodiment, the hydrogen peroxide generating module is a monomeric D-amino acid oxidase.

[0304] In another preferred embodiment, the hydrogen peroxide utilization module is a peroxidase.

[0305] In another preferred embodiment, the hydrogen peroxide utilization module is APEX2.

[0306] In another preferred embodiment, the method is used for: neural repair, brain-computer interface technology, and fabrication of neural prostheses.

[0307] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0308] An eighth aspect of the present invention provides a method for studying RNA structure, the method comprising the steps of:

[0309] (Y1) Provides an RNA located in an organelle / cell / tissue / organ containing the fusion protein of the first aspect of the present invention;

[0310] (Y2) is selected from the following group:

[0311] (y2.1) Incubate the ribonucleotide analog with the organelle / cell / tissue / organ to make the RNA contain the ribonucleotide analog, thereby obtaining RNA containing the ribonucleotide analog;

[0312] (y2.2) The organelles / cells / tissues / organs are not incubated with ribonucleotide analogs;

[0313] (Y3) Incubate the D-amino acid substrate and the labeled probe substrate carrying the reactive group with the organelle / cell / tissue / organ to perform proximity labeling;

[0314] The hydrogen peroxide generation module in the fusion protein converts the D-amino acid substrate into hydrogen peroxide. The hydrogen peroxide activates the hydrogen peroxide utilization module in the fusion protein. Under the catalysis of the activated hydrogen peroxide utilization module, the labeled probe substrate covalently links with the ribonucleotide analogs and / or ribonucleotides in the RNA containing ribonucleotide analogs obtained in (y1) that have not formed hydrogen bonds, or with the ribonucleotides in the RNA obtained in (y2) that have not formed hydrogen bonds, thereby labeling the labeled probe on the ribonucleotide analogs and / or ribonucleotides in the RNA containing ribonucleotide analogs that have not formed hydrogen bonds, or on the ribonucleotides in the RNA that have not formed hydrogen bonds, to obtain RNA labeled with the labeled probe.

[0315] (Y4) Extract the RNA labeled with the labeled probe from the organelle / cell / tissue / organ;

[0316] (Y5) is selected from the following group:

[0317] (y5.1) When the RNA obtained in step (y4) does not contain biotin labeling, the RNA is biotin-modified, specifically including the steps of: incubating the biotin-containing reaction probe targeting the labeling probe with the RNA labeled with the labeling probe to perform a click chemical reaction, thereby biotin-modifying the RNA labeled with the labeling probe.

[0318] In this process, the biotin-containing reaction probe undergoes a click chemical reaction with the labeling probe, thereby linking biotin to the RNA labeled with the labeling probe to obtain biotin-modified RNA.

[0319] (y5.2) When the RNA obtained in step (y4) contains biotin labeling, biotin modification is not performed;

[0320] (Y6) Enrich the RNA obtained in step (Y5);

[0321] (Y7) The RNA obtained in step (Y6) is reverse transcribed to obtain cDNA;

[0322] (Y8) Sequencing the cDNA to obtain a mutation profile;

[0323] (Y9) The accessibility of the RNA structure is analyzed based on the mutation map to obtain the structure of the RNA.

[0324] In another preferred embodiment, the organelle is selected from the group consisting of mitochondria, nucleus, endoplasmic reticulum, cytoplasmic matrix, or cell membrane.

[0325] In another preferred embodiment, the organelle is a mitochondria.

[0326] In another preferred embodiment, the cells comprise cells expressing mitochondrial PRADA (MITO-PRADA).

[0327] In another preferred embodiment, the cells express the amino acid sequence shown in SEQ ID NO:8.

[0328] In another preferred embodiment, the tissue includes a tumor.

[0329] In another preferred embodiment, the ribonucleotide is guanine (G).

[0330] In another preferred embodiment, the ribonucleotide analogues include: 4-thiouridine (4sU) and 6-thioguanine (6sG).

[0331] In another preferred embodiment, in step (y2.2), the incubation time is 5 ± 2 hours.

[0332] In another preferred embodiment, the D-amino acid substrate includes: D-phenylalanine, D-alanine, D-methionine, D-leucine, D-valine, D-proline, D-serine, D-valine, D-leucine, D-cysteine, D-tryptophan, or combinations thereof.

[0333] In another preferred embodiment, the D-amino acid substrate is D-phenylalanine.

[0334] In another preferred embodiment, the reactive group is selected from the group consisting of phenolic group, aniline group, phenol derivatives, aniline derivatives, or combinations thereof.

[0335] In another preferred embodiment, the labeled probe substrate carrying a reactive group includes: a labeled probe substrate carrying a phenol group, a labeled probe substrate carrying an aniline group, or a combination thereof.

[0336] In another preferred embodiment, the labeled probe substrate carrying a phenol group is selected from the group consisting of: fluorescein (FITC)-phenol, biotin-phenol (BP), biotin-xx-phenol (BxxP), alkynyl-phenol (AP), azide-phenol (NP), biomolecules containing a phenol group, or combinations thereof.

[0337] In another preferred embodiment, the substrate of the labeled probe carrying a phenol group is alkynyl-phenol.

[0338] In another preferred embodiment, the labeled probe substrate carrying an aniline group includes: biotin-aniline (BA) and a biomacromolecule containing an aniline group.

[0339] In another preferred embodiment, the hydrogen peroxide generating module is a D-amino acid oxidase.

[0340] In another preferred embodiment, the hydrogen peroxide generating module is a monomeric D-amino acid oxidase.

[0341] In another preferred embodiment, the hydrogen peroxide utilization module is a peroxidase.

[0342] In another preferred embodiment, the hydrogen peroxide utilization module is APEX2.

[0343] In another preferred embodiment, in step (Y3), the incubation time is ≥5 minutes.

[0344] In another preferred embodiment, the reaction probe comprises a biomacromolecule containing azide (azide).

[0345] In another preferred embodiment, the azide-containing biomacromolecules include: azide-biotin, azide-Cy3, and azide-fluorescein.

[0346] In another preferred embodiment, the azide-containing biomacromolecule is azide-biotin.

[0347] In another preferred embodiment, step (Y6) includes: incubating the substrate for enrichment with the RNA obtained in step (Y5) to enrich the RNA.

[0348] In another preferred embodiment, the substrate for enrichment includes: a biomacromolecule containing streptavidin, and a solid matrix containing streptavidin.

[0349] In another preferred embodiment, the solid matrix containing streptavidin is streptavidin magnetic beads.

[0350] In another preferred embodiment, in step (Y7), the reverse transcription is performed using reverse transcriptase.

[0351] In a ninth aspect, the present invention provides a kit comprising the fusion protein of the first aspect of the present invention, the polynucleotide of the third aspect of the present invention, the vector of the fourth aspect of the present invention, or the cell line of the fifth aspect of the present invention.

[0352] In another preferred embodiment, the kit further includes a pharmaceutically acceptable carrier, diluent, or excipient.

[0353] In another preferred embodiment, the kit also includes a label or instructions.

[0354] In another preferred embodiment, the label or instructions specify that the kit is used for: in vivo proximity labeling; proteomics studies; transcriptomics studies; RNA structure studies; and control of nerve cell electrical activity.

[0355] In a tenth aspect, the present invention provides the use of the fusion protein described in the first aspect of the present invention, the monomeric D-amino acid oxidase described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, the cell line described in the fifth aspect of the present invention, or the kit described in the ninth aspect of the present invention, the use including:

[0356] (I) Proximity marking in living organisms;

[0357] (II) Research in proteomics;

[0358] (III) Transcriptomics research;

[0359] (IV) Study of RNA structure;

[0360] (V) controls the electrical activity of nerve cells.

[0361] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0362] Figure 1 The development of monomeric D-amino acid oxidases and their activation of peroxidases were demonstrated.

[0363] Figure 1 A shows a schematic diagram of the D-amino acid oxidase-peroxidase cascade reaction. DAAO utilizes D-amino acid substrates to generate H2O2 in situ to activate the neighboring labeled enzyme APEX2.

[0364] Figure 1 B shows the evaluation of the RgDAAO-APEX2 enzyme cascade using the Amplex UltraRed assay. Fluorescence signal changes over time were monitored in the following cell groups: APEX2-RgDAAO fusion, RgDAAO-APEX2 fusion, APEX2 and RgDAAO co-transfected groups, or RgDAAO-only groups. Each group was treated with either 1 mM H2O2 or 10 mM D-alanine. Data are the mean ± standard deviation of two independent experiments.

[0365] Figure 1 C shows a comparison of the effects of H2O2 and D-alanine on cell viability. CellTiter-Glo assays were performed in cells expressing the APEX2-RgDAAO fusion, RgDAAO alone, or the empty vector. Cells were treated with a specified concentration of H2O2 or D-alanine for 20 min. Cell viability was assessed using a luminescent signal indicating cellular ATP levels. Data are presented as mean ± standard deviation from three independent experiments.

[0366] Figure 1 D shows a schematic diagram of the RgDAAO crystal structure (PDB ID: 1C0I). The selected area shows the flexible loop region at the homodimer interface, which has been removed to construct the Δ... 308-321 RgDAAO mutant.

[0367] Figure 1 E shows the loop region predicted by AlphaFold 3 ( 308-321 The DAAO dimerization state before and after deletion (position).

[0368] Figure 1 F shows the assay to validate the complementation of the splitting TurboID using monomeric RgDAAO. Left panel: The upper schematic shows that only dimerized RgDAAO can drive recombination of the N-terminal Tb(N) and C-terminal Tb(C) fragments of the fused TurboID; the lower flow cytometry analysis shows reduced RgDAAO dimerization after deletion of loops 308-321. Right panel: Fluorescence imaging of biotinylation in splitting TurboID. HEK293T cells co-transfected with the specified splitting TurboID-DAAO fusion construct were biotinylated for 1 hour. Tb(N) and Tb(C) expression were shown by anti-V5 (green) and anti-HA (gray) staining, respectively, and biotinylation was shown by streptavidin-Cy3 (red) staining. Scale bar: 10 μm.

[0369] Figure 1 G shows the results of PRADA reactivity. Left panel: PRADA reactivity fluorescence imaging results, HEK293T cells transfected with the specified construct were immunostained 30 minutes after being labeled with D-alanine and biotin-phenol. PRADA and labeling signals are shown by staining with anti-V5 (green) and streptavidin-Cy3 (red), respectively. Scale bar: 10 μm. Right panel: Quantitative results of PRADA reactivity.

[0370] Figure 1H shows a comparison of the properties of different D-amino acids as PRADA substrates. Cells expressing PRADA-NES were fluorescently imaged after 30 minutes of labeling with alkynylphenol and designated D-amino acids. PRADA and labeling signals were visualized by anti-V5 (green) staining and azide-Cy3 (red) click chemistry, respectively. Scale bar: 10 μm.

[0371] Figure 1 Image I shows the time progression of PRADA labeling. Cells expressing PRADA-NES are fluorescently imaged after labeling with alkynylphenol and D-phenylalanine at specified times. PRADA and the labeling signal are visualized via anti-V5 (green) staining and azide-Cy3 (red) click chemistry, respectively. Scale bar: 10 μm.

[0372] Figure 1 J shows fluorescence imaging of PRADA activity in different subcellular regions. HEK293T cells expressing the specified PRADA fusion variant were immunostained after labeling with D-phenylalanine and alkynylphenol. PRADA and the labeling signal were visualized by anti-V5 (green) staining and azide-Cy3 (red) click chemistry, respectively. DAPI was used as a nuclear marker, and endogenous Tom20 (Translocase of Outer Mitochondrial Membrane 20) and calreticulin (CRT) were used as mitochondrial and endoplasmic reticulum markers, respectively. Scale bar: 10 μm.

[0373] Figure 1 K shows that the APEX2 H2O2 reporter experiment demonstrated limited diffusion of H2O2 generated by PRADA. Left panel: Schematic diagram of the experimental design. Right panel: Localization and labeling activity fluorescence imaging of expressed peroxidase. Cells expressing the specified construct were labeled with alkynylphenol after treatment with D-phenylalanine for 30 min or H2O2 for 1 min. PRADA and APEX2 expression were shown by anti-V5 (green) and anti-Myc (gray) staining, respectively, and the labeling signal was shown by azide-Cy3 (red) click chemistry reaction. DAPI was used as a nuclear marker. Scale bar: 10 μm.

[0374] Figure 2 This demonstrates the versatile marking applications of PRADA in various life systems.

[0375] Figure 2 A shows a schematic diagram of PRADA-mediated protein and RNA tagging.

[0376] Figure 2B shows the results of RT-qPCR analysis of PRADA with RNA proximity labeling. Cells expressing NLS-PRADA were labeled with alkynylphenol and D-phenylalanine, and total RNA was extracted and enriched by azide-biotin click chemistry and streptavidin magnetic beads, showing specific enrichment of nuclear RNA relative to cytoplasmic RNA.

[0377] Figure 2 C shows the Western blot results of protein proximity labeling for PRADA. Cells expressing ERM-PRADA were labeled with alkynylphenol and D-phenylalanine, and the cell lysates were subjected to streptavidin-HRP detection (top) and Coomassie brilliant blue staining (bottom).

[0378] Figure 2 Image D shows the assembly of PRADA-mediated functional biomaterials in living cells. Top image: Schematic diagram of PRADA-catalyzed in-situ polymerization of small molecule precursors. Bottom left image: Bright-field imaging of cell membrane anchoring of PRADA-catalyzed polyaniline (PANI) and polydiaminobenzidine (PDAB). The reaction was completed by treating PRADA-expressing cells with 100 mM D-alanine or 1 mM H2O2 for 45 minutes. Scale bar: 10 μm. Bottom right image: Bright-field imaging of PRADA-catalyzed polymerization of diaminobenzidine (DAB) in living cells. HEK293T cells expressing PRADA were co-incubated with DAB with D-methionine or D-phenylalanine for 30 minutes. Scale bar: 10 μm.

[0379] Figure 2 E shows PRADA labeling in primary rat hippocampal neurons. Left panel: Neurons electrotransfected with the specified PRADA construct were labeled with alkynylphenol and D-phenylalanine for 30 minutes. PRADA and labeling signals were visualized by anti-V5 (green) staining and azide-Cy3 (red) click chemistry, respectively. WGA was used as a cell membrane marker. Scale bar: 10 μm. Right panel: Fluorescence imaging of subcellular localized PRADA labeling in different mouse cell lines. Demonstrates the labeling effects of different subcellular localized PRADA constructs in various cell lines. Scale bar: 10 μm.

[0380] Figure 2F shows PRADA labeling in live nematodes. Left: Schematic diagram of nematodes expressing PRADA in body wall muscle. Center: Fluorescent image showing PRADA localization and labeling activity in nematodes. Live nematodes were labeled by treatment with H2O2 for 1 min or D-amino acids for 60 min. PRADA expression is shown by GFP, and the labeling signal is detected by azide-Cy3 (red) click chemistry reaction. Scale bar: 50 μm. Right: Fluorescent imaging of PRADA labeling in live nematodes. Showing the PRADA labeling effect in nematodes at different substrate concentrations and incubation times, the labeling signal is shown by azide-Cy3 click chemistry reaction (red). Scale bar: 10 μm.

[0381] Figure 2 G and H show the Western blot results of PRADA-mediated protein labeling in nematodes. Nematodes expressing PRADA in their body wall muscle were labeled with alkynylphenol and D-phenylalanine, and the total lysate was then subjected to click chemistry reactions with either azide-biotin (G) or azide-fluorescein (H) before Western blotting. The right figure shows the relative intensities of each band, all normalized to the D-phenylalanine labeling group.

[0382] Figure 2 Figure I shows the comparative results of the effects of H₂O₂ and D-amino acids on the motility of nematodes. Live nematodes were incubated in a culture medium containing the specified reagents, and motility was monitored through experiments. “Non-paralyzed” refers to the proportion of nematodes exhibiting normal body movement. P-values ​​were obtained from the Log-rank test.

[0383] Figure 2 J shows a schematic diagram of transgenic fruit fly larvae expressing PRADA. Expression in muscle and brain tissues was driven using mef2-GAL4 and elav-GAL4, respectively.

[0384] Figure 2 K shows the PRADA labeling results in live Drosophila. Fluorescence imaging of PRADA labeling in Drosophila muscle and brain tissue. Complete third-instar larvae were labeled for 30 min in a medium containing alkynylphenol and D-methionine after expressing the designated PRADA construct. PRADA and labeling signals are visualized by anti-V5 (green) staining and azide-AF647 (red) click chemistry, respectively. DAPI was used as a nuclear marker. Scale bar: 50 μm.

[0385] Figure 2L shows the PRADA labeling results in live zebrafish. Top: Schematic diagram of zebrafish chimerically expressing PRADA in muscle cells via embryonic microinjection. Eighty hours post-fertilization, zebrafish were labeled for 3 hours in a medium containing alkynylphenol and D-phenylalanine. Bottom: Fluorescence imaging of PRADA localization and labeling activity. PRADA expression was shown by anti-V5 staining (magenta), and the labeling signal was shown by azide-Cy3 (red) click chemistry. Scale bar: 10 μm.

[0386] Figure 2 M shows the PRADA labeling results in live mice. Top: Schematic diagram of PRADA labeling in a subcutaneous xenograft model. The reaction was carried out by intratumoral injection of alkynylphenol and D-phenylalanine. Bottom: Western blot analysis of protein markers in tumor tissue expressing ERM-PRADA. Immunoblotting was performed with the specified antibody after the lysis buffer was reacted with azide-biotin click chemistry.

[0387] Figure 2 N shows the results of immunofluorescence imaging of highly efficient PRADA labeling throughout the tumor. Top: Schematic diagram of sampling in the tumor center and surrounding areas. Middle: Fluorescence imaging of tumor sections at designated locations. Bottom: Magnified view showing co-localization of PRADA expression and labeling signal at the subcellular level. PRADA and labeling signal are visualized by anti-V5 (green) staining and azide-Cy3 (red) click chemistry, respectively. Scale bar: 10 μm.

[0388] Figure 3 This demonstrates the quality control and structural analysis validation of the PRADA-MaP sequencing technology.

[0389] Figure 3 A, D, and I show the results of mutation rate analysis for four bases. The mutation rate distribution of the four bases in APEX-seq (A), 4sU pulse APEX-seq (D), and PRADA-MaP seq (I) are presented respectively. Sequencing data of labeled cells (red) are compared with those of unlabeled control cells (gray).

[0390] Figure 3 B, E, and J show the quantitative results of uracil reactivity in the single- and double-stranded regions of COX1 mRNA. The differences in U reactivity between the single-stranded (SS) and double-stranded (DS) regions of COX1 mRNA were compared in APEX-seq (B), 4sU pulse APEX-seq (E), and PRADA-MaP seq (J) data.

[0391] Figure 3C, F, and KM show the results of RNA structure prediction efficacy assessment. ROC curves compare the structure prediction efficacy of published COX1 mRNA structures (mitoDMS-MaP seq data) with: APEX-seq U reactivity (C), 4sU pulse APEX-seq U reactivity (F), PRADA-MaP seq U reactivity (K), G reactivity (L), and U+G combined reactivity (M).

[0392] Figure 3 G shows the validation results of PRADA labeling of 4sU pulsar RNA. Cells expressing MTS-PRADA were pretreated with 4sU and then labeled with alkynylphenol and D-phenylalanine. Total RNA was enriched by azide-biotin click chemistry and streptavidin magnetic beads, showing specific enrichment of mitochondrial matrix RNA.

[0393] Figure 3 H shows the results of the sequencing data reproducibility analysis. The left panel shows the enrichment folds between the marker group and the control group; the right panel shows the correlations between biological replicates.

[0394] Figure 3 N represents the reproducibility of PRADA reactivity across the whole mitochondrial transcriptome. G / U reactivity across the whole mitochondrial transcriptome was calculated using a 10-nucleotide sliding window, with a correlation coefficient r = 0.87 between biological replicates.

[0395] Figure 3 O displays a circular reactivity map of the mitochondrial genome. It shows the PRADA reactivity profiles of U and G bases at various nucleotide sites throughout the whole mitochondrial transcriptome.

[0396] Figure 4 This demonstrates the mapping of subcellular RNA structures in living cells using PRADA-MaP sequencing technology.

[0397] Figure 4 A shows the PRADA-MaP sequencing technology roadmap. Cells expressing PRADA are first labeled with 4sU metabolism, followed by PRADA labeling, RNA extraction, streptavidin enrichment, library construction, deep sequencing, and bioinformatics analysis.

[0398] Figure 4 B shows a volcano plot of RNA sequencing results after streptavidin enrichment. Thirteen significantly enriched mitochondrial mRNAs (red) and thirteen significantly enriched mitochondrial tRNAs (yellow) are highlighted. Cells without D-amino acid treatment serve as unlabeled negative controls.

[0399] Figure 4 C shows a mitochondrial genome circular map illustrating the unfiltered nucleotide read count distribution. Mitochondrial rRNA was removed during library construction.

[0400] Figure 4 D shows a mitochondrial genome circular plot illustrating the mutation rates of guanine (G) and uracil (U) throughout the mitochondrial transcriptome. The red and gray lines represent the mutation frequencies at each nucleotide site in the PRADA-labeled group and the unlabeled control group, respectively.

[0401] Figure 4 E shows the inter-experimental correlation analysis results for PRADA reactivity, which showed high reproducibility (correlation coefficient r = 0.96). G / U reactivity was calculated using a sliding window of 10 nucleotides on the mitochondrial transcript COX1. Rep1 and Rep2 represent biological replicates 1 and 2, respectively.

[0402] Figure 4 F shows the ROC curves comparing published mitochondrial mRNA structure data (from mitoDMS-MaP sequencing data) with the PRADA reactivity data from this study. The AUROC values ​​for all transcripts predicted based on PRADA reactivity ranged from 0.74 to 0.84.

[0403] Figure 4 G shows a representative mitochondrial RNA structural model of the PRADA-MaP sequencing reactivity profile. Nucleotides are colored according to normalized reactivity (calculated based on the G / U mutation rates of the PRADA-marked group and the control group). The figure shows partial segments of COX1, COX2, and ND1 mRNA as examples.

[0404] Figure 5 This demonstrates the ability to simultaneously map the mitochondrial proteome, transcriptome, and RNA structure in vivo using PRADA.

[0405] Figure 5 A is a schematic diagram of the experimental design: PRADA labeling, enrichment, and multi-omics analysis of mitochondrial proteins and RNA were performed simultaneously in a xenograft tumor model.

[0406] Figure 5 B shows the results of streptavidin-HRP immunoblotting analysis of MITO-PRADA-labeled proteins. Tumor lysates were subjected to an azide-biotin click chemistry reaction, and the sample volume was quantified using the BCA method to ensure consistency before immunoblotting detection.

[0407] Figure 5 C shows the volcano plot illustrating the results of MITO-PRADA-tagged proteomics analysis based on mass spectrometry.

[0408] Figure 5 D shows the gene ontology cellular component analysis of the MITO-PRADA proteomic data.

[0409] Figure 5 E shows the RT-qPCR analysis results of MITO-PRADA-labeled RNA. Total RNA enrichment via azide-biotin click chemistry and streptavidin magnetic beads showed specific enrichment of mitochondrial matrix RNA relative to cytoplasmic RNA.

[0410] Figure 5 F shows the volcano plot illustrating the sequencing analysis results of PRADA-tagged RNA. Significantly enriched mitochondrial mRNA (red) and tRNA (yellow) are highlighted.

[0411] Figure 5 G shows a volcano plot comparing the differences in total RNA expression between xenograft tumors (in vivo) and HEK293T cells in culture dishes (in vitro). Genes that are significantly upregulated (red) or downregulated (cyan) are specifically marked. Mitochondrial mRNA (orange) expression did not show significant changes.

[0412] Figure 5 H shows the gene ontology biological process analysis of differentially expressed genes in (G).

[0413] Figure 5 I shows that gene set enrichment analysis revealed a significant upregulation of mitochondrial translation-related genes under in vivo conditions.

[0414] Figure 5 J shows changes in the expression levels of mitochondrial translation-related genes (p<0.05). Mitochondrial ribosomal proteins (orange) and LRPPRC / SLIRP (red) are specifically labeled.

[0415] Figure 5 K shows a heatmap comparing the AUROC values ​​of in vivo and in vitro MITO-PRADA-MaP sequencing data (based on published mitochondrial mRNA structures).

[0416] Figure 5 L shows the association analysis between the structural differences of MT-ND5 mRNA in vitro and in vivo and ribosome / LRPPRC binding. Upper line graph: ND5 mRNA homogenization reactivity data from in vitro and in vivo PRADA-MaP sequencing data. Upper histogram (bottom): Ribosome / diribosome analysis results of mitochondria purified from HEK293T cells. Middle heatmap (bottom): Structural correlation between LRPPRC knockout and wild-type HEK293T cell purified mitochondria DMS-MaP sequencing. Lower histogram (bottom): PAR-CLIP data showing the LRPPRC binding site on ND5 mRNA. Cyan rectangle (below the x-axis): Transmembrane domain encoded by ND5 mRNA.

[0417] Figure 5M shows a Western blot analysis of the expression levels of specific mitochondrial proteins in vivo and in vitro.

[0418] Figure 6 A shows a schematic diagram of cell membrane protein labeling using PRADA.

[0419] Figure 6 B. Flow cytometry was used to detect PRADA cis labeling on the cell membrane. APEX2 and Δ 308-321 RgDAAO is simultaneously expressed in the same cell, and cell membrane protein labeling can be achieved by simultaneously administering D-Met (DM) and BxxP to the cell. The labeling signal is characterized by SA-FITC.

[0420] Figure 6 C shows the expression and labeling effect of PRADA in the mouse colon cancer cell line CT26. Scale bar: 10 μm.

[0421] Figure 6 D shows the Western blot results of protein proximity labeling with tmPRADA. After labeling with CT26 cell lines expressing tmPRADA, the cell lysates were subjected to streptavidin-HRP detection (top), expression detection (middle), and Coomassie brilliant blue staining (bottom).

[0422] Figure 6 E-mass spectrometry results showed the protein enrichment of the CT26-tmPRADA cell line after DM labeling.

[0423] Figure 6 F shows the GOCC functional analysis results of the enriched proteins in the CT26-tmPRADA cell line after DM labeling.

[0424] Figure 6 The GVenn plot shows the relationship between the proteins enriched after tmPRADA and traditional APEX2 labeling. tmPRADA labeled 166 more proteins than APEX2.

[0425] Figure 6 H shows APEX2 and DAAO transfected into different cells, co-cultured, and then labeled. A strong labeling signal was observed at the interface between the two cell types. Scale bar: 10 μm.

[0426] Figure 7 A shows the flow cytometry results of PRADA markers (using APEX2 and RgDAAO) on the cell membrane.

[0427] Figure 7 B shows the expression and labeling effect of PRADA in the mouse colon cancer cell line MC38. Scale bar: 10 μm.

[0428] Figure 7 The C-heatmap shows the differences in mass spectrometry results between the labeled and control groups. "Ctrl" represents the CT26-tmPRADA cell line without any additional compounds.

[0429] Figure 7 D-mass spectrometry results showed the protein enrichment of the CT26-tmPRADA cell line after H2O2 labeling.

[0430] Figure 7 E shows the GOCC functional analysis results of the enriched proteins in the CT26-tmPRADA cell line after H2O2 labeling.

[0431] Figure 8 Immunofluorescence results of HEK293T cell lines expressing MTS-PRADA labeled with different types of probes are shown. DAPI represents nuclear staining, V5-AF488 characterizes MTS-PRADA expression, and Cy3 represents the labeling signal. Scale bar: 10 μm. Detailed Implementation

[0432] Through extensive and in-depth experiments, the inventors have, for the first time, constructed a monomeric D-amino acid oxidase and, based on this, proposed a non-toxic proximity labeling method called PRADA (Peroxidase Reactions Activated by D-aminoAcids). By linking the monomeric D-amino acid oxidase to APEX2, or constructing the two in adjacent spatial locations, the PRADA technology enables enzymatic cascade reactions to generate H2O2 in situ in vivo, especially in vivo, avoiding the use of exogenous H2O2 and significantly reducing toxicity. Furthermore, the D-amino acid substrates used have minimal impact on biological physiology, ensuring experimental results are closer to physiological states, improving biocompatibility and data reliability, while preserving the chemical multifunctionality of peroxidases. PRADA can label not only proteins and RNA but also support the assembly of functional polymers, meeting multidimensional analytical needs and broadening its application range. This technology can be directly applied to in vivo models (Drosophila, nematodes, zebrafish, and mice) under physiological conditions, without relying on mutants or ex vivo tissues, simplifying experimental procedures, reducing costs, and improving efficiency. Furthermore, the PRADA-MaP seq technology based on PRADA has for the first time achieved the resolution of subcellular compartment-specific RNA structures in vivo, overcoming the limitations of traditional methods in spatial resolution. The main purpose of PRADA is to conduct in-depth research on the spatial organization and function of biomolecules through spatially specific molecular (including protein and RNA) labeling and RNA structure resolution in vivo. This invention is based on this foundation.

[0433] It should be understood that the specific methods and experimental conditions of the invention described below in varying degrees of detail are intended to provide a substantive understanding of the invention. Definitions of certain terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0434] the term

[0435] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value, every tenth of every intermediate integer of the value, any other intermediate value between the upper and lower limits of the range, and any other intermediate value within the specified range are included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered within the scope of this invention, but are subject to any express exclusions within the specified range. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0436] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently made of” or “made of”.

[0437] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0438] As used in this article, the term "significant" means that, in a hypothesis test, the observed effect (such as the difference between the experimental and control groups) is unlikely to be caused solely by random error. A hypothesis test includes: the null hypothesis (H0), which assumes that the observed effect does not exist (such as no difference between the experimental and control groups); the p-value, which is the probability of observing the current or more extreme effect when H0 is true; and the significance threshold (α). The significance threshold is typically used to determine whether a hypothesis test is significant. Generally, the significance threshold is 0.05. If the p-value ≤ α, then H0 is rejected, meaning the observed effect exists, and the result is called "significant."

[0439] As used in this article, the term "click chemistry" refers to a class of efficient, rapid, and highly selective chemical reactions that quickly connect small molecular modules through simple chemical reactions to form complex molecules with specific functions.

[0440] Monomeric D-amino acid oxidase

[0441] The present invention provides a monomeric D-amino acid oxidase, wherein the monomeric D-amino acid oxidase comprises or is composed of an amino acid sequence as shown in SEQ ID NO:4.

[0442] Preferably, the D-amino acid oxidase consists of an amino acid sequence that has at least 80%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:4.

[0443] Preferably, the monomeric D-amino acid oxidase has the amino acid sequence shown in SEQ ID NO:4.

[0444] Preferably, the monomeric D-amino acid oxidase is derived from wild-type D-amino acid oxidase or dimerized D-amino acid oxidase.

[0445] As used herein, the term “derived from” a region of a reference amino acid sequence includes an amino acid sequence that has at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference sequence.

[0446] In this invention, the monomeric D-amino acid oxidase is derived from wild-type D-amino acid oxidase or dimerized D-amino acid oxidase (dimer). The wild-type D-amino acid oxidase has the amino acid sequence shown in SEQ ID NO:2; the dimerized D-amino acid oxidase (dimer) refers to a D-amino acid oxidase formed by the dimerization of two wild-type D-amino acid oxidases. Based on the wild-type D-amino acid oxidase sequence, the monomeric D-amino acid oxidase is constructed by deleting a flexible loop region far from the active site in the dimer. The amino acid sequence of the flexible loop region is shown in SEQ ID NO:3.

[0447] PRADA

[0448] As used herein, the term "PRADA" refers to D-amino acid-activated peroxidase reactions, and may also refer to proximity labeling techniques or fusion proteins using PRADA.

[0449] Specifically, this invention first engineered a monomeric D-amino acid oxidase, solving the problem of wild-type D-amino acid oxidase dimerization affecting labeling efficiency. Subsequently, the monomeric D-amino acid oxidase was combined with peroxidase APEX2 to construct a fusion protein, and further, a fusion protein capable of localizing to different subcellular structures was constructed; or the monomeric D-amino acid oxidase and peroxidase APEX2 were constructed in spatially adjacent regions to achieve proximity labeling of RNA and proteins in different subcellular structures.

[0450] Specifically, the monomeric D-amino acid oxidase uses a D-amino acid substrate to convert it into hydrogen peroxide; the hydrogen peroxide activates peroxidase APEX2; the activated APEX2 uses a labeled probe to achieve proximity labeling of RNA and protein in vivo.

[0451] Those skilled in the art are familiar with methods and techniques for constructing fusion proteins, including those capable of localizing to different subcellular structures. Generally, fusion proteins may include a linker sequence and a tag sequence; the linker sequence is used to connect the various components of the fusion protein, such as linking monomeric D-amino acid oxidase and peroxidase APEX2; the tag sequence is used for protein detection, tracing, purification, etc. The linker sequence and tag sequence are typically short sequences with small molecular weights, and their presence or location has no effect on the functional components of the fusion protein. Therefore, the linker sequence and tag sequence can be located at the C-terminus or N-terminus of each functional component, such as at the C-terminus or N-terminus of monomeric D-amino acid oxidase, or at the C-terminus or N-terminus of APEX2.

[0452] Preferably, the linker sequence is a flexible linker sequence. Preferably, the linker sequence consists of 1-30 glycine (G), and / or serine (S), and / or arginine (R), and / or asparagine (N). Preferably, the linker sequence is selected from the group consisting of amino acid sequences as shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or combinations thereof. Preferably, the tag sequence is selected from the group consisting of amino acid sequences as shown in SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or combinations thereof.

[0453] Fusion proteins capable of localizing to different subcellular structures typically include various localization sequences. Preferably, the localization sequences are selected from the group consisting of: cytoplasmic matrix localization sequences, nuclear localization sequences, nucleolar localization sequences, mitochondrial matrix localization sequences, endoplasmic reticulum outer membrane localization sequences, cell membrane localization sequences, or combinations thereof. The position of some localization sequences in the fusion protein does not affect the localization process of the fusion protein; that is, the localization sequences can be located at the very front (N-terminus), the very back (C-terminus), or at both ends of the fusion protein. However, some localization sequences must be located at the very front of the fusion protein, such as mitochondrial matrix localization sequences. Those skilled in the art are familiar with various commonly used localization sequences and the characteristics of each localization sequence, and therefore can construct fusion proteins capable of localizing to different subcellular structures as described in this invention.

[0454] It is understood that all D-amino acid substrates that D-amino acid oxidase can utilize are within the scope of protection of this invention. Preferably, the D-amino acid substrate is selected from the group consisting of: D-methionine, D-phenylalanine, D-alanine, D-leucine, D-valine, D-proline, D-serine, D-valine, D-leucine, D-cysteine, D-tryptophan, or combinations thereof. Preferably, the D-amino acid substrate is D-methionine. Preferably, the D-amino acid substrate is D-phenylalanine.

[0455] The labeled probe carries a reactive group, which, under the catalysis of peroxidase, is converted into a reactive intermediate, thereby undergoing a proximity reaction with the target and attaching the labeled probe to the target. Preferably, the reactive group is selected from the group consisting of phenolic groups, aniline groups, phenol derivatives, aniline derivatives, or combinations thereof. Preferably, the labeled probe substrate carrying the reactive group is selected from the group consisting of labeled probe substrates carrying phenolic groups, labeled probe substrates carrying aniline groups, labeled probe substrates carrying phenol derivatives, labeled probe substrates carrying aniline derivatives, or combinations thereof.

[0456] Any compound carrying a phenol or aniline structure that possesses labeling probe properties falls within the scope of protection of this invention. The terms "phenol derivative" and "aniline derivative" refer to compounds formed by substitution at certain positions based on the phenol or aniline structure, which retain the activity and function of phenol or aniline. For example, adding a methyl group at the ortho position of phenol allows for adjacent labeling reactions.

[0457] Preferably, the labeled probe carrying a phenol group is selected from the group consisting of: fluorescein (FITC)-phenol, biotin-phenol (BP), biotin-xx-phenol (BxxP), alkynyl-phenol (AP), azide-phenol (NP), biomolecules containing a phenol group, or combinations thereof. Preferably, the substrate of the labeled probe carrying an aniline group includes biotin-aniline (BA) and biomolecules containing an aniline group.

[0458] A fusion protein composed of multiple localization sequences, monomeric D-amino acid oxidase, and peroxidase APEX2 can be localized to different subcellular structures. Preferably, the fusion protein is selected from the group consisting of: PRADA-NES, NLS-PRADA, NIK-PRADA, MITO-PRADA, EMR-PRADA, and MEM-PRADA. Proximity labeling can also be performed by expressing monomeric D-amino acid oxidase and peroxidase APEX2 separately on the cell membrane of the same cell, or by expressing them on the cell membranes of different cells and placing the two cell types in contact, as detailed in Example 9. Specifically, the fusion protein composed of the membrane localization sequence and monomeric D-amino acid oxidase positions D-amino acid oxidase on the inner side of the cell membrane, while the fusion protein composed of the membrane localization sequence and APEX2 positions APEX2 on the outer side of the cell membrane. Intracellularly, D-amino acid oxidase converts D-amino acid substrates into hydrogen peroxide. The hydrogen peroxide permeates the cell membrane and activates APEX2 outside the membrane. APEX2 then converts the reactive group into a reactive intermediate, which covalently links to the target, thereby completing the labeling process.

[0459] Nearest neighbor marker

[0460] As used herein, the terms "proximity labeling," "proximity labeling technology," and "proximity labeling method" are used interchangeably. Proximity labeling is an emerging technique that uses enzyme catalysis to generate a wide range of reaction intermediates for the labeling of nearby proteins or RNA biomolecules (such as biotinylation). This technique is currently used to study in vitro intermolecular interactions and molecular functions. However, the use of peroxidases (such as APEX2) for catalysis requires the use of highly toxic hydrogen peroxide, causing biotoxicity and making it unsuitable for in vivo applications. Furthermore, biotinylate ligases using non-toxic substrates (such as BioID) are currently limited to protein labeling and cannot label RNA or achieve other functional applications, thus limiting their application scope. In RNA structure resolution, traditional chemical probe-based techniques (such as DMS-MaPseq and SHAPE-MaP) only provide whole-cell-level structural information, lacking subcellular compartment specificity, resulting in insufficient understanding of RNA folding states. Therefore, it is necessary to develop an enzyme for in vivo proximity labeling, utilizing non-toxic substrates to achieve in situ controlled release and utilizing hydrogen peroxide to reduce biotoxicity, further expanding the application scenarios of proximity labeling.

[0461] In this invention, this broad reactivity is applied to in vivo labeling, enabling proximity labeling of RNA and proteins in various animal models, as well as subcellular RNA structure resolution.

[0462] The method of in vivo proximity labeling of the present invention

[0463] This invention provides a method for in vivo proximity labeling, the method comprising the steps of: (S1) providing a living organism containing the fusion protein of this invention; (S2) delivering a D-amino acid substrate and a labeled probe substrate carrying a reactive group into the living organism for proximity labeling; wherein, a hydrogen peroxide generation module in the fusion protein converts the D-amino acid substrate into hydrogen peroxide, the hydrogen peroxide activates a hydrogen peroxide utilization module in the fusion protein, and under the catalysis of the activated hydrogen peroxide utilization module, the labeled probe substrate covalently links to a target through its carried reactive group, thereby labeling the labeled probe on the target, resulting in a target labeled with the labeled probe.

[0464] Methods for incorporating or expressing the fusion protein described in this invention into an organism are well known to those skilled in the art, such as constructing transgenic animal individuals or delivering transgenic cells or transgenic vectors into specific tissues / organs. In this invention, transgenic nematodes were constructed to express cytoplasmic PRADA-NES in the body wall muscle; transgenic fruit fly strains were constructed using the UAS-GAL4 system to express PRADA in the nucleus, cytoplasm, or mitochondrial matrix in muscle or neurons.

[0465] Preferably, the delivery method includes: local, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal delivery. Preferably, the delivery method includes: injection or infusion. Preferably, the delivery method is selected from the group consisting of: intratumoral injection, intravenous injection, intradermal injection, intraperitoneal injection, transdermal delivery, or combinations thereof.

[0466] In this invention, the neighbor labeling reaction can be completed in a short time; the longer the reaction time, the more obvious the labeling effect. In some cases, the labeling effect can appear after 40 seconds of reaction. Preferably, the neighbor labeling time is ≥40 seconds, such as ≥10 minutes, ≥30 minutes, ≥60 minutes, or ≥180 minutes. Preferably, the neighbor labeling time is ≥30 minutes.

[0467] Preferably, the method further includes the step of: (S3) incubating the reactive probe targeting the labeled probe with the target labeled with the labeled probe to perform a click chemical reaction, thereby performing click chemical modification on the target labeled with the labeled probe; wherein, the reactive probe and the labeled probe undergo a click chemical reaction, thereby connecting the reactive probe to the target labeled with the labeled probe to obtain a target containing click chemical modification.

[0468] Preferably, the method further includes the step of: (S4) incubating the substrate for enriching the click-chemically modified target with the click-chemically modified target, thereby performing immunoblotting and enrichment of the click-chemically modified target. Preferably, the substrate probe for enriching the click-chemically modified target includes: a biomolecule containing streptavidin, a solid matrix containing streptavidin, and a probe containing fluorescein. Preferably, the solid matrix containing streptavidin is streptavidin magnetic beads. Various methods for enriching target RNA are well known to those skilled in the art; other methods capable of enriching the desired RNA are also within the scope of this invention. In this invention, after click-chemical modification with biotin, the desired RNA is enriched through the biotin-streptavidin system. RNA enrichment can also be achieved through other reaction systems, such as click-chemical modification with fluorescein.

[0469] The method for controlling the electrical activity of nerve cells of the present invention

[0470] This invention provides a method for controlling the electrical activity of nerve cells using the fusion protein described herein. The method includes the following steps: (Z1) providing a nerve cell, wherein the fusion protein described herein is expressed on the cell membrane of the nerve cell; (Z2) incubating the nerve cell with a D-amino acid substrate and an electrofunctional substrate, causing the electrofunctional substrate to undergo a polymerization reaction to form an electrofunctional polymer, thereby controlling the electrical activity of the nerve cell; wherein, a hydrogen peroxide generating module in the fusion protein converts the D-amino acid substrate into hydrogen peroxide, the hydrogen peroxide activates a hydrogen peroxide utilization module in the fusion protein, and the activated hydrogen peroxide utilization module catalyzes the polymerization reaction of the electrofunctional substrate to obtain the electrofunctional polymer; if the electrofunctional polymer is a conductive polymer, the cell membrane capacitance of the nerve cell decreases, and the electrical activity of the nerve cell increases; if the electrofunctional polymer is an insulating polymer, the cell membrane capacitance of the nerve cell increases, and the electrical activity of the nerve cell decreases.

[0471] Preferably, the nerve cell is a human or non-human mammalian nerve cell. The nerve cell expresses membrane-anchored PRADA. Preferably, the membrane-anchored PRADA has the amino acid sequence shown in SEQ ID NO:10.

[0472] Preferably, the electrofunctional substrate includes aniline and diaminobenzidine (DAB). Preferably, the conductive polymer includes polyaniline (PANI). Preferably, the insulating polymer includes polydiaminobenzidine (PDAB).

[0473] The method for controlling the electrical activity of nerve cells in this invention can be applied to technologies such as nerve repair, brain-computer interface technology, and the fabrication of neural prostheses.

[0474] The method for studying RNA structure of the present invention

[0475] The present invention provides a method for studying RNA structure, the method comprising the steps of: (Y1) providing an RNA located in an organelle / cell / tissue / organ containing the fusion protein of the present invention.

[0476] Natural guanine (G) ribonucleotides in RNA can be linked to the PRADA probe; natural uracil (U) ribonucleotides cannot be linked to the PRADA probe, while the ribonucleotide analog 4-thiouridine of U can be linked to the PRADA probe. Therefore, depending on the research needs, one can choose to label only G, only U, or both G and U, and then choose whether to replace the natural ribonucleotides in the original RNA with ribonucleotide analogs. Based on the above description, step (Y2) can be understood to be selected from the following group: (Y2.1) Incubating the organelle / cell / tissue / organ with the ribonucleotide analog to contain the ribonucleotide analog in the RNA, obtaining RNA containing the ribonucleotide analog, i.e., labeling only U or labeling both G and U; wherein, the natural G can also be replaced with the ribonucleotide analog 6-thioguanine of G; (Y2.2) Incubating the organelle / cell / tissue / organ without ribonucleotide analogs, i.e., labeling only G.

[0477] (Y3) The D-amino acid substrate and the labeled probe substrate carrying a reactive group are incubated with the organelle / cell / tissue / organ to perform proximity labeling; wherein, the hydrogen peroxide generation module in the fusion protein converts the D-amino acid substrate into hydrogen peroxide, the hydrogen peroxide activates the hydrogen peroxide utilization module in the fusion protein, and under the catalysis of the activated hydrogen peroxide utilization module, the labeled probe substrate covalently links with ribonucleotide analogs and / or ribonucleotides that have not formed hydrogen bonds through the reactive group it carries, thereby labeling the labeled probe on the ribonucleotide analogs and / or ribonucleotides to obtain RNA labeled with the labeled probe.

[0478] Common structures of free RNA include stem-loop structures and free single strands. In stem-loop structures, the nucleotides in the "stem" interact with their complementary nucleotides via hydrogen bonds and cannot be further linked to the labeling probe; while the nucleotides that do not form hydrogen bonds, including those in the "loop" and those in the free single strand, can be linked to the labeling probe. This property allows for the specific labeling of nucleotides that do not form hydrogen bonds.

[0479] (Y4) Extract the RNA labeled with the labeled probe from the organelle / cell / tissue / organ.

[0480] This invention achieves the enrichment of desired RNA through the biotin-streptavidin system, therefore, the RNA obtained in step (Y4) needs to be biotin-modified. If a biotin-containing labeled probe, such as biotin-phenol or biotin-aniline, is used in the aforementioned steps, the RNA obtained in step (Y4) is already biotin-linked, thus no biotin modification is required; conversely, if a biotin-containing labeled probe, such as alkynyl-phenol, is not used in the aforementioned steps, then biotin modification is necessary. Specifically, biotin modification is performed through a click chemical reaction. Based on the above description, it can be understood that step (Y5) is selected from the following group: (y5.1) When the RNA obtained in step (Y4) does not contain biotin labeling, the RNA is biotin-modified, specifically including the steps of: incubating the biotin-containing reaction probe targeting the labeling probe with the RNA labeled with the labeling probe to perform a click chemical reaction, thereby modifying the RNA labeled with the labeling probe with biotin; wherein, the biotin-containing reaction probe and the labeling probe undergo a click chemical reaction, thereby linking biotin to the RNA labeled with the labeling probe to obtain biotin-modified RNA; (y5.2) When the RNA obtained in step (Y4) contains biotin labeling, no biotin modification is performed.

[0481] (Y6) Enrich the RNA obtained in step (Y5).

[0482] (Y7) The RNA obtained in step (Y6) is reverse transcribed to obtain cDNA; preferably, the reverse transcription is performed using reverse transcriptase.

[0483] (Y8) Sequencing the cDNA to obtain a mutation profile.

[0484] (Y9) The accessibility of the RNA structure is analyzed based on the mutation map to obtain the structure of the RNA.

[0485] carrier

[0486] This invention provides a vector in which the nucleotide sequence expressing PRADA can be contained. As used herein, the term "vector" is a nucleic acid molecule used as a means of transferring exogenous nucleic acids into cells. The vector can be a vector for expressing nucleic acids in cells. Such a vector may include a promoter sequence operatively linked to a nucleotide sequence encoding the sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express the fusion protein described in this invention.

[0487] The term "operable linkage" can include a situation where a selected nucleic acid sequence is covalently linked to a regulatory nucleic acid sequence (such as a promoter and / or enhancer), thereby influencing or controlling the expression of the nucleic acid sequence (thus forming an expression cassette). Therefore, if the regulatory sequence can affect the transcription of the nucleic acid sequence, then the regulatory sequence is operably linked to the selected nucleic acid sequence.

[0488] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (such as gamma retroviral vectors (e.g., mouse leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (such as yeast artificial chromosomes). Preferably, the vector is a plasmid.

[0489] Preferably, the vector is a eukaryotic vector, such as a vector containing elements required for expressing proteins derived from the vector in eukaryotic cells.

[0490] cell lines

[0491] This invention provides a cell line that expresses the PRADA fusion protein, comprising a nucleotide sequence expressing PRADA, or a vector comprising a nucleotide sequence containing PRADA. As used herein, the term "cell line" refers to eukaryotic cells (e.g., animal cells, plant cells, fungal cells, etc.), prokaryotic cells (e.g., microbial cells, Escherichia coli, Bacillus subtilis, etc.), or cells derived from multicellular organisms (e.g., cell lines) cultured as single-celled entities. The cells serve as recipients of nucleic acids (e.g., expression vectors) and include progeny cells that have undergone nucleic acid genetic modification.

[0492] It is understandable that the offspring of a single cell can be attributed to natural, accidental, or intentional mutations and may not necessarily have the exact same morphology or genome as the original parent cell. A “recombinant cell line” (also known as a “genetically modified cell line”) is a host cell in which a heterologous nucleic acid (such as an expression vector) has been introduced.

[0493] Reagent test kit

[0494] This disclosure provides a kit comprising one, two, or more components of the fusion protein, polynucleotide, vector, or cell line described in this invention.

[0495] Preferably, the kit of the present invention further includes a label or instruction manual for using one or more components contained therein, and / or a label or instruction manual for use in combination with other necessary one or more components.

[0496] Preferably, the kit further comprises one or more buffer solutions that can be used to dissolve any of the one or more components contained therein, and / or to provide suitable reaction conditions for the one or more components. Preferably, the reaction conditions include an appropriate pH.

[0497] Sequence identity

[0498] As used herein, "sequence identity" refers to the percentage of nucleotide / amino acid residues in the host sequence that are identical to those in the reference sequence after sequence alignment, with gaps introduced where necessary to achieve the maximum percentage of sequence identity between sequences. To determine the percentage of sequence identity between two or more nucleic acid or amino acid sequences, those skilled in the art are familiar with various methods or software for performing double and multiple sequence alignments. When using such software, default parameters are used, and it is preferable to use parameters such as gap penalties and expansion penalties.

[0499] The main advantages of this invention include:

[0500] (1) A monomeric D-amino acid oxidase was constructed for the first time, and this monomeric D-amino acid oxidase was combined with APEX2 to construct a fusion protein for proximity labeling technology, namely PRADA. Compared with the protein composed of unengineered D-amino acid oxidase and APEX2, PRADA has better enzymatic activity.

[0501] (2) The PRADA technique uses D-amino acids as substrates to generate H2O2 in situ to activate APEX2. Compared with the traditional APEX2 technique that uses toxic H2O2, it significantly reduces the toxic effects of the experimental process on organisms, improves biocompatibility, and makes the experimental conditions closer to the physiological state.

[0502] (3) PRADA technology can not only achieve simultaneous labeling of proteins and RNA, but also support the assembly of conductive polymers, which significantly expands the application scenarios of proximity labeling technology.

[0503] (4) PRADA technology can be applied to a variety of cell types and can achieve proximity labeling in a variety of living organisms, including nematodes, fruit flies, zebrafish, and mice.

[0504] (5) Based on PRADA technology, this invention also proposes PRADA-MaP seq technology, which for the first time realizes the resolution of subcellular compartment-specific RNA structures in vivo, and has high accuracy (AUROC value of 0.74-0.84) compared with existing technologies.

[0505] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0506] Example 1: Construction and functional verification of engineered monomeric D-amino acid oxidase

[0507] This embodiment aims to develop a non-cytotoxic, multifunctional proximity labeling tool. An enzymatic cascade reaction system was designed to activate APEX2 by coupling it with another enzyme capable of generating H2O2 using a non-interfering substrate. The D-amino acid oxidase (RgDAAO) from the fungus *Rhodotorula gracilis* was selected as the research subject. Figure 1 A). This enzyme belongs to a diverse family of oxidases that can produce H2O2 by oxidizing small molecule metabolites and has high catalytic efficiency. At the same time, its substrate D-amino acids have little interference with mammalian cell physiology.

[0508] To preliminarily validate this strategy, RgDAAO was fused to the C-terminus or N-terminus of APEX2, expressed in HEK293T cells, and its peroxidase activity was detected using the fluorescent substrate Amplex Red.

[0509] The results showed that although the initial signal generation rate and intensity were not as high as those in the group treated with direct exogenous H2O2 addition, cells expressing APEX2-RgDAAO accumulated a strong signal comparable to that of the group treated with direct 1mM H2O2 addition after 30 minutes following the addition of 10mM D-alanine (D-Ala), with no significant adverse effects on cell viability. Figure 1 (B, 1C). These preliminary results demonstrate the potential of combining RgDAAO with APEX2 for proximity labeling, but direct ligation of the two enzymes presents problems because RgDAAO exists as a homodimer, which may lead to anomalous interactions and localization issues when fused with the target protein.

[0510] Given the limitations of RgDAAO, it was decided to engineer it into a monomer. Based on its crystal structure (PDB ID: 1C0I) ​​and existing mutation studies, AlphaFold-Multimer was used to predict a mutant (△) lacking the flexible loop region (located at the dimer interface and far from the active site in the S308-K321 segment). 308-321 The structure of RgDAAO) Figure 1 D). The prediction results show that wild-type RgDAAO tends to spontaneously dimerize (ipTM = 0.92), which is consistent with crystal structure observations; while Δ 308-321 RgDAAO is more likely to exist in monomeric form (ipTM = 0.57) Figure 1 E).

[0511] The experiment was validated in live cells using a TurboID complementation assay, where DAAO was fused to two separate fragments of TurboID. When both fragments were co-expressed in the same cell, the fragmented TurboID could only renature into the intact enzyme with biotinylation activity when DAAO dimerized. As expected, cells expressing RgDAAO-fused fragmented TurboID showed a strong biotinylation signal; while Δ 308-321 RgDAAO remains in a standalone state and fails to restore the full functionality of TurboID. Figure 1 F).

[0512] In addition, in order to detect monomerization Δ 308-321 The enzyme activity of RgDAAO was tested to assess its ability to drive APEX2 labeling. After incubation with D-Ala and the APEX2 substrate biotin-phenol (BP) for 30 minutes, APEX2-Δ was detected in the cytoplasm. 308-321 RgDAAO has a higher MFI level than APEX2-RgDAAO, indicating that APEX2-Δ 308-321 RgDAAO has a higher level of biotinylation ( Figure 1 G).

[0513] Given that this system uses D-amino acids, rather than H2O2, as the input signal to activate the peroxidase reaction, the 65kDa APEX2-Δ 308-321 The RgDAAO fusion enzyme was named PRADA.

[0514] In summary, an oxidase-peroxidase fusion system was successfully engineered, enabling proximity labeling without the use of toxic substrates.

[0515] Example 2: Characterization of PRADA markers in living cells

[0516] This embodiment relates to the systematic characterization of PRADA's performance in living cells based on its established design.

[0517] First, it was tested whether the labeling efficiency of PRADA could be modulated by changing different D-amino acid substrates, since the catalytic rate constant (K) of DAAO for different D-amino acids was different. cat ) and Michaelis constant (K m Significant differences were observed. To screen a selected set of substrates, fluorescence imaging was used to quantify the labeling efficiency of the APEX2 substrate alkynyl-phenol (AP) for clickable chemistry. AP exhibited better cell permeability than biotin-phenol (BP).

[0518] The results showed that the reactivity of D-methionine (D-Met) and D-phenylalanine (D-Phe) was higher than that of D-alanine (D-Ala). Figure 1 H). In fact, when using the more efficient substrate D-Phe, the PRADA tag signal can be detected in less than 1 minute. Figure 1 I).

[0519] Subsequently, the activity of PRADA in different subcellular regions was evaluated. To cover various subcellular environments, including membrane-bound compartments, open compartments in contact with membranes, and non-membrane organelles, PRADA was targeted to the mitochondrial matrix (MITO), nucleus (NLS), endoplasmic reticulum membrane (ERM), and nucleolus by fusing different localization signals.

[0520] Fluorescence imaging results of various PRADA constructs in HEK293T cells showed that the labeling activity was excellent after the addition of D-Phe and AP substrates, and the labeling activity was highly co-localized with the expected expression location of the enzyme. Figure 1 J).

[0521] Furthermore, the toxicity of H2O2 was assessed, and it was tested whether the H2O2 intermediate generated by DAAO could be efficiently utilized by APEX2 in the PRADA fusion protein, or whether it might diffuse into surrounding subcellular regions. To this end, a detection method was designed to co-express a nuclear-localized PRADA and a cytoplasmic-localized APEX2 in the same cell, using the cytoplasmic-localized APEX2 as a sensor for potential H2O2 diffusion.

[0522] Experimental results showed that, consistent with the expected mechanism of PRADA local generation and utilization of H2O2, the addition of D-Phe and AP triggered PRADA labeling only in the cell nucleus, without activating APEX2 in the cytoplasm. Figure 1 K).

[0523] These results demonstrate that PRADA can achieve precise labeling in a highly efficient and low-toxicity manner within gene-programmed subcellular regions.

[0524] Example 3: PRADA enables multifunctional proximity-based chemical manipulation

[0525] Peroxidases possess unique characteristics among neighboring labeling enzymes, namely their diverse chemical reactivity, including protein labeling, RNA labeling, and in situ assembly of functional polymers. Therefore, this embodiment evaluates whether these reactions can be achieved in living cells without the use of H2O2.

[0526] First, PRADA's ability to perform RNA proximity labeling was tested, a function that biotin ligase cannot achieve. Figure 2 A).

[0527] RNA was extracted from cells expressing nuclear localization (NLS)-PRADA after treatment with D-Phe and AP for 30 minutes. The labeled RNA was then enriched using azide-biotin click chemistry and streptavidin magnetic beads. Results showed that, compared to unlabeled cells without AP, the labeled group cells exhibited specific enrichment of nuclear long non-coding RNAs—X chromosome inactivation-specific transcript (XIST) and nucleoplasmic aggregation transcript 1 (NEAT1) (enrichment fold of 3.26, p<0.05), while no enrichment was observed in cytoplasmic mRNA. Figure 2 B). Similarly, mitochondrial localization (MITO)-PRADA can be used to specifically label and enrich mitochondrial mRNA.

[0528] Subsequently, endoplasmic reticulum outer membrane (ERM)-PRADA-labeled cells were analyzed by streptavidin-HRP immunoblotting, confirming the expected widespread protein biotinylation. Figure 2 C).

[0529] Furthermore, the study investigated whether PRADA could catalyze the formation of electrofunctional polymers, which could be used to manipulate cell membrane capacitance. In live cells expressing membrane-anchored (MEM)-PRADA, biopolymers with conductive or insulating properties were successfully generated via D-Ala-induced polymerization of aniline or diaminobenzidine, respectively. Optical microscopy revealed the dark deposition of the polymerization products. Figure 2 D).

[0530] Finally, the activity of PRADA in different cell types was evaluated. Consistent with the results in HEK293T cells, strong PRADA activity was also observed in primary rat hippocampal neurons and in mouse cancer cell lines derived from the colon, mammary gland, and lung. Figure 2 E).

[0531] In summary, these results demonstrate that PRADA can achieve diverse proximity-based chemical reactions in a variety of living cell types.

[0532] Example 4: Marking of PRADA in live nematodes, fruit flies, zebrafish, and mice

[0533] After observing good activity of PRADA in living cells, this example further explored whether PRADA could be labeled in living organisms—a major challenge for existing tools.

[0534] The experiment was first conducted in *C. elegans*. Transgenic nematodes were constructed to express cytoplasmic GFP-PRADA-NES in the body wall muscle via a myosin promoter. Intact nematodes were incubated for 1 hour in a medium containing AP and D-Phe, followed by click chemistry with a Cy3 fluorescent probe.

[0535] Fluorescence imaging analysis revealed a strong PRADA labeling signal, which was D-Phe dependent and highly overlapped with the GFP expression region. Figure 2 F). However, studies have shown that high levels of endogenous biotinylated proteins in nematodes may interfere with detection when performing biochemical analysis on labeled lysates. In fact, no significant differences were observed in streptavidin blot analysis following azide-biotin click (F). Figure 2 G). Labeled APs can undergo click chemistry reactions with other exogenous probes (such as fluorescein) for immunoblotting and enrichment, thereby significantly improving the signal-to-noise ratio. Figure 2 Furthermore, widely used nematode paralysis experiments have shown that even treatment with D-Phe or D-Met at a concentration of 100 mM has minimal impact on the overall physiology of nematodes, while 1 mM H2O2 can cause significant toxicity. Figure 2 I).

[0536] Subsequently, the PRADA marker was tested in live fruit flies and zebrafish. Six transgenic fruit fly strains were constructed using the UAS-GAL4 system, expressing PRADA (targeting the nucleus, cytoplasm, or mitochondrial matrix) in muscle or neurons, respectively. Figure 2 J). In all six cases, PRADA activity was successfully verified in third-instar larvae after 30 minutes of AP and D-Met treatment, while no detectable background signal was observed in the control group without substrate. Figure 2K). Meanwhile, PRADA was tested in zebrafish 80 hours after hatching, which were microinjected with a muscle-specific cytoplasmic PRADA plasmid. After incubating free-swimming zebrafish in water containing AP and D-Phe for 3 hours, specific markers were observed only in muscle cells expressing PRADA. Figure 2 L).

[0537] It is noteworthy that the achievement of peroxidase labeling in intact fruit flies and zebrafish fills an important technological gap in existing tools: previous fruit fly studies mainly relied on ex vivo tissues and the operating environment was not physiological, while the results of this paper demonstrate for the first time the achievement of peroxidase-based proximity labeling in live zebrafish.

[0538] Finally, to evaluate the suitability of PRADA in mice, a xenograft tumor model was used, in which HEK cells expressing ERM-PRADA were subcutaneously implanted into immunodeficient mice. Labeling was performed by injecting AP and D-Phe into the tumor. Subsequently, streptavidin-HRP immunoblotting analysis of the entire tumor lysate revealed the expected extensive protein biotinylation pattern. Figure 2 Furthermore, laser confocal imaging further validated that the markers at both the tumor tissue and subcellular levels possessed good specificity and intensity (M). Figure 2 N).

[0539] In summary, PRADA was successfully labeled in vivo in four model organisms—nematodes, fruit flies, zebrafish, and mice—confirming its broad potential for in vivo applications.

[0540] Example 5: Existing peroxidase labeling technology cannot provide RNA structural information.

[0541] A key feature of peroxidase reactions is their ability to perform proximity labeling of RNA, but to date, this application has been primarily limited to in vitro cultured cells. Therefore, if labeling could be achieved in vivo using PRADA, it would be possible to directly conduct spatial transcriptomics studies on subcellular RNA. However, before demonstrating this potential, it is hoped that the unique RNA reactivity of peroxidases can be further utilized to obtain more information about the labeled RNA.

[0542] The secondary structure of RNA has a significant impact on its function in specific subcellular regions, but existing chemical detection methods (such as DMS and SHAPE) can only provide overall structural information of RNA across the entire cell, lacking subcellular spatial specificity. Therefore, it is hypothesized that spatially resolved peroxidase labeling may also contain secondary structure information: sites labeled with phenoxy radicals may indicate modifiable single-stranded bases, similar to traditional chemical probes, thereby introducing mutations during reverse transcription and ultimately achieving readout through sequencing.

[0543] Mitochondria were chosen as the primary focus of this study because they serve as both the energy center and a crucial signaling regulatory hub within the cell. Furthermore, recent research has yielded high-quality and detailed annotated structural maps of mitochondrial mRNAs (mt-mRNAs) through DMS-seq (in organello) analysis after purifying mitochondria, providing valuable insights for validating hypotheses.

[0544] First, the mutation profiles in existing APEX2-labeled mitochondrial RNA sequencing datasets were re-analyzed. After processing using the ShapeMapper2 analysis pipeline, the increase in mutation rate was found to be relatively small (1.61-2.53-fold), and no significant increase in mutations of the four base pairs was detected. Figure 3 This may be due to the low yield of the APEX2-catalyzed RNA labeling reaction (AC).

[0545] To further verify this, a previous finding was reviewed, namely that the metabolic incorporation of the electron-rich nucleoside analog 4-thiouridine (4sU) can increase APEX2-mediated RNA labeling yield by more than 10-fold. After searching the dataset generated by 4sU pulse labeling, a significant increase in the U base mutation rate was indeed observed in the labeled group compared to the unlabeled control group (5.49-fold). Figure 3 D).

[0546] However, when using cytochrome c oxidase 1 (COX1), the most abundant mRNA in mitochondria, as a representative to further examine whether the mutation profile conforms to the structural model determined by DMS-seq, the results showed that the reactivity difference between single-stranded regions and base-pairing regions was extremely small (1.14-fold, p = 1.389 × 10⁻⁶). -2 ()( Figure 3 E and 3F). Therefore, it is speculated that the loss of structural information may be due to the rapid increase in phenolic radical concentration during the H2O2-triggered peroxidase reaction, leading to the indiscriminate labeling of all U bases regardless of their accessibility. Furthermore, it is believed that this limitation could be overcome by employing enzymatic cascade reactions with stepwise catalytic properties, such as PRADA, because their phenolic radical generation rate is relatively slow (…). Figure 1 B). PRADA's unique reaction properties promise to avoid non-selective labeling and preferentially label more accessible bases. Therefore, current methods are not yet able to resolve the RNA folding state of the subcellular transcriptome in living cells.

[0547] Example 6: Development of PRADA-MaP seq for mapping subcellular RNA structures in living cells

[0548] Based on the above results, this embodiment designs a method combining 4sU metabolic incorporation and PRADA labeling to achieve spatiotemporal resolution RNA structure analysis in living cells, namely, mutation spectrum sequencing (PRADA-MaP seq). Figure 4 A).

[0549] The specific method is as follows: In PRADA cells expressing specific subcellular regions, a 5-hour 4sU metabolic pulse treatment was first performed, followed by 30 minutes of PRADA labeling with D-Phe and AP. The labeled RNA was modified via an azide-biotin click chemistry reaction, enriched using streptavidin magnetic beads, and then synthesized into cDNA using high-fidelity reverse transcriptase for high-throughput sequencing. Subsequently, the accessibility of RNA structure was deduced based on mutation profile data, thereby modeling the RNA folding state.

[0550] To evaluate this workflow, PRADA-MaP seq experiments were performed in live cells stably expressing MITO-PRADA. Using the unlabeled control group without D-Phe as a reference, the selection criteria were set as an enrichment fold >4-fold and an adjusted p-value <0.05. All 13 mitochondrial mRNAs (mt-mRNAs) were reproducibly identified with high sequencing coverage, validating effective RNA labeling and enrichment at the transcriptome level. Figure 4 B, 4C, 3G, and 3H). Due to rRNA removal during library construction, the number of sequencing reads for the two mitochondrial rRNAs was insufficient. Similar to the 4sU-APEX2 marker analysis, a significantly higher mutation rate was observed in the PRADA-marked samples than in the unmarked control group. Figure 3 I).

[0551] To investigate whether PRADA markers can reflect RNA structural accessibility, we again analyzed the reactivity of U bases using COX1 mRNA as an example. The results showed that the enrichment of U mutations in single-stranded regions was significantly higher than in double-stranded regions (2.27-fold, p = 1.747 × 10⁻⁶). -15 ()( Figure 3 J). Further analysis using the area under the receiver operating characteristic (AUROC) curve confirmed that the reactivity of the U base was highly consistent with the COX1 structural model constructed by DMS-seq (AUROC = 0.77). Figure 3 K).

[0552] To further improve the accuracy and sensitivity of structure mapping, the analysis was expanded to include the G base—currently the only known natural base that can directly react with phenoxy radicals generated by peroxidase. Although the mutation rate and AUROC of G are lower than those of U, G still provides supplementary structural information. Combined with GU base reactivity, the AUROC of COX1 mRNA increased to 0.78. Figure 3 KM). In PRADA-labeled cells, GU reactivity was significantly increased and highly correlated among independent biological replicates (r = 0.96), further validating the reproducibility of the PRADA mutation profile analysis. Figure 4 D, 4E, 3N, and 3O).

[0553] Based on this analytical workflow incorporating GU reactivity, the AUROC values ​​of all mRNAs encoded by the mitochondrial heavy chain in live-cell PRADA-labeled data were further calculated and compared with published purified mitochondrial structural models. The results showed that the AUROC values ​​of all transcripts were between 0.74 and 0.84. Figure 4 F) indicates that PRADA-MaP seq can not only perform high-quality RNA structure detection, but also verify that the mitochondrial RNA structure in live cells is highly consistent with that in purified mitochondria (in organello). Consistent with AUROC analysis, when GU label reactivity was plotted on various mitochondrial RNA structure models, high reactivity was observed mainly in exposed regions such as single strands, loops, and protrusions, while unreachable base-pairing regions showed low reactivity, consistent with expectations. Figure 4 G).

[0554] In summary, the PRADA-MaP seq technology was established, achieving for the first time subcellular spatial resolution RNA structure mapping in living cells.

[0555] Example 7: Simultaneous in vivo mapping of mitochondrial proteome and transcriptome using PRADA markers

[0556] To fully leverage PRADA's advantages in proximity-dependent protein labeling, RNA labeling, RNA structure detection, and in vivo applications, this embodiment further applies PRADA to the mapping of subcellular multi-omics atlases in live mice. Figure 5 A).

[0557] In tumors induced by subcutaneous implantation of MITO-PRADAHEK cells, RNA metabolic labeling was first performed via intraperitoneal injection of 4sU, followed by intratumoral injection of PRADA substrate to initiate the labeling reaction. Since both protein and RNA labeling can be accomplished using the same substrate probe AP, the tissue lysis process was optimized. TRIzol was used to simultaneously isolate and enrich labeled protein and RNA components, enabling the simultaneous mapping of spatial proteome, transcriptome, and RNA structure in a single experiment.

[0558] In decoding the mitochondrial proteome, streptavidin-HRP analysis was first used to verify the presence of a strong, widespread biotinylation signal in the labeled mouse tissue samples. Figure 5B). An unlabeled control group (without D-amino acid treatment) was included in the experiment to exclude interference from endogenous biotinylated proteins and nonspecific magnetic bead conjugates. Two mice were used as biological replicates for each condition. Subsequently, biotinylated proteins were captured using streptavidin magnetic beads and digested with trypsin on the beads. Label-free mass spectrometry analysis identified a total of 3010 proteins in all samples. Figure 5 C). Using a log2 fold change > 1 and a p-value < 0.05 as the screening criteria, 306 proteins were finally selected for Gene Ontology (GO) analysis.

[0559] The results showed that proteins related to mitochondria and the mitochondrial matrix were most enriched in the cellular component category, highlighting the effectiveness of PRADA markers in achieving subcellular spatial proteomics studies in vivo. Figure 5 D).

[0560] Meanwhile, biotinylated RNA was enriched from the aqueous phase separated by TRIzol using streptavidin magnetic beads. As preliminary validation of the mitochondrial-specific marker, quantitative real-time PCR detection revealed that mitochondrial mRNA showed significant enrichment relative to cytoplasmic control RNA only in MITO-PRADA-labeled mice. Figure 5 E).

[0561] Furthermore, sequencing analysis of RNA enriched in independently labeled mice revealed a high correlation between transcript enrichment levels across biological replicates (r = 0.985), indicating good reproducibility. Differential enrichment analysis between the PRADA-labeled group and the unlabeled control group showed that PRADA sensitively and specifically recovered transcripts encoded by the mitochondrial genome, including significant enrichment of 12 mt-mRNAs and 18 mt-tRNAs (log2 fold change > 0.8 and p < 0.05). Figure 5 F).

[0562] In summary, it has been demonstrated that PRADA enables spatial transcriptome mapping of subcellular regions defined for specific genes in living animals, a feat previously only achievable in in vitro cell systems.

[0563] Example 8: Mitochondrial RNA structural remodeling regulates gene expression in vivo

[0564] Although it is generally believed that the environment of cells in vivo differs significantly from that of cells in a culture dish, whether these environmental differences affect dynamic RNA structure remains to be studied in detail. In MITO-PRADAHEK cells (in vitro, Figure 4 B) and its mouse xenograft model ( Figure 5The PRADA mutation profile data obtained in F) provides a good opportunity for a systematic analysis of changes in mitochondrial RNA folding patterns.

[0565] Therefore, the differences in gene expression under the two conditions were first assessed by sequencing the total RNA extracted. Figure 5 G). The results showed that, compared with 2D culture conditions, the transcriptional program involving multiple processes such as signal transduction and cell metabolism in in vivo tumor tissues was significantly altered, reflecting differences in extracellular environment, nutrient supply, and mechanical forces. Figure 5 H). In particular, components related to the mitochondrial translation mechanism, including mitochondrial ribosomal proteins and the mitochondrial RNA remodeling complex—the leucine-rich pentapeptide repeat-SRA stem-loop binding protein (LRPPRC-SLIRP)—were significantly upregulated in vivo (p < 0.05), as evidenced by gene set enrichment analysis (GSEA). Figure 5 I and 5J). Notably, the expression levels of mitochondrial RNAs (mtRNAs) did not differ significantly between the two conditions (I and 5J). Figure 5 Based on this observation, PRADA-MaP seq data were further used to assess the differences in mitochondrial RNA structure between in vivo and in vitro.

[0566] Based on validated mitochondrial intracellular RNA structures, the in vivo MITO-PRADA reactivity curves were found to be highly consistent with known structures, with most mitochondrial mRNAs exhibiting AUROC values ​​greater than 0.7, and similar to intracellular PRADA-MaP sequencing results. Figure 5 K). However, the ND5 gene (the longest mitochondrial mRNA encoding the most transmembrane domains) is a notable exception. It has the lowest matching degree to the in organello structure (AUROC = 0.60), suggesting a unique folding pattern in vivo.

[0567] Further focusing on the ND5 gene, its PRADA reactivity distribution in vitro and in vivo was compared. Figure 5Analysis showed that the sequence encoding the 14th transmembrane region of ND5 exhibited significantly higher reactivity in vivo, implying that this region is more flexible and less structurally rigid in vivo. To identify potential structural regulators, it was hypothesized that upregulation of LRPPRC expression might play a role, as LRPPRC is known to remodel mitochondrial mRNA folding. Supporting this hypothesis are the high overlap between the structurally altered regions and reported LRPPRC binding sites, and previous studies showing that LRPPRC knockout leads to significant structural changes in these regions. Furthermore, these regions also coincide with enrichment sites in mitochondrial ribosome and disome translation, suggesting the presence of mRNA-encoded translational pauses in this region in cultured cells.

[0568] To further verify whether the simple structure of in vivo transcripts is associated with the upregulation of ND5 protein levels, the immunoblotting results of mitochondrial-encoded proteins in HEK cells were compared under in vitro and in vivo conditions. The results showed that ND5 protein levels were most significantly increased in vivo. Figure 5 M), which is highly consistent with the results of RNA structure profile analysis.

[0569] In summary, these data provide concrete examples of how PRADA can be used to gain specific biological insights in living animals.

[0570] Example 9: Labeling and identification of cell membrane proteins in vitro using PRADA labeling

[0571] DAAO and APEX2 are anchored to the cell membrane via localization peptides. The cells are then given D-amino acids to catalyze the production of H2O2 from DAAO, thereby activating APEX2 catalytic probes (such as BxxP) to achieve the labeling reaction. Figure 6 A). Because it is a PRADA label on the cell membrane, it is called "tmPRADA". Flow cytometry analysis revealed that when both DAAO and APEX2 are simultaneously anchored to a cell, simultaneous administration of D-amino acids and BxxP to the cell can achieve a good labeling response. Figure 6 B and Figure 7 A).

[0572] Therefore, stable cell lines co-expressing DAAO and APEX2 were successfully constructed in mouse colon cancer cell lines CT26 and MC38, and labeling was achieved. Figure 6 C, 6D and Figure 7 B). The CT26-tmPRADA cell line was used for labeling and enrichment, followed by mass spectrometry to detect the types of enriched proteins. Figure 6 E and Figure 7 C Figure 7 D). GOCC analysis of the significantly enriched proteins revealed that the vast majority of the enriched proteins were cell membrane proteins. Figure 6 F, Figure 6 G Figure 7 E).

[0573] APEX2 and DAAO were anchored to the cell membranes of different cells and then co-cultured. After administering D-amino acids and BxxP to the mixed cells and reacting for 5 minutes, a strong labeling signal was observed in the region where the two cell types were in close contact. Figure 6 H).

[0574] Therefore, PRADA labeling can be used to label single-cell membranes and the contact areas between two types of cells.

[0575] Example 10: PRADA labeling and identification using multiple labeled probe substrates

[0576] This embodiment involves PRADA labeling using biotin-aniline (BA), alkynyl-phenol (AP), biotin-phenol (BP), and azido-phenol (NP), with results as follows: Figure 8 As shown.

[0577] As shown in the figure, all of the above-mentioned labeled probe substrates can be used for PRADA labeling.

[0578] sequence

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A fusion protein, characterized in that, The fusion protein has a structure as shown in formula (I), formula (II), formula (III) or formula (IV): (Z1-Z2)-Z3-(Z4-Z5)(I); (Z4-Z5)-Z3-(Z1-Z2)(II); Z1-Z2-Z5(III); or Z1-Z4-Z5(IV); In the formula, Z1 is none, or is selected from the following group: location sequence, connection sequence, label sequence, or a combination thereof; Z2 is the hydrogen peroxide utilization module; Z3 is a connection sequence; Z4 is the hydrogen peroxide generation module; Z5 is absent, or selected from the following group: positioning sequence, connection sequence, or a combination thereof; "-" indicates a peptide bond that connects the above sequences; (Z1-Z2) is selected from the following group: Z1-Z2 or Z2-Z1; (Z4-Z5) is selected from the following group: Z4-Z5 or Z5-Z4.

2. A monomeric D-amino acid oxidase for constructing the fusion protein of claim 1, characterized in that, The monomeric D-amino acid oxidase comprises or is composed of an amino acid sequence as shown in SEQ ID NO:

4.

3. A polynucleotide, characterized in that, The polynucleotide encodes the fusion protein of claim 1.

4. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 3.

5. A cell line, characterized in that, The cell line expresses the protein of claim 1, has the polynucleotide of claim 3 integrated into its genome, or contains the vector of claim 4.

6. A method for performing proximity labeling in vivo, characterized in that, The method includes the following steps: (S1) Provide an organism containing the fusion protein of claim 1; (S2) Delivering a D-amino acid substrate and a labeled probe substrate carrying a reactive group into the organism for proximity labeling; Specifically, the hydrogen peroxide generation module in the fusion protein converts the D-amino acid substrate into hydrogen peroxide. The hydrogen peroxide activates the hydrogen peroxide utilization module in the fusion protein. Under the catalysis of the activated hydrogen peroxide utilization module, the labeled probe substrate is covalently linked to the target through its carried reactive groups, thereby labeling the labeled probe on the target and obtaining a target labeled with the labeled probe.

7. A method for controlling the electrical activity of nerve cells, characterized in that, The method includes the following steps: (Z1) Provides a nerve cell, wherein the cell membrane of the nerve cell expresses the fusion protein of claim 1; (Z2) The D-amino acid substrate and the electrofunctional substrate are incubated with the nerve cells to cause the electrofunctional substrate to undergo a polymerization reaction to form an electrofunctional polymer, thereby controlling the electroactivity of the nerve cells; The hydrogen peroxide generation module in the fusion protein converts the D-amino acid substrate into hydrogen peroxide, which activates the hydrogen peroxide utilization module in the fusion protein. The activated hydrogen peroxide utilization module catalyzes the polymerization reaction of the electrofunctional substrate to obtain an electrofunctional polymer. If the electrofunctional polymer is a conductive polymer, the cell membrane capacitance of the nerve cell decreases and the electrical activity of the nerve cell increases; if the electrofunctional polymer is an insulating polymer, the cell membrane capacitance of the nerve cell increases and the electrical activity of the nerve cell decreases.

8. A method for studying RNA structure, characterized in that, The method includes the following steps: (Y1) Provides an RNA located in an organelle / cell / tissue / organ containing the fusion protein of claim 1; (Y2) is selected from the following group: (y2.1) Incubate the ribonucleotide analog with the organelle / cell / tissue / organ to make the RNA contain the ribonucleotide analog, thereby obtaining RNA containing the ribonucleotide analog; (y2.2) The organelles / cells / tissues / organs are not incubated with ribonucleotide analogs; (Y3) Incubate the D-amino acid substrate and the labeled probe substrate carrying the reactive group with the organelle / cell / tissue / organ to perform proximity labeling; The hydrogen peroxide generation module in the fusion protein converts the D-amino acid substrate into hydrogen peroxide. The hydrogen peroxide activates the hydrogen peroxide utilization module in the fusion protein. Under the catalysis of the activated hydrogen peroxide utilization module, the labeled probe substrate covalently links with the ribonucleotide analogs and / or ribonucleotides in the RNA containing ribonucleotide analogs obtained in (y1) that have not formed hydrogen bonds, or with the ribonucleotides in the RNA obtained in (y2) that have not formed hydrogen bonds, thereby labeling the labeled probe on the ribonucleotide analogs and / or ribonucleotides in the RNA containing ribonucleotide analogs that have not formed hydrogen bonds, or on the ribonucleotides in the RNA that have not formed hydrogen bonds, to obtain RNA labeled with the labeled probe. (Y4) Extract the RNA labeled with the labeled probe from the organelle / cell / tissue / organ; (Y5) is selected from the following group: (y5.1) When the RNA obtained in step (y4) does not contain biotin labeling, the RNA is biotin-modified, specifically including the steps of: incubating the biotin-containing reaction probe targeting the labeling probe with the RNA labeled with the labeling probe to perform a click chemical reaction, thereby biotin-modifying the RNA labeled with the labeling probe. In this process, the biotin-containing reaction probe undergoes a click chemical reaction with the labeling probe, thereby linking biotin to the RNA labeled with the labeling probe to obtain biotin-modified RNA. (y5.2) When the RNA obtained in step (y4) contains biotin labeling, biotin modification is not performed; (Y6) Enrich the RNA obtained in step (Y5); (Y7) The RNA obtained in step (Y6) is reverse transcribed to obtain cDNA; (Y8) Sequencing the cDNA to obtain a mutation profile; (Y9) The accessibility of the RNA structure is analyzed based on the mutation map to obtain the structure of the RNA.

9. A reagent kit, characterized in that, The kit comprises the fusion protein of claim 1, the polynucleotide of claim 3, the vector of claim 4, or the cell line of claim 5.

10. The use of the fusion protein of claim 1, the monomeric D-amino acid oxidase of claim 2, the polynucleotide of claim 3, the vector of claim 4, the cell line of claim 5, or the kit of claim 9, characterized in that, The uses include: (I) Proximity marking in living organisms; (II) Research in proteomics; (III) Transcriptomics research; (IV) Study of RNA structure; (V) controls the electrical activity of nerve cells.

Images