Methods of enhancing translocation of charged analytes through transmembrane protein pores

Patent metadata

Field	Value
Publication number	US-9562887-B2
Application number	US-201414334285-A
Country	US
Kind code	B2
Filing date	Jul 17, 2014
Priority date	Nov 14, 2008
Publication date	Feb 7, 2017
Grant date	Feb 7, 2017

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.

First claim

The invention claimed is:

1. A method of decreasing translocation speed of a nucleic acid strand through a transmembrane beta-barrel protein pore, comprising:
(a) increasing the net positive charge of the barrel of the pore by (i) substituting one or more positively charged amino acids into the barrel of the pore or (ii) substituting one or more negatively charged amino acids into the barrel of the pore with one or more uncharged amino acids, non-polar amino acids or aromatic amino acids; and
(b) passing the nucleic acid strand through the pore, wherein increasing the net positive charge decreases the translocation speed of the nucleic acid strand through the pore, and
wherein said transmembrane beta-barrel protein pore is selected from the group consisting of β-toxin, α-hemolysin, leukocidin, and outer membrane porin F (OmpF).

2. A method according to claim 1 , wherein increasing the net positive charge further:
(a) increases the frequency of translocation of the nucleic acid strand through the pore;
(b) decreases the threshold voltage for translocation of the nucleic acid strand through the pore; or
(c) decreases the number of non-translocation interactions between the nucleic acid strand and the pore.

3. A method according to claim 1 , wherein the pore is α-hemolysin, or a variant thereof, and comprises:
(a) seven subunits each comprising SEQ ID NO: 2 or a variant thereof that is at least 95% homologous to the entire length of the amino acid sequence of SEQ ID NO: 2 based on amino acid identity and retains pore forming activity; or
(b) seven subunits each comprising SEQ ID NO: 4 or a variant thereof that is at least 95% homologous to the entire length of the amino acid sequence of SEQ ID NO: 4 based on amino acid identity and retains pore forming activity.

4. A method according to claim 1 , wherein the one or more positively-charge amino acids are histidine (H), lysine (K) and/or arginine (R).

Assignees

• Isis Innovation
• Univ Oxford Innovation Ltd

Inventors

• Maglia Giovanni
• Bayley John Hagan Pryce

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• G01N33/6872

  Intracellular protein regulatory factors and their receptors, e.g. including ion channels · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title

• G01N2333/705

  Assays involving receptors, cell surface antigens or cell surface determinants · CPC title

• G01N27/447

  using electrophoresis · CPC title