Methods of enhancing translocation of charged analytes through transmembrane protein pores

A technology for transmembrane protein pores and analytes, which is applied in the field of enhancing the translocation of charged analytes through transmembrane protein pores, and can solve problems such as laboriousness

Active Publication Date: 2012-01-11
OXFORD UNIV INNOVATION LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the course of these studies, attempts to engineer α-HL to directly bind small organic analytes were found to be laborious and with little success (Guan, X., Gu, L.-Q. , Cheley, S., Braha, O., and Bayley, H. (2005) ChemBioChem 6, 1875-1881)

Method used

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  • Methods of enhancing translocation of charged analytes through transmembrane protein pores
  • Methods of enhancing translocation of charged analytes through transmembrane protein pores
  • Methods of enhancing translocation of charged analytes through transmembrane protein pores

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0199] In this example, by increasing the internal positive charge of the α-hemolysin pore and altering its distribution, the inventors increased the capture rate of 92-nt ssDNA at +120mV by approximately 10-fold compared to wild-type pore and significantly decreased the voltage threshold for DNA translocation across the pore, e.g. for 1 event s -1 mM -1 reduced by 50mV. Furthermore, events where DNA entered the pore but was not translocated were virtually eliminated. These genetically engineered nanopores provide the basis for improved nucleic acid analysis and could facilitate ultrahigh-speed nanopore sequencing.

[0200] 1. Materials and Methods

[0201] 1.1 Protein preparation

[0202] α-HL was generated as described elsewhere (Cheley S, Braha O, Lu X, Conlan S, Bayley H: A functional protein pore with a "retro" transmembrane domain. Protein Sci. 1999, 8: 1257-1267). The sequence of one subunit of α-HL is shown in SEQ ID NO:2. Briefly, the protein in [ 35 [S]Met...

Embodiment 2

[0265] In this example, the inventors demonstrated that the translocation of DNA molecules through the α-HL bionanopore can be reduced by two orders of magnitude by introducing a positive charge within the cavity of the pore. Although the ionic currents during DNA translocation are almost completely suppressed, the inventors propose that these nanopores can be used to control the speed of DNA translocation, which in combination with emerging DNA analysis techniques could lead to the development of a robust platform for nucleic acid sequencing.

[0266] 1. Materials and Methods

[0267] The same materials and methods as used in Example 1 were used in this example.

[0268] 2. Results and Discussion

[0269] 2.1. Ion flow through the α-HL nanopore

[0270] Individual channels were examined by single-channel current recordings on planar lipid bilayers in 1 M KCl, 25 mM Tris.HCl, pH 8.0, containing 100 [mu]M EDTA. At +120mV, all channels exhibited a steady open pore curre...

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Abstract

The invention relates to enhancing translocation of a charged analyte through a transmembrane protein pore. Translocation is enhanced by increasing the net opposing charge of the barrel or channel and/or entrance of the pore. The invention also relates to pores enhanced in accordance with the invention.

Description

technical field [0001] The present invention relates to enhanced translocation of charged analytes across transmembrane protein pores. The displacement is enhanced by increasing the net opposite charge of the barrel or channel and / or inlet of the pore. The invention also relates to pores reinforced according to the invention. Background technique [0002] Stochastic detection is a sensing method that relies on the observation of individual binding events between analyte molecules and receptors. Stochastic sensors can be formed by embedding nanometer-sized single pores in insulating membranes and measuring voltage-driven ion transport through the pores in the presence of analyte molecules. The frequency with which current fluctuations occur can reveal the concentration of analyte bound in the pore. The type of analyte is revealed by its characteristic current profile, especially the duration and degree of current blockage (Braha, O., Walker, B., Cheley, S., Kasianowicz, J....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31C12N15/31C12Q1/68G01N33/68G01N27/06
CPCC07K14/245G01N33/6872G01N2333/705C12Q1/6869G01N33/48721C12Q2565/631G01N27/447
Inventor G·玛格里亚J·H·P·贝利
Owner OXFORD UNIV INNOVATION LTD
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