Nanoparticles having oligonucleotides attached thereto and uses therefor

A method of detecting one or more nucleic acid targets, each having at least two portions, said method comprising:

• providing a substrate having one or more types of oligonucleotides attached thereto, at least one type of oligonucleotides having a sequence complementary to a first portion of the sequence of a specific nucleic acid target to be detected;
• providing one or more types of aggregate probes, each type of aggregrate probes comprising at least two types of nanoparticles having oligonucleotides attached thereto, the nanoparticles of each type of aggregate probe being bound to each other as a result of the hybridization of some of the oligonucleotides attached to them, at least one of the types of nanoparticles of each type of aggregate probe having oligonucleotides attached thereto which have a sequence complementary to a second portion of the sequence of said specific nucleic acid target to be detected;
• contacting one or more nucleic acid targets, the substrate and one or more types of aggregate probes under conditions effective to allow hybridization of said nucleic acid targets with the oligonucleotides on the aggregate probes and with the oligonucleotides on the substrate, wherein in the presence of said nucleic acid targets and under hybridization conditions, the nanoparticles having oligonucleotides bound thereto form complexes with said nucleic acid targets, the resulting nanoparticle-nucleic acid target complexes having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of analogous complexes formed with said nucleic acid targets and an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the nanoparticles, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid targets under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complexes;
• and
• observing a detectable change.

A method of detecting one or more nucleic acids, each having at least two portions, said method comprising:

• providing a substrate having oligonucleotides attached thereto;
• providing one or more types of aggregate probes, each type of aggregrate probes comprising at least two types of nanoparticles having oligonucleotides attached thereto, the nanoparticles of the aggregate probe being bound to each other as a result of the hybridization of some of the oligonucleotides attached to them, at least one of the types of nanoparticles of each type of aggregate probe having oligonucleotides attached thereto which have a sequence complementary to a first portion of the sequence of a specific nucleic acid target to be detected;
• providing one or more types of nanoparticles, each type of nanoparticles having at least two types of oligonucleotides attached thereto, the first type of oligonucleotides having a sequence complementary to a second portion of the sequence of said nucleic acid target to be detected, the second type of oligonucleotides having a sequence complementary to at least a portion of the sequence of the oligonucleotides attached to the substrate;
• contacting one or more nucleic acid targets, one or more types of aggregate probes, one or more types of nanoparticles and the substrate, the contacting taking place under conditions effective to allow hybridization of said nucleic acid targets with the oligonucleotides on the aggregate probes and on the nanoparticles and hybridization of the oligonucleotides on the nanoparticles with the oligonucleotides on the substrate, wherein in the presence of said nucleic acid targets and under hybridization conditions, the nanoparticles having oligonucleotides bound thereto form complexes with said nucleic acid targets, the resulting nanoparticle-nucleic acid target complexes having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of analogous complexes formed with said nucleic acid targets and an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the nanoparticles, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid targets under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complexes;
• and
• observing a detectable change.

A kit for detecting one or more nucleic acid targets, each nucleic acid target having at least two portions, said kit comprising:

• nanoparticles having oligonucleotides attached thereto, at least a portion of the oligonucleotides have a sequence complementary to the sequence of a first portion of a binding oligonucleotide;
• and
• one or more types of binding oligonucleotides, each type of binding oligonucleotides having a sequence comprising at least two portions, the first portion being complementary to the sequence of the oligonucleotides on the nanoparticles and the second portion being complementary to the sequence of a portion of a nucleic acid target to be detected;
• wherein in the presence of one or more nucleic acid targets and under hybridization conditions, the binding oligonucleotides form complexes with said nanoparticles having oligonucleotides bound thereto and said nucleic acid targets, the resulting nanoparticle-binding oligonucleotide-nucleic acid target complexes having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of analogous complexes formed with said nucleic acid targets, binding oligonucleotides and an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the nanoparticles, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid targets under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complexes.

A kit for detecting a nucleic acid target comprising:

• a container holding one type of nanoparticles having oligonucleotides attached thereto and one or more types of binding oligonucleotides, each of the types of binding oligonucleotides having a sequence comprising at least two portions, the first portion being complementary to the sequence of the oligonucleotides on the nanoparticles, whereby the binding oligonucleotides are hybridized to the oligonucleotides on the nanoparticles, and the second portion being complementary to the sequence of one or more portions of a nucleic acid target, wherein in the presence of said nucleic acid target and under hybridization conditions, the nanoparticles having binding oligonucleotides bound thereto form a complex with said nucleic acid target, the resulting nanoparticle-nucleic acid target complex having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of an analogous complex formed with said nucleic acid target and an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the binding oligonucleotides bound to the nanoparticles, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid target under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complex.

A kit for detecting a nucleic acid target having at least two portions comprising:

• a substrate having oligonucleotides attached thereto which have a sequence complementary to the sequence of a first portion of a specific nucleic acid target;
• a first container holding nanoparticles having oligonucleotides attached thereto, some of which have a sequence complementary to the sequence of a second portion of the nucleic acid target;
• and
• a second container holding nanoparticles having oligonucleotides attached thereto which have a sequence complementary to at least a portion of the sequence of the oligonucleotides attached to the nanoparticles in the first container, wherein in the presence of said nucleic acid target and under hybridization conditions, the nanoparticles having oligonucleotides bound thereto form a complex with said nucleic acid target, the resulting nanoparticle-nucleic acid target complex having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of an analogous complex formed with said nucleic acid target and an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the nanoparticles, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid target under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complex.

A kit for detecting a nucleic acid target having at least two portions comprising:

• a substrate, the substrate having attached thereto nanoparticles, the nanoparticles having oligonucleotides attached thereto which have a sequence complementary to the sequence of a first portion of a nucleic acid target;
• and
• a first container holding an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, the nanoparticles of the aggregate probe being bound to each other as a result of the hybridization of some of the oligonucleotides attached to them, at least one of the types of nanoparticles of the aggregate probe having oligonucleotides attached thereto which have a sequence complementary to a second portion of the sequence of the nucleic acid target, wherein in the presence of said nucleic acid target and under hybridization conditions, the nanoparticles having oligonucleotides bound thereto form a complex with said nucleic acid target, the resulting nanoparticle-nucleic acid target complex having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of an analogous complex formed with said nucleic acid target and an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the nanoparticles, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid target under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complex.

A kit for detecting a nucleic acid target having two portions comprising:

• a substrate, the substrate having oligonucleotides attached thereto, the oligonucleotides having a sequence complementary to the sequence of a first portion of a nucleic acid target;
• and
• a first container holding an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, the nanoparticles of the aggregate probe being bound to each other as a result of the hybridization of some of the oligonucleotides attached to them, at least one of the types of nanoparticles of the aggregate probe having oligonucleotides attached thereto which have a sequence complementary to a second portion of the sequence of the nucleic acid target, wherein in the presence of said nucleic acid target and under hybridization conditions, the nanoparticles having oligonucleotides bound thereto and the oligonucleotides bound to the substrate form a complex with said nucleic acid target, the resulting nanoparticle-nucleic acid target complex having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of an analogous complex formed with said nucleic acid target and an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the nanoparticles, and said oligonucleotides bound to the substrate, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid target under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complex.

A kit for detecting a nucleic acid target having at least two portions comprising:

• a substrate having oligonucleotides attached thereto;
• a first container holding an aggregate probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, the nanoparticles of the aggregate probe being bound to each other as a result of the hybridization of some of the oligonucleotides attached to them, at least one of the types of nanoparticles of the aggregate probe having oligonucleotides attached thereto which have a sequence complementary to a first portion of the sequence of the nucleic acid target;
• and
• a second container holding nanoparticles having at least two types of oligonucleotides attached thereto, the first type of oligonucleotides having a sequence complementary to a second portion of the sequence of the nucleic acid target, and the second type of oligonucleotides having a sequence complementary to at least a portion of the sequence of the oligonucleotides attached to the substrate, wherein in the presence of said nucleic acid target and under hybridization conditions, the nanoparticles having oligonucleotides bound thereto and the oligonucleotides bound to the substrate form a complex with said nucleic acid target, the resulting nanoparticle-nucleic acid target complex having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of an analogous complex formed with said nucleic acid target and an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the nanoparticles, and said oligonucleotides bound to the substrate, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid target under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complex.

A kit for detecting a nucleic acid target having a least two portions comprising:

• a substrate having oligonucleotides attached thereto which have a sequence complementary to the sequence of a first portion of a nucleic acid target;
• a first container holding nanoparticles having oligonucleotides attached thereto, some of which have a sequence complementary to the sequence of a second portion of the nucleic acid target;
• and
• a second container holding nanoparticles having oligonucleotides attached thereto which have a sequence complementary to at least a portion of the sequence of the oligonucleotides attached to the nanoparticles in the first container, wherein in the presence of said nucleic acid target and under hybridization conditions, the nanoparticles having oligonucleotides bound thereto and the oligonucleotides bound to the substrate form a complex with said nucleic acid target, the resulting nanoparticle-nucleic acid target complex having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of an analogous complex formed with said nucleic acid target and an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the nanoparticles, and said oligonucleotides bound to the substrate, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid target under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complex.

A kit for detecting a nucleic acid target having at least two portions comprising:

• a first container holding a type of microspheres having oligonucleotides attached thereto, the oligonucleotides having a sequence complementary to a first portion of the sequence of a nucleic acid target and being labeled with a fluorescent molecule;
• and
• a second container holding a type of nanoparticles having oligonucleotides attached thereto, the oligonucleotides having a sequence complementary to a second portion of the sequence of the nucleic acid target, wherein in the presence of said nucleic acid target and under hybridization conditions, the nanoparticles having oligonucleotides bound thereto and the oligonucleotides bound to the microspheres form a complex with said nucleic acid target, the resulting nanoparticle-nucleic acid target complex having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of an analogous complex formed with said nucleic acid target and an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the nanoparticles, and said oligonucleotides bound to the substrate, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid target under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complex.

A kit for detecting one or more nucleic acid targets, each nucleic acid target having at least two portions, said kit comprising:

• one or more types of first metallic or semiconductor nanoparticles having oligonucleotides attached thereto, the oligonucleotides attached each type of metallic or semiconductor nanoparticles having a sequence complementary to a first portion of the sequence of a specific nucleic acid target to be detected and being labeled with a fluorescent molecule;
• and
• one or more types of second metallic or semiconductor nanoparticles having oligonucleotides attached thereto, the oligonucleotides attached to each type of metallic or semiconductor nanoparticles having a sequence complementary to a second portion of the sequence of the specific nucleic acid target to be detected and being labeled with a fluorescent molecule;
• wherein in the presence of one or more nucleic acid targets and under hybridization conditions, the first and second metallic or semiconductor nanoparticles having oligonucleotides bound thereto form complexes with said nucleic acid targets, the resulting nanoparticle-nucleic acid target complexes having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of analogous complexes formed with said nucleic acid targets and first unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the first type of nanoparticles and a second unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the second type of nanoparticles, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid targets under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complexes.

A kit for detecting one or more nucleic acid targets, each nucleic acid target having at least two portions, the kit comprising:

• a core probe, the core probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, the nanoparticles of the core probe being bound to each other as a result of the hybridization of some of the oligonucleotides attached to them;
• one or more types of nanoparticles having two types of oligonucleotides attached thereto, the first type of oligonucleotides attached to each type of nanoparticles having a sequence complementary to a second portion of the nucleic acid target to be detected, and the second type of oligonucleotides having sequence complementary to a portion of the sequence of the oligonucleotides attached to at least one of the types of nanoparticles of the core probe;
• and
• a substrate having one or more types of oligonucleotides attached thereto, each type of oligonucleotides having a sequence complementary to a first portion of the sequence of a nucleic acid target to be detected;
• wherein in the presence of one or more nucleic acid targets and under hybridization conditions, complexes are formed between the nucleic acid targets with the oligonucleotides bound to the substrate, between the nucleic acid targets and the first type of oligonucleotides bound to the nanoparticles having two types of oligonucleotides attached thereto, between the second type of oligonucleotides bound to the nanoparticles having two types of oligonucleotides attached thereto and oligonucleotides attached to at least one of the types of nanoparticles of the core probe, the resulting nanoparticle-nucleic acid target complexes having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of analogous complexes formed with said nucleic acid targets and said oligonucleotides bound to the substrate and an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the first type of oligonucleotides bound to the type of nanoparticles having two types of oligonucleotides attached thereto, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid targets under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complexes.

A kit for detecting one or more nucleic acid targets, each nucleic acid target having at least two portions, the kit comprising:

• a core probe, the core probe comprising at least two types of nanoparticles having oligonucleotides attached thereto, the nanoparticles of the core probe being bound to each other as a result of the hybridization of some of the oligonucleotides attached to them;
• a substrate having one or more types of oligonucleotides attached thereto, each type of oligonucleotides having a sequence complementary to a first portion of the sequence of a specific nucleic acid target to be detected;
• and
• one or more types of linking oligonucleotides, each type of linking olignucleotides comprising a sequence complementary to a second portion of the sequence of the specific nucleic acid target to be detected and a sequence complementary to a portion of the sequence of the oligonucleotides attached to at least one of the types of nanoparticles of the core probe;
• wherein in the presence of one or more nucleic acid targets and under hybridization conditions, complexes are formed between the targets and the oligonucleotides bound to the substrate, the targets and the linker oligonucleotides, and the linker oligonucleotides and the oligonucleotides attached to at least one of the types of nanoparticles of the core probes, the resulting nanoparticle-nucleic acid target complexes having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of analogous complexes formed with said nucleic acid targets, the oligonucleotides bound to the substrate, the linker oligonucleotides, and an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides attached to at least of one the types of nanoparticles of the core probes, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid target under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complexes.