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Intron-source circular RNA molecules and application of cyclization key nucleotide sequences of intron-source circular RNA molecules

A nucleic acid sequence and intron technology, applied in the field of nucleic acid research, can solve the problems of no reports, and the discovery of key nucleic acid sequences that have no reports on the looping function, etc., to achieve the effect of enriching knowledge

Active Publication Date: 2015-03-18
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the discovery of circular RNAs is mainly derived from back-spliced ​​exon sequences. However, circular RNAs from other sequences have not been reported in this field. What is more important is the discovery of key nucleic acid sequences for the formation of circular RNAs and their formation. Functional role not yet reported

Method used

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  • Intron-source circular RNA molecules and application of cyclization key nucleotide sequences of intron-source circular RNA molecules
  • Intron-source circular RNA molecules and application of cyclization key nucleotide sequences of intron-source circular RNA molecules
  • Intron-source circular RNA molecules and application of cyclization key nucleotide sequences of intron-source circular RNA molecules

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Discovery and Identification of Intron-derived Circular RNA

[0037] 1. Experimental materials

[0038] 1) Reagents

[0039] TRIzol, oligo(dT) magnetic beads, RiboMinus kit, and CDP-STAR reagent were purchased from Invitrogen; Dig wash and block buffer set, 10×Dig RNA labeling mix, Anti-Digoxigenin, Fab Fragment were purchased from Roche; AmpliScribe TM T7, T3, and SP6 High Yield Transcription Kits, RNase R were purchased from Epicentre; Illumina TruSeq Stranded Total RNA HT Sample Prep Kit was purchased from Illumina; RNA Reverse Kit was purchased from Takara; 2×QPCR mix was purchased from Toyobo; DNase Ⅰ was purchased from Ambion Company; EcoR Ⅰ and Not Ⅰ were purchased from NEB Company; α-amanitin was purchased from Sigma Company.

[0040] 2) Cell lines and vectors

[0041] Stem cells H9 were cultured on Martigel-coated culture dishes using CM (fibroblast-conditioned medium) medium, which was MEF supplemented with 4 ng / mL fibroblast growth factor (bFGF,...

Embodiment 2

[0087] Example 2 Discovery and Identification of Key Nucleic Acid Sequences for Circular RNA from Intron Origin

[0088] 1. Reagents

[0089] RNA Marker Ⅲ was purchased from Roche Company; Nhe Ⅰ, BamH Ⅰ, Hind Ⅲ were purchased from NEB Company; agarose gel recovery kit, common DNA product recovery kit were purchased from Tiangen Company.

[0090] 2. Cell lines and vectors

[0091] PA-1 cells were adherently cultured in MEMα medium (Gibco) supplemented with 10% fetal bovine serum (Gbico), 1% L-glutamine, and 1‰ double antibody. The vector used to construct the plasmid is pcDNA3 and the modified pEGFP-C1 plasmid, namely pZW1, which divides egfp into two parts, with a splicing site and a restriction site in the middle. Green fluorescence formation (Systematic Identification and Analysis of Exonic Splicing Silencers, Wang et al., 2004).

[0092] 3. Experimental method

[0093] 3.1 Reconstruction of ciRNAs

[0094] 3.1.1 Construction of expression plasmid

[0095] In order to ...

Embodiment 3

[0126] Example 3 New materials and new methods for looping key sequences to induce looping of linear RNA molecules

[0127] 1. Reagents

[0128] Nhe Ⅰ, BamH Ⅰ, Hind Ⅲ were purchased from NEB Company; QuikChange Site-Directed Mutagenesis Kit was purchased from Stratagene Company; Agarose Gel Recovery Kit and Common DNA Product Recovery Kit were purchased from Tiangen Company; AmpliScribe TM T7, T3, and SP6 High Yield Transcription Kits were purchased from Epicentre.

[0129] 2. Cell lines and vectors

[0130] PA-1 cells were adherently cultured in MEMα medium (Gibco) supplemented with 10% fetal bovine serum (Gbico), 1% L-glutamine, and 1‰ double antibody. The vector is pcDNA3 and pZW1 after transformation.

[0131] 3. Experimental method

[0132] The purpose of this embodiment is to explore whether the key sequence of ciRNAs has wide applicability. The inventor selected an intron that cannot form circular RNA under natural conditions, that is, the fifth intron (intron5) of...

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Abstract

The invention relates to intron-source circular RNA molecules and an application of cyclization key nucleotide sequences of intron-source circular RNA molecules. The invention specifically discloses sequences of circular RNA novel molecules and genome mapping and the cyclization key nucleotide sequences of the circular RNA novel molecules and further discloses a set of plasmids which contain the cyclization key nucleotide sequences for generating the circular RNA molecules comprising any nucleotide sequences. The invention discloses the intron-source circular RNA molecules and the application of cyclization key nucleotide sequences of intron-source circular RNA molecules for the first time, thereby providing a brand-new concept and method for generating the circular RNA molecules comprising any nucleotide sequences.

Description

technical field [0001] The invention relates to the field of nucleic acid research, in particular to a circular intron RNA and its application. Background technique [0002] The completion of the genome sequencing project and the application of next-generation deep sequencing technology revealed that more than 95% of the transcribed sequences in mammalian cells are noncoding RNA (noncoding RNA, ncRNA). Among them, in addition to the well-known "housekeeping" non-coding RNAs (such as ribosomal RNAs, transfer RNAs and small nucleolar RNAs, etc.) Coding RNAs (long noncoding RNAs, lncRNAs). Long non-coding RNAs are a class of RNAs longer than 200 nucleotides that do not have the ability to encode proteins. [0003] At present, most long non-coding RNAs are discovered by enriching poly(A)+ components through oligo(dT) magnetic beads for deep sequencing. Therefore, such long non-coding RNAs end with poly(A) tails like mRNA, including Natural antisense transcripts (nature antise...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10
Inventor 陈玲玲杨力张杨张晓鸥
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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