Encoded Nanopore Sensor for Multiplex Nucleic Acids Detection

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.

1 - 52 . (canceled) 
     
     
         53 . A set of probe molecules including a first probe molecule and a second probe molecule, each comprising:
 a sequence partially complementary or fully complementary to a target nucleic acid;   at least one extension covalently attached to the sequence; and   at least one label covalently attached to the sequence or to an internal residue of the extension;   wherein the sequence of the first probe molecule is partially complementary or fully complementary to a first target nucleic acid, and the sequence of the second probe molecule is partially complementary or fully complementary to a second target nucleic acid; and   wherein the label of the first probe molecule is different from the label of the second probe molecule, such that the set of probe molecules provides distinct signals corresponding to the respective first target nucleic acid and second target nucleic acid in a nanopore system.   
     
     
         54 . The set of probe molecules of  claim 53 , wherein the first probe molecule comprises a first extension covalently attached to a 3′ terminus of the sequence, and the first extension comprises a label covalently attached to the first extension within nine residues of the 3′ terminus of the sequence. 
     
     
         55 . The set of probe molecules of  claim 54 , wherein the first extension comprises a homopolymer, and the label of the first extension is covalently attached to the second residue of the extension from the 3′ terminus of the sequence. 
     
     
         56 . The set of probe molecules of  claim 54 , wherein the first probe molecule comprises a second extension covalently attached to a 5′ terminus of the sequence, the second extension including a label. 
     
     
         57 . The set of probe molecules of  claim 53 , wherein the sequence of the first probe molecule comprises a peptide nucleic acid, and the label of the first probe molecule is covalently attached to the peptide nucleic acid. 
     
     
         58 . The set of probe molecules of  claim 53 , wherein the label of the first probe molecule comprises a polymer. 
     
     
         59 . The set of probe molecules of  claim 58 , wherein the label of the second probe molecule comprises a polymer having a different length than the polymer of the label of the first probe molecule. 
     
     
         60 . The set of probe molecules of  claim 53 , wherein the label of at least one of the first probe molecule or the second probe molecule comprises a polyglycol, a polyamine, a peptide, an oligonucleotide, or an oligosaccharide. 
     
     
         61 . The set of probe molecules of  claim 53 , wherein the sequence of the first probe molecule is a DNA sequence, and the sequence of the second probe molecule is a DNA sequence different than the DNA sequence of the first probe molecule. 
     
     
         62 . The set of probe molecules of  claim 53 , wherein the DNA sequence of the first probe molecule is partially complementary or fully complementary to a bacterial nucleic acid or a viral nucleic acid. 
     
     
         63 . A set of probe molecules comprising a first probe molecule that comprises:
 a sequence partially complementary or fully complementary to a target nucleic acid;   a first extension covalently attached to a 3′ terminus of the sequence, the first extension comprising a homopolymer, a di-nucleotide heteropolymer, or a tri-nucleotide heteropolymer; and
 a label covalently attached to an internal residue of the first extension within nine residues of the 3′ terminus of the sequence. 
   
     
     
         64 . The set of probe molecules of  claim 63 , wherein the label comprises a polyglycol chosen from polyethylene glycol (PEG), methoxypolyethylene glycol (MPEG), polypropylene glycol (PPG), polybutylene glycols (PBG), and copolymers thereof. 
     
     
         65 . The set of probe molecules of  claim 63 , wherein the first extension comprises poly(dC), and the label is covalently attached to the second residue of the extension from the 3′ terminus of the sequence. 
     
     
         66 . The set of probe molecules of  claim 66 , wherein the label comprises polyethylene glycol. 
     
     
         67 . The set of probe molecules of  claim 63 , wherein the first probe molecule further comprises a second extension covalently attached to a 5′ terminus of the sequence, and a label covalently attached to an internal residue of the second extension within nine residues of the 5′ terminus of the sequence. 
     
     
         68 . The set of probe molecules of  claim 63 , wherein the sequence of the first probe molecule is partially complementary or fully complementary to a bacterial nucleic acid or a viral nucleic acid. 
     
     
         69 . The set of probe molecules of  claim 63 , wherein the target nucleic acid is a first target nucleic acid, and further comprising a second probe molecule that comprises:
 a sequence partially complementary or fully complementary to a second target nucleic acid;   at least one extension covalently attached to the sequence of the second probe molecule; and   at least one label covalently attached to the sequence of the second probe molecule or to an internal residue of the extension of the second probe molecule;   wherein the label of the first probe molecule is different from the label of the second probe molecule, such that the set of probe molecules provides distinct signals corresponding to the respective first target nucleic acid and second target nucleic acid in a nanopore system.   
     
     
         70 . A set of probe molecules comprising:
 a first probe molecule comprising:
 a DNA sequence, an RNA sequence, or a peptide nucleic acid sequence partially complementary or fully complementary to a first target nucleic acid; and 
 at least one label covalently attached to the sequence; and 
   at least one second probe molecule comprising:
 a DNA sequence, an RNA sequence, or a peptide nucleic acid sequence partially complementary or fully complementary to a second target nucleic acid; 
   wherein the set of probe molecules provides distinct signals corresponding to the respective first target nucleic acid and second target nucleic acid in a nanopore system.   
     
     
         71 . The set of probe molecules of  claim 70 , wherein the first probe molecule comprises a first extension covalently attached to a 3′ terminus of the sequence of the first probe molecule, the first extension including a nucleic acid homopolymer that comprises 5 to 40 nucleotides. 
     
     
         72 . The set of probe molecules of  claim 70 , wherein the sequence of the first probe molecule comprises a peptide nucleic acid, and the label of the first probe molecule comprises polyethylene glycol covalently attached to the peptide nucleic acid sequence.