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Novel method of intracellular site-specific covalent RNA labeling

A site-specific, cell-based technology used in biochemistry to address site- and sequence-specific labeling issues

Active Publication Date: 2016-10-05
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Currently, combining translational / post-translational modification mechanisms with bioorthogonal chemistry has enabled protein labeling with unprecedented precision and versatility, but there is no suitable method for site- and sequence-specific RNA in mammalian cells. sex marker

Method used

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  • Novel method of intracellular site-specific covalent RNA labeling
  • Novel method of intracellular site-specific covalent RNA labeling
  • Novel method of intracellular site-specific covalent RNA labeling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Chemical Synthesis of Agmatine (AGM, Compound 1) Analogs

[0060] 1. Synthesis of compound 2 (N-(4-aminobutyl)-2-azidoacetamide, AGN) ( figure 2 ):

[0061] Dissolve 656mg of BOC-1,4-butanediamine hydrochloride and 1.48g of sodium carbonate in 80mL, 1:1 (v / v) mixture of ethyl acetate and water, and place in an ice bath at 0°C. After 1 hour, 10 ml of ethyl acetate solution in which 702 mg of 2-bromoacetyl bromide was dissolved was added to the reaction solution, and stirred at room temperature for 2 hours. The upper organic phase was taken, dried with a rotary evaporator, and then purified by silica gel chromatography on silica gel (containing ethyl acetate:petroleum ether in a volume ratio of 1:1) to obtain 320 mg of white intermediate product 1: tert-butyl (4- (2-Bromoacetamido)butyl)carbamate (Tert-butyl(4-(2-bromoacetamido)butyl)carbamate).

[0062] Take 230 mg of intermediate product 1 and dissolve it in 5 mL of acetone, and place it in an ice bath at...

Embodiment 2

[0067] Embodiment 2: the chemical synthesis of fluorescent dye

[0068] 1. Synthesis of BCN-FITC ( Figure 4 ):

[0069] Take 15.0mL of 1,5-cyclooctadiene and 281mg of dipolyrhodium acetate and mix them into 10mL of dichloromethane solution, then add 10mL of dichloromethane solution in which 15mmol of ethyl diazoacetate is dissolved dropwise within 3 hours, while the temperature Keep at 0°C. After reacting at room temperature for 15 hours, the reaction system was placed back in an ice-water bath to keep the temperature at 0°C, and 10 mL of dichloromethane solution in which 15 mmol ethyl diazoacetate was dissolved was added within 3 hours, and the reaction was continued at room temperature for 21 hours . Subsequently, dichloromethane was removed by rotary evaporation, and silica gel column separation (petroleum ether: ethyl acetate=20:1, v / v) gave 2.52g of intermediate product 1: (1R, 8S, 9r, Z)-ethyl bicycle[6.1. 0] non-4-ene-9-carboxylate (endo product) and 2.08g intermed...

Embodiment 3

[0081] Embodiment 3: the recombinant expression of RNA modifying enzyme Tias

[0082] The inventor purchased the archaebacterium Archaeoglobus fulgidus (purchased from ATCC Company, registration number 49558), cloned, expressed and purified the active RNA modifying enzyme Tias (nucleotide sequence shown in SEQ ID NO: 1, The amino acid sequence is shown in SEQ ID NO: 2).

[0083] The specific operation method is: transform the pET22b Af Tias plasmid into Escherichia coli BL21(DE3), pick a single clone in LB liquid medium containing ampicillin antibiotic, shake the bacteria overnight at 37°C; Inoculate in LB liquid medium containing ampicillin antibiotics, shake the bacteria at 37°C until OD600=0.6, add 1mM IPTG, induce for 4 hours at 37°C, collect the bacteria by centrifugation, discard the medium, and wash with 50mM Tris, 500mM NaCl, 5% Glycerol, 10mM imidazole, pH 8.0 solution resuspended, sonicated, and then purified with a His tag purification column (purchased from Nanjin...

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Abstract

The invention relates to a novel method of in-vitro or intracellular site-specific covalent RNA labeling. In the method, a tRNAlle2-agmatidine synthesis enzyme (Tias for short, wherein the nucleotide sequence is represented as the SEQ ID No.1, and the amino acid sequence is represented as the SEQ ID No.2), and achaeoglobus fulgidus tRNAlle2 (SEQ ID No. 3) or a derived nucleotide sequence thereof are introduced in in-vitro or intracellular manner to enable the Tias to be capable of specifically recognizing a special sequence of AftRNAlle2 in a transcriptome, so that sequence and site-specific combination between a small molecule, which contains an azide or an alkynyl functional group, and a target RNA containing a tRNAlle2 label, and further specific labeling and imaging on the target RNA are achieved through a chemical reaction between a fluorescent dye and the functional groups. The invention also relates to a kit for covalently labeling the target RNA in an intracellular site-specific manner, wherein the kit includes the tRNAlle2-agmatidine synthesis enzyme and the tRNAlle2-3-5.

Description

technical field [0001] The invention belongs to the field of biochemistry. Specifically, the present invention provides a novel method for site-specific covalent labeling of RNA in vitro or in cells. More specifically, the invention introduces tRNA in vitro or in cells Ile2 -agmatidine synthetase (tRNA Ile2 -agmatidine synthetase, referred to as Tias, its nucleotide sequence as shown in SEQ ID NO: 1, amino acid sequence as shown in SEQ ID NO: 2) and Archaeoglobus fulgidus (Archaeoglobus fulgidus, referred to as Af) tRNA Ile2 (The nucleotide sequence is shown in SEQ ID NO: 3), so that Tias specifically recognizes Af tRNA in the transcriptome Ile2 The specific sequence of the small molecule with azide or alkynyl functional group and the tRNA containing Ile2 Sequence and site-specific binding occurs between the tagged target RNAs, and then the specific labeling and imaging of the target RNAs is achieved through the chemical reaction between the fluorescent dye and the functio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 王江云李发慧
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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