Translocation control for sensing by a nanopore
Translocation control for sensing by a nanopore, as well as methods and products related to the same, are provided. Such methods optimize duplex stability to provide high fill rate (of the hybridization sites) but do not prevent rapid dissociation required for high read rates, as well as controlling the translocation of a target molecule for sensing by a nanopore by use of a selective pulsed voltage. Products related to the same include a reporter construct comprising two or more phosphoramidites.
1. A method for controlling the translocation of a target molecule for sensing by a nanopore, comprising passing the target molecule through the nanopore subjected to a base line voltage and a pulsed voltage, wherein the target molecule comprises two or more features which provide translocation control by steric hindrance, and wherein the periodicity of the pulsed voltage ranges from between greater than 100 Hz (10 milliseconds) to 4,000 Hz (250 microseconds).
2. The method of claim 1 , wherein the pulsed voltage is sufficient to allow translocation of the feature engaged with the nanopore by steric hindrance, while leaving the next feature of the target molecule free to engage with the nanopore.
3. The method of claim 2 , wherein the feature of the target molecule engaged with the nanopore by steric hindrance translocates upon each pulse of the pulsed voltage.
4. The method of claim 2 , wherein the feature of the target molecule engaged with the nanopore by steric hindrance translocates upon multiple pulses of the pulsed voltage.
5. The method of claim 1 , wherein the target molecule is sensed by the nanopore during the time period between pulses of the pulsed voltage.
6. The method of claim 1 , wherein the periodicity of the pulsed voltage is 500 Hz (2 milliseconds).
7. The method of claim 1 , wherein the pulsed voltage has a duration of less than 100 microseconds.
8. The method of claim 1 , wherein the pulsed voltage has a duration of less than 50 microseconds.
9. The method of claim 1 , wherein the pulsed voltage has a duration of less than 10 microseconds.
10. The method of claim 1 , wherein the pulsed voltage has a duration of less than 5 microseconds.
11. The method of claim 1 , wherein the pulsed voltage has a duration of less than 1 microsecond.
12. The method of claim 1 , wherein the pulsed voltage has a voltage of greater than 0.2 volts.
13. The method of claim 1 , wherein the pulsed voltage has a voltage of greater than 0.5 volts.
14. The method of claim 1 , wherein the pulsed voltage is 1 volt.
15. The method of claim 1 , wherein the pulsed voltage has a voltage of greater than 1 volt.
16. The method of claim 1 , wherein the pulsed voltage has a voltage of greater than 10 volts.
17. The method of claim 1 , wherein the target molecule comprises a reporter construct.
18. The method of claim 17 , wherein the reporter construct comprises a low impedance polymer that produces an I/I o value of 0.27 or greater upon translocation through the nanopore, and a high impedance polymer that produces an I/I o value of less than 0.27 upon translocation through the nanopore.
19. The method of claim 17 , wherein the reporter construct comprises triethylene glycol (X), hexaethylene glycol (D), ethane (L), hexane (P), dodecane (Z), or abasic (Q), or combinations thereof.
20. The method of claim 17 , wherein the reporter construct comprises deoxyadenosine (A), deoxythymine (T), deoxycytosine (C), or deoxyguanodine (G), or combinations thereof.
21. The method of claim 17 , wherein the reporter construct comprises two or more phosphoramidites selected from the group consisting of triethylene glycol (X), hexaethylene glycol (D), ethane (L), hexane (P), dodecane (Z), deoxyadenosine (A), deoxythymine (T), deoxycytosine (C), deoxyguanodine (G) and abasic (Q), and wherein the reporter construct comprises at least one of X, D, L, P, Z or Q.