High contrast signal to noise ratio device components
Provided are device components, devices and methods characterized by a high contrast signal to noise ratio (CNR).
1. A device comprising a nanopore comprising a modified Staphylococcus aureus alpha hemolysin polypeptide, wherein:
non-native amino acids are substituted for native amino acids at positions M113, E111, K147 and T145 of a wild-type Staphylococcus aureus alpha hemolysin polypeptide, wherein the amino acid positions of the wild-type Staphylococcus aureus alpha hemolysin polypeptide align with corresponding positions of the amino acid sequence of the Staphylococcus aureus alpha hemolysin polypeptide of SEQ ID NO. 1; and
the modified alpha hemolysin polypeptide permits measurement of a residual current of the nanopore, as a polymer translocates through the nanopore, with a contrast signal to noise ratio (CNR) of two (2) or greater.
2. The device of claim 1 , wherein the nanopore further comprises a non-native amino acid substitution at position L135 of a wild-type Staphylococcus aureus alpha hemolysin polypeptide, wherein the amino acid position of the wild-type Staphylococcus aureus alpha hemolysin polypeptide aligns with a corresponding position of the amino acid sequence of the Staphylococcus aureus alpha hemolysin polypeptide of SEQ ID NO. 1.
3. The device of claim 1 , wherein the non-native amino acids are chosen from: an amino acid that reduces the charge compared to the native amino acid, an amino acid that eliminates the charge of the side chain of the native amino acid, an amino acid that reduces hydrogen bonding between the amino acid and the polymer compared to the native amino acid and the polymer, an amino acid smaller in size than the native amino acid or an amino acid that changes hydrophobic interactions between the amino acid and the polymer, wherein the substitution increases the measured CNR.
4. The device of claim 1 , wherein at least one of the non-native amino acids that are substituted for native amino acids comprise serine.
5. The device of claim 2 , wherein the non-native amino acid is isoleucine.
6. The device of claim 2 , wherein amino acid substitution at position L135 is isoleucine and the amino substitutions at positions M113, E111, K147 and T145 are serine.
7. The device of claim 1 , wherein non-native amino acids are further substituted for native amino acids at one or more positions comprising N121, N123, T125, D127, T129, K131, G133, G137 and N139 of a wild-type Staphylococcus aureus alpha hemolysin polypeptide, wherein the amino acid positions of the wild-type Staphylococcus aureus alpha hemolysin polypeptide align with corresponding positions of the amino acid sequence of the Staphylococcus aureus alpha hemolysin polypeptide of SEQ ID NO. 1.
8. The device of claim 1 , wherein the CNR is 3.0 or greater.
9. The device of claim 1 , wherein the polymer is DNA or RNA.
10. The device of claim 1 , wherein the polymer is single stranded DNA.
11. The device of claim 1 , wherein the polymer is a homopolymer.
12. The device of claim 1 , comprising a lipid bilayer in which the nanopore resides.
13. The device of claim 1 , comprising a membrane in which the nanopore resides.
14. The device of claim 1 , wherein the rate of translocation of a polymer through the nanopore is controlled.
15. The device of claim 14 , wherein the device comprises a polymerase, an exonuclease, a single stranded binding protein, a helicase and/or one or more hybridizing segments of complementary strands of DNA with a portion of the polymer, to control the rate of translocation of the polymer through the nanopore.
16. The device of claim 14 , wherein the device comprises an electrolyte with an additive, to control the rate of translocation of the polymer through the nanopore.
17. The device of claim 14 , wherein the device comprises a DC measurement system able to apply a DC bias across the nanopore.
18. The device of claim 14 , wherein the device comprises an AC measurement system able to apply an AC bias across the nanopore.