Methods of Detecting Target Nucleic Acids

Abstract

The present disclosure relates to methods of identifying target nucleic acids by using coded molecules and its analysis by translocation through a nanopore. Generally, coded molecules are subject to a target polynucleotide dependent modification. The modified coded molecule is detected by isolating the modified coded molecules from the unmodified coded molecules prior to analysis through the nanopore or by detecting a change in the signal pattern of the coded molecule when analyzed through the nanopore.

1 . A method of detecting a target polynucleotide, comprising:
 a) contacting a coded molecule with a target polynucleotide, wherein the coded molecule comprises
 (i) one or more block polymer regions, and 
 (ii) a target probe capable of hybridizing to the target polynucleotide; 
   b) modifying the target probe with a modifying agent, wherein the modification is dependent on the target polynucleotide hybridized to the target probe; and   c) translocating the coded molecule through a nanopore and detecting a signal pattern associated with the coded molecule.   
     
     
         2 . The method of  claim 1 , further comprising the step of rendering the coded molecule single-stranded prior to translocation through the nanopore. 
     
     
         3 . The method of  claim 1 , further comprising comparing the detected signal pattern to a signal pattern of an unmodified coded molecule, wherein a difference in the detected signal pattern of the modified coded molecule compared to the signal pattern of the unmodified coded molecule indicates the presence of the target polynucleotide. 
     
     
         4 . The method of  claim 1 , further comprising associating the detected signal pattern to the target probe. 
     
     
         5 . The method of  claim 1  in which the target polynucleotide comprises a 5-prime region and a 3-prime region, and the target probe comprises a 3-prime terminal sequence that hybridizes to the 5-prime region of the target polynucleotide, and wherein the modifying agent is a template-dependent polymerase and the modification is extension of the hybridized 3-prime region of the target probe. 
     
     
         6 . The method of  claim 5  in which the 3-prime terminal sequence of the target probe comprises a 3-prime terminal nucleotide that interrogates a site of nucleotide polymorphism on the target polynucleotide. 
     
     
         7 . The method of  claim 5  in which the target polynucleotide comprises a circular nucleic acid, wherein the circular nucleic acid is a ligated open circle probe (OCP). 
     
     
         8 . The method of  claim 7  in which the ligated OCP is formed by hybridization of the OCP terminal regions to a nucleic acid of interest. 
     
     
         9 . The method of  claim 5  in which the primer extension is carried out in the presence of one or more nucleotide triphosphates. 
     
     
         10 . The method of  claim 9  in which all four of the nucleotide triphosphates are present. 
     
     
         11 . The method of  claim 1  in which the target polynucleotide comprises adjacent first and second regions, and the method further comprises hybridizing a ligation probe to the target polynucleotide, wherein the ligation probe hybridizes to the first region and the target probe hybridizes to the second region of the target polynucleotide such that a terminus of the ligation probe and a terminus of the target probe are adjacent, and wherein the modifying agent is a ligase and the modification is ligation of the ligation probe to the target probe. 
     
     
         12 . The method of  claim 11  in which the ligase is a DNA ligase. 
     
     
         13 . The method of  claim 11  in which the terminus of the target probe comprises a terminal nucleotide that interrogates a site of nucleotide polymorphism on the target polynucleotide. 
     
     
         14 . The method of  claim 13  in which the target probe terminal nucleotide is a 5-prime or 3-prime terminal nucleotide. 
     
     
         15 . The method of  claim 11  in which the terminus of the ligation probe comprises a terminal nucleotide that interrogates a site of nucleotide polymorphism on the target polynucleotide. 
     
     
         16 . The method of  claim 15  in which the ligation probe terminal nucleotide is a 5-prime or 3-prime terminal nucleotide. 
     
     
         17 . The method of  claim 11  in which the ligation probe further comprises a signal generating segment. 
     
     
         18 . The method of  claim 1  in which the target polynucleotide comprises adjacent first and second regions, and the target probe comprises a 5-prime region and a  3 -prime region, and wherein the method further comprises hybridizing a FLAP probe to the target polynucleotide, wherein the FLAP probe comprises a 3-prime segment that hybridizes to the first region, and the 3-prime region of the target probe hybridizes to the second region such that the 3-prime segment of the FLAP probe and the 3-prime region of the target probe are adjacently hybridized to the target polynucleotide to form a FLAP substrate, and wherein the modifying agent is a FLAP endonuclease and the modification is cleavage of the target probe. 
     
     
         19 . The method of  claim 18  in which the 5-prime region of the target probe is non-complementary to the target polynucleotide. 
     
     
         20 . The method of  claim 18  in which the FLAP probe further comprises a 3-prime unpaired segment that overlaps with the 5-prime region of the target probe in the FLAP substrate, thereby forming a double FLAP substrate. 
     
     
         21 . The method of  claim 20  in which the 3-prime unpaired segment of the FLAP probe is one nucleotide. 
     
     
         22 . The method of  claim 21 , further comprising contacting with a ligase subsequent to the modification of the target probe by the FLAP endonuclease. 
     
     
         23 . The method of  claim 18  in which the FLAP endonuclease comprises an archaea FEN-1 endonuclease. 
     
     
         24 . The method of  claim 18  in which the target probe interrogates a site of nucleotide polymorphism on the target polynucleotide. 
     
     
         25 . The method of  claim 18  in which the FLAP probe interrogates a site of nucleotide polymorphism on the target polynucleotide. 
     
     
         26 . The method of  claim 18  in which the 5-prime region of the target probe further comprises a signal generating segment. 
     
     
         27 . The method of  claim 1  in which the target polynucleotide comprises a 3-prime region, and the target probe comprises a first, second, third, and fourth regions, wherein
 (i) the first region is located 3-prime and the fourth region is positioned 5-prime relative to each other,   (ii) the second and third regions are located between the first and fourth regions and are complementary such that the second and third regions hybridize to one another, and   (iii) the first region is adjacent to the second region and hybridizes to the 3-prime region of the target polynucleotide such that the hybridized 3-prime region of the target polynucleotide is adjacent to the hybridized third region of the target probe to form a FLAP substrate; and   wherein the modifying agent is a FLAP endonuclease and the modification is cleavage of the target probe.   
     
     
         28 . The method of  claim 27  in which the fourth region of the target probe is non-complementary to the target probe. 
     
     
         29 . The method of  claim 27  in which the 3-prime region of the target polynucleotide further comprises a 3-prime unpaired segment that overlaps with the fourth region of the target probe in the FLAP substrate, thereby forming a double FLAP substrate. 
     
     
         30 . The method of  claim 29  in which the 3-prime unpaired segment of the target polynucleotide is one nucleotide. 
     
     
         31 . The method of  claim 30 , further comprising contacting with a ligase subsequent to the modification of the target probe by the FLAP endonuclease. 
     
     
         32 . The method of  claim 27  in which the fourth region of the target probe further comprises a signal generating segment. 
     
     
         33 . The method of  claim 1  in which the hybridization of the target probe to the target polynucleotide forms an endonuclease recognition site and a corresponding enodonuclease cleavage site, and wherein the modifying agent is an endonuclease that recognizes the recognition site and the modification is cleavage of the target probe. 
     
     
         34 . The method of  claim 33  in which the endonuclease recognition site is a sequence-specific endonuclease recognition site and wherein the modifying agent is a sequence specific endonuclease active on the recognition site. 
     
     
         35 . The method of  claim 34  in which the sequence specific endonuclease recognition site is a site of nucleotide polymorphism. 
     
     
         36 . The method of  claim 33  in which the endonuclease recognition site is a nucleotide mismatch and wherein the modifying agent is a mismatch specific endonuclease. 
     
     
         37 . The method of  claim 36  in which the site of nucleotide mismatch is a site of nucleotide polymorphism. 
     
     
         38 . The method of  claim 36  in which the mismatch specific endonuclease is CEL-1. 
     
     
         39 . The method of  claim 33  in which the target probe further comprises a signal generating segment capable of being released from the coded molecule by activity of the endonuclease. 
     
     
         40 . The method of  claim 1  in which the modifying agent is a double-stranded specific exonuclease suitable to act on the target probe hybridized to the target polynucleotide and the modification is degradation of all or a portion of the target probe. 
     
     
         41 . The method of  claim 40  in which the double-stranded specific exonuclease is a 5-prime to 3-prime exonuclease. 
     
     
         42 . The method of  claim 41  in which the 5-prime to 3-prime exonuclease is X exonuclease. 
     
     
         43 . The method of  claim 40  in which the double-stranded specific exonuclease is a 3-prime to 5-prime exonuclease. 
     
     
         44 . The method of  claim 43  in which the 3-prime to 5-prime exonuclease is exonuclease III. 
     
     
         45 . The method of  claim 1  in which the coded molecule comprises a chimeric polymer. 
     
     
         46 . The method of  claim 45  in which the chimeric polymer comprises nucleobase and non-nucleobase polymers. 
     
     
         47 . The method of  claim 1  in which the coded molecule comprises at least two block polymers, and wherein the two block polymers are separated by a non-block polymer segment. 
     
     
         48 . The method of  claim 47  in which the non-block polymer segment is a random polymer. 
     
     
         49 . The method of  claim 1  in which one or more of the block polymers is a polypurine. 
     
     
         50 . The method of  claim 49  in which one or more of the block polymers is a polypyrimidine. 
     
     
         51 . The method of  claim 1  in which one or more block polymers is a block copolymer. 
     
     
         52 . The method of  claim 51  in which the block copolymer comprises an alternating copolymer. 
     
     
         53 . The method of  claim 52  in which the alternating copolymer is alternating purines. 
     
     
         54 . The method of  claim 52  in which the alternating copolymer is alternating pyrimidines. 
     
     
         55 . The method of  claim 52  in which the alternating copolymer is a polymer of alternating purine and pyrimidine. 
     
     
         56 . The method of  claim 1  in which the block polymer comprises dinucleotide repeats. 
     
     
         57 . The method of  claim 1  in which the block polymer comprises trinucleotide repeats. 
     
     
         58 . The method of  claim 1  in which the block polymer comprises tetranucleotide repeats. 
     
     
         59 . The method of  claim 1  in which at least one of the block polymers is a peptide nucleic acid. 
     
     
         60 . The method of  claim 1  in which at least one of the block polymers is a deoxyribonucleic acid. 
     
     
         61 . The method of  claim 1  in which the coded molecule comprises a plurality of block polymers. 
     
     
         62 . The method of  claim 61  in which at least two of the plurality of block polymers comprise different nucleobase polymers. 
     
     
         63 . The method of  claim 1  in which the detecting is by measuring current blockade. 
     
     
         64 . The method of  claim 1  in which the detecting is by measuring electron tunneling current. 
     
     
         65 . The method of  claim 1  in which the detecting is by imaging charged induced field effects. 
     
     
         66 . The method of  claim 1  in which the nanopore is selective for translocation of a single-stranded nucleobase polymer. 
     
     
         67 . The method of  claim 1  in which the nanopore comprises a biological nanopore. 
     
     
         68 . The method of  claim 1  in which the nanopore comprises a solid state nanopore. 
     
     
         69 . A method of detecting target polynucleotides, comprising:
 a) contacting at least a first coded molecule and a second coded molecule with a plurality of target polynucleotides, wherein
 (i) the first coded molecule comprises a first one or more block polymer regions and a first target probe capable of hybridizing to a first target polynucleotide, wherein the first coded molecule has a detectable first signal pattern; and 
 (ii) the second coded molecule comprises a second one or more block polymer regions and a second target probe capable of hybridizing to a second target polynucleotide, wherein the second coded molecule has a detectable second signal pattern distinguishable from the first signal pattern; 
   b) modifying the first and second target probes with a modifying agent, wherein the modification is dependent on the target polynucleotide hybridized to the target probe;   c) translocating the coded molecules through a nanopore and detecting the signal pattern of each of the translocated coded molecules; and   d) associating the detected signal pattern to the first or second coded molecule.   
     
     
         70 . The method of  claim 69 , wherein the first and second target probes hybridize to different target polynucleotides. 
     
     
         71 . The method of  claim 69 , further comprising comparing the detected signal pattern to the signal pattern of the associated coded molecule, wherein a difference in the detected signal pattern compared to the signal pattern of the associated coded molecule indicates the presence of the first or second target polynucleotide in the plurality of target polynucleotides. 
     
     
         72 . A method of detecting a plurality of target polynucleotides, comprising:
 a) contacting a population of coded molecules with a plurality of target polynucleotides, wherein the population of coded molecules comprises a plurality of subpopulations and each coded molecule of each subpopulation comprises
 (i) one or more block polymer regions, and a target probe capable of hybridizing to a target polynucleotide, wherein the target probe of each subpopulation hybridizes to a different target polynucleotide; and 
 (ii) a detectable signal pattern distinguishable amongst the plurality of subpopulations; 
   b) modifying the target probe with a modifying agent, wherein the modification is dependent on the target polynucleotide hybridized to the target probe;   c) translocating the coded molecules through a nanopore and detecting the signal pattern; and   d) associating the detected signal pattern to a specific subpopulation of coded molecules in the plurality of subpopulations.   
     
     
         73 . The method of  claim 72  in which a difference in the detected signal pattern compared to the signal pattern of the specific subpopulation indicates the presence of the target polynucleotide.