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Genetic coding sensor for endogenous RNA (Ribonucleic Acid) imaging as well as preparation method and application of genetic coding sensor

A genetically encoded, endogenous technology, applied in biochemical equipment and methods, fusion with RNA binding domains, DNA/RNA fragments, etc., to achieve the effect of improving sensitivity and signal amplification ability

Pending Publication Date: 2021-11-09
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this fluorescent protein strategy requires genetic modification of the target DNA by inserting RNA motifs that bind to degraders, and it remains challenging to develop fluorescent protein methods for direct imaging of endogenous RNA.

Method used

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  • Genetic coding sensor for endogenous RNA (Ribonucleic Acid) imaging as well as preparation method and application of genetic coding sensor
  • Genetic coding sensor for endogenous RNA (Ribonucleic Acid) imaging as well as preparation method and application of genetic coding sensor
  • Genetic coding sensor for endogenous RNA (Ribonucleic Acid) imaging as well as preparation method and application of genetic coding sensor

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1: A degApt having a stem-loop conformation of the present invention is one of degAptE1, degAptE2, degAptE3, and degAptE4.

[0045] Wherein the nucleotide sequence of degAptE1 is shown in SEQ ID NO.1, specifically: GCU CGU UGA GCU CAUUAG CUC CGA GC C ACC GCA U GA AUG UAG AGA UGC GGU GGC.

[0046] The nucleotide sequence of degAptE2 is shown in SEQ ID NO.2, specifically: GCU CGU UGA GCU CAU UAGCUC CG A GC C ACC GCA GAA UGU AGA GA U GCG GUG GCU.

[0047] The nucleotide sequence of degAptE3 is shown in SEQ ID NO.3, specifically: GCU CGU UGA GCU CAU UAGCUC CGA GC C ACC G AA UGU AGA GAU GCG GUG G CU CG.

[0048] The nucleotide sequence of degAptE4 is shown in SEQ ID NO.4, specifically: GCU CGU UGA GCU CAU UAGC UC CGA GCC AC G AAU GUA GAG AUG CG G UGG CUC GGA.

[0049] Among them, GCU CGU UGA GCU CAU UAG CUC CGA GC is degApt, GA AUG UAG AGA UGCGGU GGC is the survivin mRNA recognition sequence, and the underlined part is th...

Embodiment 2

[0052] Example 2: A csiFP sensor of the present invention includes an RNA sensing module and a fluorescent protein reporter module fused with a degradant.

[0053] Wherein the RNA sensing module is degAptE 3 in Example 1, degAptE 3 has a stem-loop conformation, and its stem region is the key to binding with the Tat peptide-based degrader (tDeg).

[0054] The fluorescent protein reporter module is the fluorescent protein EGFP-tDeg fused with tDeg. The amino acid sequence of EGFP-tDeg is shown in SEQ ID NO.9. Among them, SGPRPRGTRGKGRRI: Tat peptide; RRRG: degradant; RPRGTRGKGR: nucleic acid sequence combined with the aptamer, the structural diagram is shown in image 3 .

[0055] The working principle of the csiFP sensor can be found in Figure 4 . It can be seen from the figure that when there is no target RNA (target survivin: GAC CAC CGC AUC UCU ACA UUC A), because the target RNA binding sequence expands degApt and destroys its active conformation, degApt loses the fluor...

Embodiment 3

[0062] Example 3: A csiFP sensor of the present invention includes an RNA sensing module and a fluorescent protein reporter module fused with a degradant. The RNA sensing module is a tRNA lys Scaffold-protected degAptE 3(tRNA lys -degAptE3), its nucleotide sequence is as shown in SEQ ID NO.5, specifically: (Double underlined part indicates degAptE 3 sequence, single underlined part indicates tRNA lys stand). The fluorescent protein reporter module is the fluorescent protein EGFP-tDeg fused with tDeg. Its preparation method is consistent with embodiment 2.

[0063] Experiment 2: Investigate the effect of stent protection.

[0064] Using tRNA in Example 3 lys - No tRNA in degAptE 3 and Example 1 lys Scaffold-protected degAptE 3 for fluorescence imaging of MCF-7 cells. See results Figure 7 .

[0065] Figure 7 For expression of EGFP-tDeg(none), expression of EGFP-tDeg and no tRNA lys Protected sensing module (noscaffold), expresses EGFP-tDeg and has tRNA lysFlu...

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Abstract

The invention discloses a genetic coding sensor for endogenous RNA (Ribonucleic Acid) imaging. The genetic coding sensor comprises an RNA sensing module and a fluorescent protein report module, wherein the RNA sensing module is degApt with stem-loop conformation, and the fluorescent protein report module is fused with fluorescent protein tDeg. According to the invention, target RNA can recover the active conformation of the degApt in the RNA sensing module so as to stabilize the fluorescent protein, so that the target RNA is imaged, and the expression and the dynamic state of the survivin mRNA and the lncRNA MALAT-1 in the living mammalian cell can be imaged. The genetic coding sensor for endogenous RNA imaging can be widely applied to RNA imaging of living cells and animals in biomedical research and clinical treatment.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a genetically coded sensor for endogenous RNA imaging and its preparation method and application. Background technique [0002] RNAs such as mRNA, microRNAs and long noncoding RNAs (long noncoding RNAs, lncRNAs) play important regulatory roles in a variety of biological processes. The abundance and subcellular distribution of RNAs are tightly regulated, and their aberrant expression and localization are associated with the development of different diseases. Imaging and dynamic tracking of RNA abundance and localization are critical for understanding their cellular function and disease treatment. There are two main traditional methods for imaging RNA dynamics, fluorescent proteins that label RNA-binding proteins and DNA-based hybridization probes. However, these methods have certain limitations, such as overexpression of fluorescent protein fusion proteins leading to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65C12N15/62C12N15/115
CPCC12N15/85C12N15/65C07K14/43595C12N15/115C12N2800/107C07K2319/10C07K2319/60C07K2319/85C07K2319/95C12N2310/16C12N2310/531Y02A50/30
Inventor 蒋健晖周文静张可可汪凤林楚霞唐丽娟
Owner HUNAN UNIV
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