Target detection with nanopore
Provided are methods and compositions for detecting a target molecule or particle suspected to be present in a sample using a polymer scaffold, a fusion molecule, and a pore.
1. A method of detecting the presence or absence of a plurality of distinct target molecules in a sample, comprising:
a) contacting a sample containing a plurality of distinct target molecules with a plurality of polymer scaffold-bound fusion molecules, wherein each fusion molecule comprises a distinct target molecule binding domain capable of selectively binding to a distinct target molecule, and further comprising a polymer scaffold binding domain, wherein said polymer scaffold binding domain is permanently bound to said polymer scaffold, and wherein each of the polymer scaffolds comprises a scaffold barcode for distinguishing between each of the polymer scaffolds;
b) loading the collection of polymer scaffolds into a device comprising a pore that separates an interior space of the device into two volumes, and configuring the device to pass each of the polymer scaffolds through the pore from one volume to the other volume, wherein the device comprises a sensor for identifying objects passing through the pore; and
c) determining, with the sensor, whether each distinct target molecule binding domain of each of the fusion molecules is bound to the distinct target molecule upon translocation through the pore, thereby detecting the presence or absence of the plurality of distinct target molecules in the sample.
2. The method of claim 1 , wherein the polymer scaffold comprises a polynucleotide or a polypeptide.
3. The method of claim 2 , wherein said polynucleotide comprises a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or a peptide nucleic acid (PNA).
4. The method of claim 1 , wherein the target molecule is selected from the group consisting of: a protein, a peptide, a polynucleotide, a chemical compound, an ion, and an element.
5. The method of claim 1 , wherein the determining step is performed 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 or more times for each sample.
6. The method of claim 1 , wherein said polymer scaffold is covalently linked to said fusion molecule.
7. The method of claim 1 , wherein said polymer scaffold is cross-linked to said fusion molecule.
8. The method of claim 1 , wherein the fusion molecule comprises a polymer scaffold binding domain.
9. The method of claim 8 , wherein said polymer scaffold comprises a polynucleotide.
10. The method of claim 9 , wherein said polymer scaffold binding domain binds to the backbone of the polynucleotide.
11. The method of claim 9 , wherein said polymer scaffold binding domain binds a chemically modified region of said polymer scaffold.
12. The method of claim 11 , wherein said chemical modification is selected from the group consisting of: acetylation, methylation, summolation, glycosylation, phosphorylation, biotinylation, and oxidation.
13. The method of claim 9 , wherein said polymer scaffold binding domain recognizes and binds a sequence of no more than 6, 5, 4, 3, or 2 nucleotides.
14. The method of claim 9 , wherein said polymer scaffold binding domain recognizes and binds a sequence of the polymer scaffold that is present in an average probability frequency of at least once every 20,000, 10,000, 5,000, 2,000, 1,000, 500, 200, or 100 base pairs.
15. The method of claim 9 , wherein said polymer scaffold binding domain recognizes and binds a sequence of the polymer scaffold that is present in naturally occurring frequency of at least once every 20,000, 10,000, 5,000, 2,000, 1,000, 500, 200, or 100 base pairs.
16. The method of claim 1 , wherein said polymer scaffold comprises a non-engineered binding domain recognized by the polymer scaffold binding domain of the fusion molecule.
17. The method of claim 1 , wherein the sensor comprises electrodes configured to apply a voltage differential between the two volumes and measure current flow through the pore.
18. The method of claim 1 , wherein the device comprises an upper chamber, a middle chamber and a lower chamber,
wherein the upper chamber is in communication with the middle chamber through a first pore, and the middle chamber is in communication with the lower chamber through a second pore;
wherein the first pore and second pore are configured to simultaneously translocate the same polymer scaffold; and
wherein each of the chambers comprises an electrode for connecting to a power supply.
19. The method of claim 1 , further comprising applying independent voltages across each pore with a common ground in the middle chamber to capture the polymer first into both pores, and subsequently using voltage control logic to move and control the polymer scaffold in any direction after the fusion molecule bound to the polymer scaffold passes through the pore, to detect and re-detect whether the fusion molecule bound is bound to a target molecule or particle.