Encoded Nanopore Sensor for Multiplex Nucleic Acids Detection

• US 20140309129A1
• Filed: 03/14/2014
• Published: 10/16/2014
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Curators of the University of Missouri (University of Missouri)
• Original Assignee: Curators of the University of Missouri (University of Missouri)
• Inventors: Gu, Li-Qun, Zhang, Xinyue
• Granted Patent: US 9,732,379 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68

Abstract

The present invention provides a new and improved multiplexed oligonucleotide detection method based on the nanopore technology with one or more probes containing a sequence with complementarity to the target oligonucleotide, a terminal extension at the probe'"'"'s 3′ terminus, 5′ terminus, or both termini and a label attached to the terminus. The improved probes and probe sets enable sensitive, selective, and direct multiplex detection, differentiation and quantification of distinct target oligonucleotides such as miRNAs. The inventive detection method may also be employed as a non-invasive and cost-effective diagnostic method based on miRNA levels in the patient'"'"'s tissue sample.

Claims

1. A set of probe molecules comprising at least a first probe molecule and a second probe molecule, wherein at least two probe molecules comprise:

a) a capture domain that comprises a sequence with complementarity to a target nucleic acid;

b) a terminal extension that is covalently linked to the 5′

end, the 3′

end, or both the 5′

end and the 3′

end of the capture domain; and

c) at least one polymer label attached to at least one of the terminal extension(s),

wherein the nucleic acid capture domain of the first probe molecule comprises a sequence with complementarity to first target nucleic acid and the nucleic acid capture domain of the second probe molecule comprises a sequence with complementarity to a second target nucleic acid,

and wherein the polymer label of the first probe molecule is different from the polymer label of the second probe molecule and provide for independent detection of the first and second target nucleic acids in a nanopore system.

2. The probe set of claim 1, wherein the polymer labels of the first and second probe molecules provide for distinct signature conductance blocks when hybridized with their respective targets and subjected to an applied voltage in a nanopore system.

3. The probe set of claim 1, wherein the polymer label is a hydrophilic homopolymer or a hydrophilic heteropolymer.

4. The probe set of claim 1, wherein the polymer label is selected from the group consisting of a polyglycol, a polyamine, a peptide, an oligonucleotide, and an oligosaccharide.

5. The probe set of claim 1, wherein the polyglycol is selected from the group consisting of polyethylene glycol (PEG), methoxypolyethylene glycol (MPEG), polypropylene glycol (PPG), polybutylene glycols (PBG), and copolymers thereof.

6. The probe set of claim 1, wherein the capture domain and terminal extension are independently selected from the group consisting of nucleic acids and peptide nucleic acids.

7. The probe set of claim 1, wherein the terminal extension is covalently linked to the 3′

terminus of the capture domain.

8. The probe set of claim 1, wherein the terminal extension is selected from the group consisting of poly(dC)_(27-33), poly(dG)_(27-33), poly(dA)_(27-33), poly(dT)_(27-33), and poly(dN)_(27-33), where N is any combination of cytosine, guanosine, adenosine, thymine, an abase, inosine, xanthosine, 7-methylguanosine, dihydrouridine, and 5-methylcytidine.

9. The probe set of claim 1, wherein the probe set comprises at least one additional probe molecule that comprises:

a) a capture domain that comprises a sequence with complementarity to a target nucleic acid;

b) a terminal extension that is covalently linked to the 5′

or 3′

end of the capture domain; and

c) at least one polymer label attached to at least one of the terminal extension(s),

wherein the nucleic acid capture domain of the additional probe molecule(s) comprises a sequence with complementarity to a target nucleic acid that is distinct from the first and second target nucleic acids and wherein the polymer label of each additional probe molecule(s) is different from the polymer label of the first probe molecule, second probe molecule, and any other additional probe molecules and provides for independent detection of the first, second target, and additional target nucleic acids in a nanopore system.

15. The probe set of claim 1, wherein the label attached to the terminal extension of the first probe molecule is a polyethylene glycol of a certain length and the label attached to the extension of the second probe molecule is a polyethylene glycol of a different length than the polyethylene glycol attached to the first probe molecule.

16. The probe set of claim 1, further comprising a probe with a capture domain comprising a sequence with complementarity to an additional distinct target nucleic acid and, optionally, a terminal domain, wherein said probe lacks a polymer label and provides for independent detection of the additional distinct target nucleic acid.

17. The probe set of claim 1 wherein the polymer label is attached to a residue of the terminal extension that is located within nine residues of 5′

or 3′

covalent linkage of the terminal extension to the capture domain.

18. The probe set of claim 17, wherein the polymer label is attached to the second to the fifth residue of the terminal extension that is located 5′

or 3′

from the covalent linkage to the capture domain.

35. The method of claim 1, wherein the polymer label is attached to a residue of the terminal extension that is located within nine residues of 5′

or 3′

covalent linkage of the terminal extension to the capture domain.

36. The method of claim 35, wherein the polymer label is attached to the second to the fifth residue of the terminal extension that is located 5′

or 3′

from the covalent linkage to the capture domain.

10-14. -14. (canceled)

19. A method for detecting at least two distinct single stranded target nucleic acids in a sample with a nanopore system, the method comprising:

a) contacting the sample with a set of at least two probe molecules and allowing the probe molecules to hybridize with any target nucleic acids present in the sample to form a hybridized sample, wherein the set of probe molecules comprises at least a first probe molecule and a second probe molecule, both of which comprise;

(i) a capture domain that comprises a sequence with complementarity to a target nucleic acid;

(ii) a terminal extension that is covalently linked to the 5′

end 3′

end, or both the 5′

end and the 3′

end of the capture domain; and

(ii) at least one polymer label attached to at least one of the terminal extension(s),

wherein the nucleic acid capture domain of the first probe molecule hybridizes with a first target nucleic acid and the nucleic acid capture domain of the second probe molecule hybridizes with a second target nucleic acid, and wherein the polymer label of the first probe molecule is different from the polymer label of the second probe molecule;

b) applying a voltage to said hybridized sample mixture in a cis compartment of a dual chamber nanopore system sufficient to trap a hybridized probe/target nucleic acid complex in the nanopore and drive translocation of said hybridized probes and target nucleic acids through a nanopore of said system by an unzipping process, and,

c) analyzing an electrical current pattern in said nanopore system over time, wherein presence of said distinct single stranded target nucleic acids in the sample is indicated by occurrence of two distinct signature electrical current blocks corresponding to trapping of each distinct hybridized probe and target nucleic acids in the nanopore.

20. The method of claim 19 wherein the presence a first target nucleic acid and the presence of a second target nucleic acid in the sample result in distinct signature electrical current blocks that are distinct signature conductance blocks.

34. The method of claim 19, wherein the label attached to the terminal extension of the first probe molecule is a polyethylene glycol of a certain length and the label attached to the extension of the second probe molecule is a polyethylene glycol of a different length than the polyethylene glycol attached to the first probe molecule.

21-33. -33. (canceled)

37. A probe molecule comprising:

(i) a capture domain that comprises a sequence with complementarity to a target nucleic acid;

(ii) a terminal extension that is covalently linked to the 5′

end, the 3′

end, or both the 5′ and

3′

end of the capture domain, and

(ii) at least one polymer label attached to at least one terminal extension, wherein said probe molecule provides for detection of the target nucleic acid in a nanopore system.

52. A method for detecting at least one distinct single stranded target nucleic acids in a sample with a nanopore system, the method comprising:

a) contacting the sample with a set of at least one probe molecule of claim 37 and allowing the probe molecule to hybridize with any target nucleic acid present in the sample to form a hybridized sample, wherein the set of probe molecules comprises at least a first probe molecule and a second probe molecule;

b) applying a voltage to said hybridized sample mixture in a cis compartment of a dual chamber nanopore system sufficient to trap a hybridized probe/target nucleic acid complex in the nanopore and drive translocation of said hybridized probe and target nucleic acid through a nanopore of said system by an unzipping process, and,

c) analyzing an electrical current pattern in said nanopore system over time, wherein presence of said distinct single stranded target nucleic acid in the sample is indicated by occurrence of a distinct signature electrical current block corresponding to translocation of the hybridized probe and target nucleic acid through the nanopore.

38-51. -51. (canceled)