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Enzyme mutant

a technology of enzymolysis and mutants, applied in the field of enzymolysis mutants, can solve the problems of slow and expensive technology of existing

Inactive Publication Date: 2012-04-26
OXFORD NANOPORE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Existing technologies are slow and expensive mainly because they rely on amplification techniques to produce large volumes of nucleic acid and require a high quantity of specialist fluorescent chemicals for signal detection.

Method used

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1 Materials and Methods

1.1 Overview

[0368]One method of sequencing nucleic acids using stochastic sensing involves the use of an exonuclease attached to the α-HL pore. This can be done by introducing a chemical linkage between the two proteins. The chemical linkage joins one cysteine on the exonuclease to one cysteine on the hemolysin, either directly (a disulphide bond), or via a linker molecule between the two cysteine residues.

[0369]In order to introduce the chemical linkage in a controlled and directed approach it was necessary to replace 5 naturally occurring cysteine residues found within Exonuclease I with other residues. An additional mutation would then be made in order to introduce the cysteine residue attachment point. Computer modeling was used to identify suitable attachment positions (see below).

1.2 Modelling Exo I CYS Knockout Mutations

[0370]Molecular modelling has been used in an attempt to predict which mutations of the 5 wild type cysteine residues of E. coli Exo I ...

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Abstract

The invention relates to constructs comprising a nucleic acid binding protein and a surface. At least one native accessible cysteine residue is removed from the binding protein. The binding protein is attached to the surface via one or more accessible cysteine residues. The removal of other accessible cysteine residues from the protein allows control attachment to the surface. The constructs can be used to generate transmembrane pores having a nucleic acid binding protein attached thereto. Such pores are particularly useful for sequencing nucleic acids. The enzyme handles the nucleic acid in such a way that the pore can detect each of its component nucleotides by stochastic sensing.

Description

FIELD OF THE INVENTION[0001]The invention relates to constructs comprising a nucleic acid binding protein and a surface. At least one native accessible cysteine residue is removed from the binding protein. The binding protein is attached to the surface via one or more accessible cysteine residues. The removal of other accessible cysteine residues from the protein allows control attachment to the surface. The constructs can be used to generate transmembrane pores having a nucleic acid binding protein attached thereto. Such pores are particularly useful for sequencing nucleic acids. The enzyme handles the nucleic acid in such a way that the pore can detect each of its component nucleotides by stochastic sensing.BACKGROUND OF THE INVENTION[0002]Stochastic detection is an approach to sensing that relies on the observation of individual binding events between analyte molecules and a receptor. Stochastic sensors can be created by placing a single pore of nanometer dimensions in an insulat...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12N9/16C07H21/04C12N11/00
CPCC07K14/31C07K2319/03C12N9/90C12N9/22C12N9/1241C07K19/00C07K1/00C12N15/11
Inventor MOYSEY, RUTHKNAGGS, MICHAELJAYASINGHE, LAKMALWHITEMCKEOWN, BRIANMILTON, JOHN
Owner OXFORD NANOPORE TECH LTD
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