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Modified nanopores, compositions comprising the same, and uses thereof

A technology of nanopore and pancreatic juice, applied in the direction of nanotechnology, nanotechnology, nanotechnology, etc. for materials and surface science

Pending Publication Date: 2019-09-20
OXFORD NANOPORE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, translocation of double-stranded or single-stranded DNA through nanopores with inner surfaces facing negatively charged amino acids is ineffective

Method used

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  • Modified nanopores, compositions comprising the same, and uses thereof
  • Modified nanopores, compositions comprising the same, and uses thereof
  • Modified nanopores, compositions comprising the same, and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0296] Example 1: For expression and purification of modified secretin nanopore subunit polypeptides such as modified InvG nano Exemplary Methods of Pore Subunit Polypeptides

[0297] A secretin nanopore subunit polypeptide (for example, amino acid sequence as shown in SEQ ID NO: 1 or 2 containing coding modification, which has one or more (for example, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 25, 30 or more and up to 50 of the amino acid modifications described herein)) of the ampicillin-resistant pT7 vector with a C-terminal hexahistidine (His) tag C43DE3pLysS cells were cotransformed with a kanamycin-resistant pRham vector containing a gene encoding InvH protein (a 20Kd protein that will enhance InvG expression), and in the presence of ampicillin (100 μg / ml) and kanamycin (30 μg / ml ml) agar plates and grown overnight at 37°C. A single colony was used to inoculate a 100 ml starter culture of TB medium containing ampicillin (100 μg / ml) and kanamycin (30 μg / ml). The cultu...

Embodiment 2

[0299] Example 2 for expression and purification of a modified secretin nanopore subunit polypeptide comprising an endopeptidase cleavage site Exemplary method for

[0300]Ampicillin-resistant pT7 vector containing a gene encoding a modified secretin nanopore subunit polypeptide - which contains an endopeptidase cleavage site, such as tobacco etch virus (TEV) protease cleavage site - with a C-terminal hexahistidine tag position (for example, the amino acid sequence shown in SEQ ID NO: 3, which has one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 , 30 or more and up to 50 amino acid modifications described herein)) - transformed into C43DE3pLysS cells and plated on agar plates containing ampicillin (100 μg / ml). A single colony was used to inoculate a 100 ml starter culture of TB medium containing ampicillin (100 μg / ml). The culture was stirred at 250 rpm for 18 hours at 37°C. 15 ml starter culture was used to inoculate 500 ml TB medium containing ampici...

Embodiment 3

[0302] Design, expression and purification of embodiment 3 GspD mutants

[0303] Using the V. cholerae GspD sequence shown in SEQ ID NO: 32 as a starting sequence, GspD mutants were designed as shown in the table below.

[0304] Table 1: DNA capture mutants

[0305]

[0306]

[0307] Table 2: Increase the shrink size of the central door

[0308]

[0309] *Selected as background for further mutational design

[0310] Table 3: Stabilizing Central Doors

[0311]

[0312]

[0313] Table 4: Stabilizing Capgate: Removing Charge

[0314]

[0315] Table 5: Stabilizing Cap Doors

[0316]

[0317] Table 6: Shrinkage and cap extreme mutants

[0318]

[0319]

[0320] GspD mutants were expressed and purified in vitro using the NEB Pure Expression Kit. Set up the reaction as shown below.

[0321] Table 7: Reaction Mixture

[0322] components Volume (μL) Solution A 10 Solution B 7.5 35S methionine 1 rifampicin 0.8 ...

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Abstract

Provided herein relate to modified or mutant forms of secretin and compositions comprising the same. In particular, the modified or mutant forms of secretin permits efficient capture and / or translocation of an analyte through the modified or mutant secretin nanopores. Methods for using unmodified secretin or the modified or mutant forms of secretin and compositions, for example, for characterizing an analyte, e.g., a target polynucleotide, are also provided.

Description

technical field [0001] Provided herein are modified or mutated forms of secretin and compositions comprising the same. Also provided are methods of using modified or mutated forms of secretin and compositions, eg, for characterizing a target analyte, eg, a target polynucleotide. Also provided herein are compositions comprising secretin and an enzyme provided in the lumen of secretin. Background technique [0002] Transmembrane pores such as nanopores have been used to identify small molecules or folded proteins and to monitor chemical or enzymatic reactions at the single-molecule level. Electrophoretic translocation of DNA across nanopores reconstituted into artificial membranes holds great promise for practical applications such as DNA sequencing and biomarker identification. However, translocation of double-stranded or single-stranded DNA through nanopores with inner surfaces facing negatively charged amino acids was ineffective. Contents of the invention [0003] The...

Claims

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Application Information

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IPC IPC(8): C07K14/195
CPCC07K14/195C12Q1/6869B82Y30/00
Inventor 拉科马·贾亚辛格伊丽莎白·杰恩·华莱士普拉提克·拉吉·辛格
Owner OXFORD NANOPORE TECH LTD
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