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Probe for capturing RNAm<1>A modified binding protein and method thereof

A technology for binding proteins and probes, applied in the field of molecular biology, can solve problems such as increasing decay and reducing the stability of m6A-modified mRNA, and achieve the effects of preventing pollution, saving experimental costs and saving time.

Active Publication Date: 2020-04-10
SHANGHAI FIRST MATERNITY & INFANT HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

YTH domain-containing protein 3 (YTHDF3) and YTHDF1 promote protein synthesis by interacting with protein translation machinery, increasing the decay of mA-containing RNA, but YTHDF2 reduces the stability of mA-modified mRNA and inhibits mRNA translation

Method used

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  • Probe for capturing RNAm&lt;1&gt;A modified binding protein and method thereof
  • Probe for capturing RNAm&lt;1&gt;A modified binding protein and method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1 is used for RNAm 1 A Design Method for Probes Captured by Modified Binding Proteins

[0043] It is characterized in that, comprising the following steps:

[0044] 1) Base sequence design of the methylation probe:

[0045] 5′-biotin-ACCCGUCUUG(m 1 A) AACACGGCCGUUG(m 1 A) UCACGUC-3';

[0046] Base sequence design of control non-methylated probe:

[0047] 5′-biotin-ACCCGUCUUGAAACACGGCCGUUGAUCACGUC-3′;

[0048] 2) The sequence inserted between the 5' end of the probe and the first stem, between the 3' end and the second stem, and between the first stem and the second stem is a human genome irrelevant sequence, and the inserted The sequence is not complementary to other sequences in the probe.

[0049] The probe is composed of a base stem-loop structure, and the 3' or 5' of the probe is labeled with biotin, which can bind to the avidin on the magnetic beads; the length of the probe is 32bp; the probe The length from the 5' end of the probe to the first ste...

Embodiment 2

[0054] A kind of embodiment 2 utilizes the probe in implementing 1 to capture RNAm 1 A method of modifying the binding protein

[0055] Specific steps are as follows:

[0056] (1) Preparation of probe-magnetic bead complex: wash 80 μL streptavidin-labeled magnetic beads with 1 mL 1×TBS, remove the TBS washing solution, and then add 1 mL 1×TBS containing BSA and tRNAs to the magnetic beads, After incubation at room temperature for 30 min, the TBS solution was removed. Add 2-5μg DNA probe to the pretreated magnetic beads, add 100μL 1×TBS, 5μL 10U / μL RNase inhibitor, mix gently at 4°C for 30-60min, remove the supernatant; wash the magnetic beads with 1×TBS Beads twice to remove TBS.

[0057] (2) Extract and pretreat total cell protein: wash 2×10 with 1×PBS 7 Each cell (Raw264.7 and 293T) was centrifuged twice at 4°C and 1200rpm for 5 min each time, and the supernatant was discarded; 800 μL of pre-cooled lysis and binding buffer, 8 μL of 10 mg / mL PMSF solution, 5 μL of 10 U / μL...

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Abstract

The invention discloses a probe for capturing RNAm<1>A modified binding protein and a method thereof. The probe is composed of a base stem-loop structure, the length of the probe is 32-632 base, and 3'or 5 'of the probe is provided with a biotin label, and the probe can be bound with avidin on a magnetic bead. The probe is coupled to the magnetic beads by desulfurized biotin labelling and streptavidin and is in affinity binding with desulfurized biotin. The cell extraction total protein is incubated with a magnetic bead-probe compound and the acting protein can be specifically bound with an RNAm<1>A modified probe; the non-specific binding protein can be removed through washing; and finally, eluting is performed with an eluent to obtain a probe-protein compound. According to the specific RNAm<1>A modified protein and a secondary structure, the RNAm<1>A modified binding protein can be captured effectively and the enrichment of the specific binding protein is remarkably improved.

Description

【Technical field】 [0001] The invention belongs to the technical field of molecular biology, in particular to a method for RNAm 1 A probe and method for capturing modified binding proteins. 【Background technique】 [0002] More than 100 post-transcriptional modifications can occur in eukaryotic RNA, which can regulate RNA splicing, localization, stability, binding and translation (Gilbert WV, Bell TA, Schaening C. Messenger RNA modifications: Form, distribution, and function. Science 352, 1408-1412(2016)). Adenosine (m6A) N6 methylation is involved in the epigenetic regulation of gene expression. N1-methyladenosine (m1A) is a common methylation modification at the N1 position on RNA, which is found in many tRNAss. Recent transcriptome maps also show that m1A modification also exists in human mRNA, suggesting that m1A plays a potential role in regulating mRNAs splicing and translation. At the same time, m1A modification will also undergo dynamic changes under external heat o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/02C07K1/14
CPCC07H21/02C07K1/14
Inventor 郑青亮金莉萍
Owner SHANGHAI FIRST MATERNITY & INFANT HOSPITAL
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