Embedding adapters, anchor molecules, molecular membranes, devices, methods and applications

A molecular membrane and molecular technology, applied in chemical instruments and methods, biochemical equipment and methods, and microbial determination/inspection, etc., can solve problems such as affecting the detection effect, the length of effective current data output, and the difficulty of detecting biomolecules. To achieve the effect of increasing the output time

Active Publication Date: 2022-04-15
QITAN TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing anchor molecules are usually cholesterol with tethers, and the cholesterol is evenly distributed on the molecular membrane, while the nanopore is only set at the amphiphilic molecular membrane of the molecular membrane, such as a phospholipid membrane or a polymer membrane, causing some parts to be far away from the nanometer membrane. The biomolecules in the pores are not easy to be detected, especially when the concentration of biomolecules is low, the effective current data output time is short, which affects the detection effect

Method used

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  • Embedding adapters, anchor molecules, molecular membranes, devices, methods and applications
  • Embedding adapters, anchor molecules, molecular membranes, devices, methods and applications
  • Embedding adapters, anchor molecules, molecular membranes, devices, methods and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1: Preparation of an anchor molecule

[0100] Design and synthesize linker, its nucleotide sequence is as follows:

[0101] GGTCGGTGCTGGACTTTTTTTTTTTTTTTTTTTTT-DBCO;

[0102] Design and synthesize the embedded linker, its amino acid sequence is as follows:

[0103] N3-WERNLP- SVSGLLKIIGFSTSVTALGFVLY -KYKLL PRS.

[0104] In this embodiment, the connection linker is connected to the analyte through the capture chain, the insertion linker is a polypeptide, and the connection linker and the insertion linker are connected through "DBCO" and "N3". see Figure 4 , is the predicted map of the polypeptide transmembrane region, from Figure 4 It can be seen that the 7th to 29th amino acids in the amino acid sequence (underlined in the amino acid sequence) are transmembrane regions, indicating that the anchor molecule can penetrate the amphiphile membrane.

[0105] The connecting adapter is incubated with the embedding adapter, and the incubated product is collected...

Embodiment 2

[0106] Example 2: Preparation and detection of an embedded joint

[0107] Design and synthesize the embedded linker, its amino acid sequence is as follows:

[0108] DBCO-YIK- LFIMIVGGLVGLRIVFAVLSIVN -RVRQ.

[0109] see Figure 7 , is the prediction map of the transmembrane region of the polypeptide, wherein amino acids 4 to 26 (underlined in the amino acid sequence) are transmembrane regions.

[0110] Connect the fluorophore FAM or Cy5 to the embedding adapter, add a buffer solution (500 mM KCl, 20 mM HEPES, pH 8.0) into the reservoir chamber of the biochip of the gene sequencer QNome-9604 of Qitan Technology Co., Ltd., and then Add 40 μl of silicone oil and phosphatidylcholine (PC, 10mg / mL) mixed oil, and finally add the aforementioned buffer solution to prepare a molecular film, the structure of which is as follows: figure 2 Shown; Mix the embedded linker with the fluorescent group and the molecular membrane, so that the concentration of the embedded linker in the solu...

Embodiment 3

[0112] Example 3: Nanopore sequencing of analytes using anchor molecules

[0113] (1) Set up an experimental group and a control group. In the experimental group and the control group, the same biochip was used, and the molecular film was prepared according to the method in Example 2;

[0114] (2) On the molecular membrane of the experimental group, add the anchor molecule prepared in Example 1, so that the concentration of the embedded linker in the solution on the adding side of the molecular membrane is 0.5 μM, and incubate at room temperature for 60 minutes; on the molecular membrane of the control group , without adding any anchor molecules, incubate at room temperature for 60 minutes;

[0115] (3) Samples of the same concentration were added to both the experimental group and the control group, and the gene sequencer QNome-9604 of Qitan Technology Co., Ltd. was used to sequence the ssDNA in the samples to obtain the via signal map.

[0116] see Figure 10 , Figure 11...

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Abstract

The application discloses an embedding linker, an anchor molecule, a molecular membrane, a device, a method and an application. Among them, the embedded joint is used to be embedded in the amphiphilic molecular membrane, and the embedded joint includes a first hydrophilic segment, a hydrophobic segment and a second hydrophilic segment connected in sequence, the hydrophobic segment is used to span the hydrophobic region of the amphiphilic molecular membrane, and the hydrophobic The segment is rigid; the first hydrophilic segment and / or the second hydrophilic segment are used to attach to the analyte. The embedded linker provided by this application can be enriched in the amphiphilic molecular membrane, increasing the effective detection time of the analyte.

Description

technical field [0001] The present application belongs to the field of gene sequencing technology, and in particular relates to a device for embedding adapters, anchor molecules, molecular membranes, and nanopores to characterize biomolecules, a method for assembling embedded adapters in amphiphilic molecular membranes, and enriching biomolecules into transmembrane nanopores. Methods for analytes, methods and applications for nanopore characterization of analytes. Background technique [0002] In existing nanopore sequencing, in a cavity filled with electrolyte, a molecular membrane with nanoscale pores divides the cavity into two small chambers. When a voltage is applied to the electrolyte, ions or other small molecular substances can pass through the small chambers. The pores form a stable and detectable ionic current, and different types of biomolecules can be detected according to the current information. [0003] Anchor molecules are provided in existing molecular memb...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12Q1/6869
CPCC07K14/00C12Q1/6869C12Q2525/191C12Q2565/631
Inventor 刘少伟张子朋朱丽梅
Owner QITAN TECH LTD
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