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 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 at least a first level and a second level within 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 computed at a predetermined filter frequency;
which polymer comprises two or more sections, each of which sections comprises at least a portion of a monomer;
which CNR is calculated as a contrast signal divided by a total noise value; and
which contrast signal is calculated as the absolute difference between the first level and the second level, wherein:
each level used for calculating the CNR correlates to a composition of a section of the polymer,
each level is an average of measurements within the residual current for each section of the polymer,
the first level and the second level are measurably distinct, and
at least one of the first level and the second level has an average duration of one millisecond or less; and
which total noise value is the square root of the sum of the squares of a first average root mean square noise value of the first level at the predetermined filter frequency and a second average root mean square noise value of the second level at the predetermined filter frequency.
2. The nanopore of claim 1 , further comprising 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 nanopore of claim 1 , wherein the average of measurements of the first level and the second level are computed at the predetermined filter frequency and one half of a time period of the predetermined filter frequency is removed from the beginning and end of first levels and second levels prior to computing the first level and second level.
4. The nanopore of claim 1 , which permits measurement of the first level and the second level for at least two sets of two distinct sections within the polymer with a CNR of 2 or greater.
5. The nanopore of claim 1 , wherein the CNR is 3.0 or greater.
6. The nanopore of claim 1 , wherein the predetermined filter frequency is 10 kHz.
7. The nanopore of claim 1 , wherein data is filtered to the predetermined filter frequency using a low pass 3 pole Bessel filter.
8. The nanopore of claim 1 , wherein the polymer is DNA or RNA.
9. The nanopore of claim 8 , wherein the polymer is single stranded DNA.
10. The nanopore of claim 1 , wherein the average duration is at most 500 microseconds.
11. The nanopore 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 DNA, wherein the substitution increases the CNR.
12. The nanopore of claim 1 , wherein the first level and the second level are computed from at least 50 measurements.
13. The nanopore of claim 1 , wherein the CNR at the predetermined filter frequency is calculated under test conditions comprising an electrolyte, a known polymer, an applied bias and a buffer, and wherein the electrolyte is 3 M sodium chloride, the buffer is 10 mM Tris and 1 mM EDTA, the known polymer is a polyA40C80A40 single stranded DNA and the applied bias is 120 mV.
14. The nanopore of claim 1 , wherein the first level and the second level are correlated to each corresponding composition of the respective sections of the polymer by measuring the residual current of a first homopolymer and a second homopolymer translocating the nanopore where the first homopolymer comprises the same monomer or monomers in a first section of the polymer that produced the first level used to compute the CNR and the second homopolymer comprises the same monomer or monomers in a second section of the polymer that produced the second discrete level used to compute the CNR.
15. The nanopore of claim 1 , wherein the discrete levels are correlated to each corresponding composition of the sections of the polymer by measuring the residual current of a first homopolymer and a second homopolymer immobilized within the nanopore where the first homopolymer is comprised of the same monomer or monomers that comprises the first section of the polymer that produced the first discrete level used to compute the CNR and the second homopolymer is comprised of the same monomer or monomers that comprises the second section of the polymer that produced the second discrete level used to compute the CNR.
16. The nanopore of claim 1 , wherein the first level and the second level are correlated to each corresponding composition of the respective sections of the polymer by mapping the nanopore with an immobilized polymer that comprises the same monomer or monomers in the sections of the polymer that produce the first discrete level used to compute the CNR and the second discrete level used to compute the CNR.
17. The nanopore of claim 11 , wherein the substituted non-native amino acid comprises an amino acid that reduces the charge compared to the native amino acid.
18. The nanopore of claim 11 , wherein the substituted non-native amino acid comprises an amino acid that eliminates the charge of the side chain of the native amino acid.
19. The nanopore of claim 11 , wherein the substituted non-native amino acid comprises an amino acid that reduces hydrogen bonding between the amino acid and the polymer compared to the native amino acid and the polymer.
20. The nanopore of claim 11 , wherein the substituted non-native amino acid comprises an amino acid smaller in size than the native amino acid.
21. The nanopore of claim 11 , wherein the substituted non-native amino acid comprises an amino acid that changes hydrophobic interactions between the amino acid and the DNA.
22. The nanopore of claim 1 , wherein at least one of the non-native amino acids that are substituted for native amino acids comprise serine.
23. The nanopore of claim 22 , wherein all the non-native amino acids that are substituted for native amino acids comprises serine.
24. The nanopore of claim 23 , further comprising 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.
25. The nanopore of claim 24 , wherein the non-native amino acid is isoleucine.
26. The nanopore 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.
27. The nanopore of claim 2 , 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.
28. The nanopore of claim 14 , wherein the first section does not comprise the same monomer and the second section does not comprise the same monomer.
29. The nanopore of claim 14 , wherein the first and the second levels of the residual current are each associated with the measurement of a single monomer.
30. The nanopore of claim 14 , wherein the first section and the second section of the polymer comprise different monomers.