Nanopore biosensors for detection of proteins and nucleic acids
Described herein are nanopore biosensors based on a modified cytolysin protein. The nanopore biosensors accommodate macromolecules including proteins and nucleic acids, and may additionally comprise ligands with selective binding properties.
1. A nanopore sensor comprising a modified ClyA pore, wherein the modified ClyA pore comprises at least 12 subunits, each subunit comprising a polypeptide represented by an amino acid sequence with at least 80% identity to SEQ ID NO:1 wherein the amino acid at position 103 (D) is substituted with a cysteine residue and wherein the position of the amino acid substitution is numbered with reference to an amino acid sequence as set forth in SEQ ID NO:1, starting from the second amino acid residue.
2. The nanopore sensor according to claim 1 , wherein the nanopore sensor further comprises a ligand that confers selective binding properties to an analyte.
3. The nanopore sensor according to claim 2 , wherein the ligand is an aptamer.
4. The nanopore sensor according to claim 3 , wherein the aptamer is a nucleic acid aptamer or a peptide aptamer that is selected to bind to a specific target protein analyte.
5. The nanopore sensor according to claim 2 , wherein the ligand is an antibody.
6. The nanopore sensor according to claim 2 , wherein the ligand is a receptor.
7. The nanopore sensor according to claim 2 , wherein the ligand is a peptide.
8. The nanopore sensor according to claim 4 , wherein the protein analyte binds within the lumen of the modified ClyA pore.
9. The nanopore sensor according to claim 4 , wherein the protein analyte is a protein with a molecular weight in the range of 15-70 kDa.
10. The nanopore sensor according to claim 8 , wherein the protein analyte is a protein with a molecular weight in the range of 15-70 kDa.
11. The nanopore sensor according to claim 1 , wherein the lumen of the modified ClyA pore is modified to alter the size, binding properties, and/or structure of the pore.
12. The nanopore sensor according to claim 2 , wherein the modified ClyA pore is attached to the ligand.
13. The nanopore sensor according to claim 12 , wherein the modified ClyA pore is attached to the ligand via a disulfide linkage.
14. The nanopore sensor according to claim 12 , wherein the modified ClyA pore is attached to the ligand via cross-linking.
15. The nanopore sensor according to claim 12 , wherein the modified ClyA pore is attached to the ligand via chemical ligation.
16. The nanopore sensor according to claim 2 , wherein the modified ClyA pore is not attached to the ligand.