Novel labeling method for messenger RNA and circular RNA based on bimolecular fluorescence complementation

A bimolecular and messenger technology, applied in the field of cell biology, can solve problems such as difficult sequence distinctions

Inactive Publication Date: 2018-09-04
PEKING UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Since each circular RNA and its corresponding linear isoform are spliced ​​from the same precursor RNA, circular RNA, linear RNA and precursor RNA share part of the nucleotide sequence, and it is difficult to target the sequence specific markers to distinguish the three

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  • Novel labeling method for messenger RNA and circular RNA based on bimolecular fluorescence complementation
  • Novel labeling method for messenger RNA and circular RNA based on bimolecular fluorescence complementation
  • Novel labeling method for messenger RNA and circular RNA based on bimolecular fluorescence complementation

Examples

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Embodiment 1

[0024] Example 1: Bimolecular fluorescent complementary labeling of intracellular messenger RNA based on self-ligation tags. Take the messenger RNA of the split HaloTag to mark CFP as an example:

[0025] (1) The forward and reverse strands of MS2 and PP7 were directly synthesized, mixed in equal proportions, annealed, and connected to the 3' end of the non-coding region of the CFP gene to obtain the vector pcDNA3.1(+)-CFP-MS2-PP7.

[0026] (2) The corresponding DNA fragments were amplified from the plasmids containing tdMCP and tdPCP by PCR. Add restriction enzyme sites to the amplified primers. Purified DNA fragments were recovered by DNA agarose gel electrophoresis and a gel purification kit to obtain tdMCP and tdPCP fragments. The PCR amplification system used is as follows:

[0027]

[0028] (3) The tdMCP and tdPCP recovered in the previous step and the eukaryotic expression vector pcDNA3.1(+) were digested overnight with restriction enzymes respectively, and purifi...

Embodiment 2

[0039] Example 2: Bimolecular fluorescent complementary labeling of intracellular circular RNA based on self-ligation tags. Take the circular RNA of ZKSCAN1(548-1047) marked by the split HaloTag used as an example:

[0040] (1) Directly synthesize the positive and negative strands of MS2 and PP7 sequences, mix them in equal proportions, anneal, and connect them into ZKSCAN1 (548-1047) between the splicing donor and acceptor to obtain the vector pcDNA3.1(+)-CircRNA Mini Vector- MS2-ZKSCAN1(548-1047)-PP7.

[0041] (2) The human breast cancer cell line MDA-MB-231 was digested with trypsin, replanted in a 35mm Petri-Dish dedicated for imaging at a density of 50-60%, and cultured for 24 hours.

[0042] (3) The above cells were co-transfected with 1 μg of vectors pcDNA3.1(+)-CircRNAMini Vector-MS2-ZKSCAN1(548-1047)-PP7 and pcDNA3.1(+)-HaloTag N58 using 6mL liposome Lipo-2000 -tdPCP, and pcDNA3.1(+)-tdMCP-HaloTag C58. The medium used for transfection was serum-free Opti-MEM. After ...

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Abstract

RNA molecules are an important member of an intracellular genetic information delivery system. Conventional RNA labeling methods cannot simultaneously have the characteristics of living cells, no invasion, high temporal and spatial resolution, single molecules, etc., so a novel method for labeling and tracking RNA need to be developed. The invention discloses a novel labeling method for messengerRNA and circular RNA based on self-ligation label bimolecular fluorescence complementation. A RNA aptamer subsystem is cooperatively used, aptamers are inserted at proper positions in the RNA, and twoparts of a self-ligation label are respectively fused with the binding proteins of the RNA aptamers. When the fusion proteins do not bind to RNA, RNA molecules do not fluoresce; when and only when the fusion proteins bind to RNA, the RNA molecules fluoresce, so the fluorescence background of an intracellular RNA marker is reduced. The novel RNA labeling method based on self-ligation label bimolecular fluorescence complementation can be used for long-term interference-free single-molecule imaging observation and tracking of messenger RNA and circular RNA of living cells and fixed cells, and has good application prospects in cell biology.

Description

technical field [0001] The present invention relates to a novel intracellular messenger RNA and circular RNA labeling method and its application in cell biology. in the field of cell biology. Background technique [0002] Ribonucleic acid (RNA) molecules are carriers of genetic information present in biological cells and some viruses and viroids. A ribonucleotide monomer molecule is composed of a molecule of phosphate, ribose sugar and base. There are four main bases of ribonucleotide monomers, namely adenine (A), guanine (G), cytosine (C), and uracil (U). RNA is a long-chain polymer formed by the condensation of ribonucleotides through phosphodiester bonds. The central dogma states that DNA is transcribed to form RNA, and RNA is translated into protein to function. RNA is a single strand formed by transcription based on a strand of DNA as a template and based on the principle of complementary base pairing. Its main function is to realize the transfer of genetic informat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C09K11/06G01N21/64
CPCC09K11/06C09K2211/1044C09K2211/1088C12N15/113C12N2310/10C12N2310/51G01N21/6486
Inventor 邵世鹏孙超英孙育杰
Owner PEKING UNIV
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