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Difunctional marking method of 4-sulfydryl uracil and application of difunctional marking method in sequence enrichment and single base resolution sequencing

A technology of mercaptouracil and labeling method, applied in biochemical equipment and methods, microbial determination/inspection, organic chemistry, etc., can solve problems such as non-enrichment, and achieve the effect of high reaction efficiency and good biocompatibility

Pending Publication Date: 2022-04-15
XI AN JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a dual-functional labeling method for 4-mercaptouracil and its application in sequence enrichment and single-base resolution sequencing, which can solve the problem of only enrichment without single-base resolution or single-base resolution in the prior art. Pitfalls of performing single-base resolution without enrichment

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  • Difunctional marking method of 4-sulfydryl uracil and application of difunctional marking method in sequence enrichment and single base resolution sequencing
  • Difunctional marking method of 4-sulfydryl uracil and application of difunctional marking method in sequence enrichment and single base resolution sequencing
  • Difunctional marking method of 4-sulfydryl uracil and application of difunctional marking method in sequence enrichment and single base resolution sequencing

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Embodiment 1

[0036] Using the nucleophilic substitution reaction of E-IAA to 4-mercaptouracil, the dual-functional labeling of 4-mercaptouracil and its nucleic acid samples can be realized, which can not only realize the enrichment of target sequences but also perform single-base resolution on them.

[0037] In vitro transcription of RNA with 4-mercaptouracil, the specific process is: sequence cDNA Sigle A or cDNASigle G or cDNA mult A or cDNA mult G and cDNA T7 for DNA extension, followed by transcription under the action of T7 RNA polymerase containing 4- The RNA of mercaptouracil, the nucleophilic substitution reaction of RNA and E-IAA. The reaction conditions are 10% DMSO aqueous solution as a solvent, sodium phosphate buffer with a pH value equal to 8, and reaction at 50° C. for 30 min. After the reaction, the RNA was recovered by alcohol precipitation. The labeled RNA was reacted with rSAP and RNAse I at 37°C for 12 hours to degrade the nucleic acid into nucleosides. Finally, vario...

Embodiment 2

[0039] The nucleophilic substitution reaction of 4-mercaptouracil by using E-IAA described in the present invention is used to perform single base resolution on the nucleic acid sequence of 4-mercaptouracil.

[0040] The RNA with 4-mercaptouracil was transcribed in vitro, and the RNA was reacted with E-IAA. The reaction conditions were 10% DMSO aqueous solution as solvent, sodium phosphate buffer with a pH value equal to 8, and reacted at 50° C. for 30 minutes. After the reaction, the RNA was recovered by alcohol precipitation. The primers used for reverse transcription of labeled RNA are RT primer, and the primers used for PCR are PCRfp1, PCR rp1, PCR fp2 and PCR rp2. PCR products were characterized by 15% PAGE and sequencing. A mutation from T to C has occurred at the original position of 4-mercaptouracil. Therefore, this method realizes the single base recognition of 4-mercaptouracil. Such as figure 2 As shown, 15% PAGE and one-generation sequencing characterize the sin...

Embodiment 3

[0042] After enrichment of low-abundance 4sU-RNA by using the E-IAA described in the present invention to 4-mercaptouracil bifunctional chemical labeling, single-base resolution can still be performed.

[0043] RNAs containing 4-mercaptouracil and uracil were transcribed in vitro, and the sequences used to transcribe uracil RNA were q-pcr-U-cDNA mult-A and q-pcr-U-cDNAT7. The two RNAs were mixed at a ratio of 1 / 10, 1 / 100, 1 / 1000, 1 / 10000 and 1 / 20000, respectively, and the mixed samples were subjected to E-IAA reaction under standard conditions. Next, the purified RNA was subjected to click chemistry with an azide compound with disulfide bond biotin, so that 4sU-RNA was biotinylated. Streptavidin magnetic beads were used to enrich the RNA, and finally the DTT eluent was used to release the RNA from the magnetic beads. By calculating the ratio of the two RNAs after enrichment, the enrichment factor of 4sU-RNA by this method can be obtained. The sequences used for reverse trans...

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Abstract

The invention discloses a bifunctional labeling method of 4-sulfydryl uracil and application of the bifunctional labeling method in sequence enrichment and single base resolution sequencing, and belongs to the technical field of nucleic acid chemistry. N-hexyl-5-alkynyl-2-iodoacetamide (E-IAA) is utilized to perform nucleophilic substitution reaction on 4-sulfydryl uracil so as to realize labeling of double functions, and the double functions are reflected as follows: alkynyl at the tail end can be efficiently crosslinked with an azide group with biotin under a mild condition, so that enrichment of a sequence containing 4-sulfydryl uracil is realized; the N-H bond at the near end can change the original base hydrogen bond pairing mode of 4-sulfydryl uracil, and mutation from T to C is generated at the position of 4-sulfydryl uracil, so that single base distinguishing of 4-sulfydryl uracil and nucleic acid derivatives is realized. The bifunctional labeling reaction efficiency is high, and the biological compatibility is good.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid chemistry, and in particular relates to a dual-function labeling method of 4-mercaptouracil and its application in sequence enrichment and single-base resolution sequencing. Background technique [0002] RNA modifications are ubiquitous in living organisms and have important regulatory functions in fundamental biological processes. Since the abundance of different types of RNA modifications is very different, more and more sequencing technologies are used to detect RNA modifications in the transcriptome. Among these RNA modifications, the corresponding derivative of 4-mercaptouracil, 4-mercaptouridine, naturally exists in Escherichia coli. In addition, 4-mercaptouridine can be widely used in metabolic labeling of mammalian cells to study the dynamics of cellular transcriptomes. Among them, during the metabolic labeling process, only less than 1% of the total RNA or less 4-mercaptouridine is...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C07D403/12C07D239/56
Inventor 赵永席陈锋苏丽
Owner XI AN JIAOTONG UNIV
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