NANOREPORTERS AND METHODS OF MANUFACTURING AND USE THEREOF
The present invention relates to compositions and methods for detection and quantification of individual target molecules in biomolecular samples. In particular, the invention relates to coded, labeled probes that are capable of binding to and identifying target molecules based on the probes' label codes. Methods of making and using such probes are also provided. The probes can be used in diagnostic, prognostic, quality control and screening applications.
1 . A nucleic acid probe comprising:
(i) a first label attachment region, comprising a first DNA sequence of about 300 to about 1500 nucleotides in length having a G/C content of about 40% to about 60% and having a regularly repeated base every about 4 to about 25 bases, and comprising at least a first RNA molecule having a regularly repeated base every about 4 to about 25 bases which is hybridized to the first label attachment region, to the first RNA molecule is attached at said regularly repeated base one or more label monomers that emit light constituting a first signal;
(ii) a second label attachment region, which is non-overlapping with the first label attachment region, comprising a second DNA sequence of about 300 to about 1500 nucleotides in length having a G/C content of about 40% to about 60% and having a regularly repeated base every about 4 to about 25 bases, and comprising at least a second RNA molecule having a regularly repeated base every about 4 to about 25 bases which is hybridized to the second label attachment region, to the second RNA molecule is attached at said regularly repeated base one or more label monomers that emit light constituting a second signal;
(iii) a third label attachment region, which is non-overlapping with the first label attachment region and is non-overlapping with the second label attachment region, comprising a third DNA sequence of about 300 to about 1500 nucleotides in length having a G/C content of about 40% to about 60% and having a regularly repeated base every about 4 to about 25 bases, and comprising at least a third RNA molecule having a regularly repeated base every about 4 to about 25 bases which is hybridized to the third label attachment region,
wherein the third RNA molecule is not attached to a label monomer and lacks a signal; and
(iv) a first target-specific sequence capable of binding to a target nucleic acid.
2 . The nucleic acid probe of claim 1 , further comprising a first moiety that is capable of selectively binding to a substrate or to a first binding moiety on the substrate.
3 . The nucleic acid probe of claim 2 , wherein the first moiety selectively binds to the substrate or to a first binding moiety on the substrate via one or more non-covalent bonds.
4 . The nucleic acid probe of claim 2 , wherein the first moiety selectively binds to the substrate or to a first binding moiety on the substrate via one or more covalent bonds.
5 . The nucleic acid probe of claim 2 , wherein the first moiety is selected from the group consisting of a ligand, an antigen, a carbohydrate, a nucleic acid, a receptor, a lectin, an antibody, a succinamide, an amine, an aldehyde, an epoxy, a thiol, biotin, digoxigenin, FITC, avidin, streptavidin, antidigoxigenin, and anti-FITC.
6 . The nucleic acid probe of claim 5 , wherein the first moiety is biotin.
7 . The nucleic acid probe of claim 5 , wherein the first moiety is avidin or streptavidin.
8 . A pair of nucleic acid probes, comprising:
(a) a first nucleic acid molecule comprising:
(i) a first label attachment region, comprising a first DNA sequence of about 300 to about 1500 nucleotides in length having a G/C content of about 40% to about 60% and having a regularly repeated base every about 4 to about 25 bases, and comprising at least a first RNA molecule having a regularly repeated base every about 4 to about 25 bases which is hybridized to the first label attachment region, to the first RNA molecule is attached at said regularly repeated base one or more label monomers that emit light constituting a first signal;
(ii) a second label attachment region, which is non-overlapping with the first label attachment region, comprising a second DNA sequence of about 300 to about 1500 nucleotides in length having a G/C content of about 40% to about 60% and having a regularly repeated base every about 4 to about 25 bases, and comprising at least a second RNA molecule having a regularly repeated base every about 4 to about 25 bases which is hybridized to the second label attachment region, to the second RNA molecule is attached at said regularly repeated base one or more label monomers that emit light constituting a second signal;
(iii) a third label attachment region, which is non-overlapping with the first label attachment region and is non-overlapping with the second label attachment region, comprising a third DNA sequence of about 300 to about 1500 nucleotides in length having a G/C content of about 40% to about 60% and having a regularly repeated base every about 4 to about 25 bases, and comprising at least a third RNA molecule having a regularly repeated base every about 4 to about 25 bases which is hybridized to the third label attachment region,
wherein the third RNA molecule is not attached to a label monomer and lacks a signal;
(iv) a first target-specific sequence capable of binding to a target nucleic acid; and
(v) a first moiety that is capable of selectively binding to a substrate or to a first binding moiety on the substrate; and
(b) a second nucleic acid molecule comprising:
(i) a second target-specific sequence capable of binding to the target nucleic acid, wherein the first target-specific sequence and the second target-specific sequence bind to different regions of the target nucleic acid; and
(ii) a second moiety that is capable of selectively binding to the substrate or to a second binding moiety on the substrate.
9 . The pair of nucleic acid probes of claim 8 , wherein the first or second moiety selectively binds to the substrate or to a first or second binding moiety on the substrate via one or more non-covalent bonds.
10 . The pair of nucleic acid probes of claim 8 , wherein the first or second moiety selectively binds to the substrate or to a first or second binding moiety on the substrate via one or more covalent bonds.
11 . The pair of nucleic acid probes of claim 8 , wherein the first moiety and the second moiety are independently selected from the group consisting of a ligand, an antigen, a carbohydrate, a nucleic acid, a receptor, a lectin, an antibody, a succinamide, an amine, an aldehyde, an epoxy, a thiol, biotin, digoxigenin, FITC, avidin, streptavidin, antidigoxigenin, and anti-FITC.
12 . The pair of nucleic acid probes of claim 11 , wherein the first moiety or the second moiety is biotin.
13 . The pair of nucleic acid probes of claim 11 , wherein the first moiety or the second moiety is avidin or streptavidin.
14 . A population of probe pairs, wherein each probe pair in the population comprises:
(a) a first nucleic acid molecule comprising
(i) a first label attachment region, comprising a first DNA sequence of about 300 to about 1500 nucleotides in length having a G/C content of about 40% to about 60% and having a regularly repeated base every about 4 to about 25 bases, and comprising at least a first RNA molecule having a regularly repeated base every about 4 to about 25 bases which is hybridized to the first label attachment region, to the first RNA molecule is attached at said regularly repeated base one or more label monomers that emit light constituting a first signal;
(ii) a second label attachment region, which is non-overlapping with the first label attachment region, comprising a second DNA sequence of about 300 to about 1500 nucleotides in length having a G/C content of about 40% to about 60% and having a regularly repeated base every about 4 to about 25 bases, and comprising at least a second RNA molecule having a regularly repeated base every about 4 to about 25 bases which is hybridized to the second label attachment region, to the second RNA molecule is attached at said regularly repeated base one or more label monomers that emit light constituting a second signal;
(iii) a third label attachment region, which is non-overlapping with the first label attachment region and is non-overlapping with the second label attachment region, comprising a third DNA sequence of about 300 to about 1500 nucleotides in length having a G/C content of about 40% to about 60% and having a regularly repeated base every about 4 to about 25 bases, and comprising at least a third RNA molecule having a regularly repeated base every about 4 to about 25 bases which is hybridized to the third label attachment region,
wherein the third RNA molecule is not attached to a label monomer and lacks a signal;
(iv) a first target-specific sequence capable of binding to a target nucleic acid; and
(v) a first moiety that is capable of selectively binding to a substrate or to a first binding moiety on the substrate; and
(b) a second nucleic acid molecule comprising:
(i) a second target-specific sequence capable of binding to the target nucleic acid, wherein the first target-specific sequence and the second target-specific sequence bind to different regions of the target nucleic acid; and
(ii) a second moiety that is capable of selectively binding to the substrate or to a second binding moiety on the substrate.
15 . The population of probe pairs of claim 14 , wherein each first or second moiety selectively binds to the substrate or to a first or second binding moiety on the substrate via one or more non-covalent bonds.
16 . The population of probe pairs of claim 14 , wherein each first or second moiety selectively binds to the substrate or to a first or second binding moiety on the substrate via one or more covalent bonds.
17 . The population of probe pairs of claim 14 , wherein each first moiety and second moiety is independently selected from the group consisting of a ligand, an antigen, a carbohydrate, a nucleic acid, a receptor, a lectin, an antibody, a succinamide, an amine, an aldehyde, an epoxy, a thiol, biotin, digoxigenin, FITC, avidin, streptavidin, antidigoxigenin, and anti-FITC.
18 . The population of probe pairs of claim 17 , wherein each first moiety or second moiety is biotin.
19 . The population of probe pairs of claim 17 , wherein each first moiety or second moiety is avidin or streptavidin.
20 . A kit comprising the population of probe pairs of claim 14 .