Stable nanoreporters
View Patent ↗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 improved, stable nanoreporter probes that are capable of binding to and identifying target molecules based on the probes' uniquely detectable signal. Methods for identifying target-specific sequences for inclusion in the probes are also provided, as are methods of making and using such probes. Polynucleotide sequences of certain nanoreporter components are also provided. The probes can be used in diagnostic, prognostic, quality control and screening applications.
1. A population of uniquely labeled nanoreporter probes, wherein each uniquely labeled nanoreporter probe of the population comprises:
i) a unique target-specific region; and
ii) a region comprising a single-stranded nucleic acid synthetic backbone, said single-stranded nucleic acid backbone comprising a plurality of label attachment regions covalently attached together in a unique linear combination;
wherein each label attachment region of each single-stranded nucleic acid backbone is different from the other label attachment regions in that same single-stranded nucleic acid backbone;
wherein each label attachment region of each single-stranded nucleic acid backbone is selected from a population of designed polynucleotide sequences;
wherein each designed polynucleotide sequence is hybridized to a complementary polynucleotide sequence having attached thereto one or more detectable molecules;
wherein each complementary polynucleotide sequence has designated a specific detectable molecule;
wherein each label attachment region has a G/C content of about 50%, a thymine content of about 35-45%, and wherein each complementary polynucleotide sequence has a G/C ratio of about 1/1 or, optionally, of about 3/2;
wherein each uniquely labeled nanoreporter probe of the population has a detectable signal that distinguishes it from the other uniquely labeled nanoreporter probes in said population;
wherein the single-stranded nucleic acid synthetic backbone comprises less than about 1% of inverted repeats or direct repeats, wherein the inverted repeats or direct repeats are 9 nucleotides or greater; and
wherein each single-stranded nucleic acid synthetic backbone contains less than 85% homology to another single-stranded nucleic acid synthetic backbone in the population of nanoreporters or to any sequence described in the REFSEQ public database.
2. The population of claim 1 wherein at least one of said label attachment regions comprises about 800 to 1300 nucleotide bases.
3. The population of claim 1 wherein said complementary polynucleotide sequence has a G/C ratio of about 3/2.
4. The population of claim 1 wherein each uniquely labeled nanoreporter probe of the population further comprises a constant region, wherein the constant region comprises a plurality of repeat nucleotide sequences.
5. The population of claim 1 wherein the label attachment regions comprise a regularly repeated pattern of adenine bases.
6. The population of claim 5 wherein the adenine bases are spaced about every 8 to 16 nucleotide bases.
7. The population of claim 1 wherein the complementary polynucleotide sequence comprises a RNA polynucleotide sequence.
8. The population of claim 7 wherein the RNA polynucleotide sequence comprises at least one amino-allyl modified uracil base.
9. The population of claim 8 wherein the detectable molecule is attached to the amino-allyl modified uracil base.
10. The population of claim 7 wherein the RNA polynucleotide sequence comprises a plurality of amino-allyl modified uracil bases that are spaced at about an average of every 8 to 16 bases in said RNA polynucleotide sequence.
11. The population of claim 10 wherein the detectable molecule is attached to each of the allyl modified uracil bases.
12. The population of claim 1 wherein the detectable molecules are fluorescent dyes.
13. The population of claim 1 , wherein when said population of uniquely labeled nanoreporter probes is used to detect a population of target molecules in a sample by a method comprising (a) forming at least one molecular complex with each uniquely labeled nanoreporter probe comprising at least one target molecule and at least one nanoreporter probe and (b) individually counting the presence of one or more molecules of each of said molecular complex in said sample, the numbers of counts above background of each of said molecular complex after normalization of the sample is at least two fold higher when compared to a nanoreporter probe comprising M13 DNA.
14. The population of claim 1 where the melting temperature (Tm) of said complementary polynucleotide sequences when hybridized to its label attachment regions is about 80° C. or higher.
15. The population of claim 1 wherein each single-stranded nucleic acid synthetic backbone contains less than 50% homology to another single-stranded nucleic acid synthetic backbone in the population of nanoreporters or to any sequence described in the REF SEQ public database.
16. The population of claim 1 wherein each label attachment region lacks a direct or inverted repeat that is greater than 12 nucleotides in length.
17. The population of claim 16 wherein each label attachment region lacks a direct or inverted repeat that is greater than about 7 nucleotides in length.
18. A uniquely labeled nanoreporter probe comprising:
i) a unique target-specific region; and
ii) a region comprising a plurality of designed label attachment regions covalently attached together in a synthetic linear combination, wherein each label attachment region comprises about 800 to 1300 nucleotide bases, has a G/C content of about 50%, a thymine content of about 35-45%, wherein each label attachment region is different from the other label attachment region in the uniquely labeled nanoreporter probe;
wherein each label attachment region is hybridized to a complementary polynucleotide sequence having attached thereto one or more detectable molecules, wherein the complementary polynucleotide sequence has a G/C ratio of at least 1/1;
wherein the region comprising a plurality of designed label attachment regions covalently attached together in a synthetic linear combination comprises less than about 1% of inverted repeats or direct repeats, wherein the inverted repeats or direct repeats are 9 nucleotides or greater; and
wherein the region comprising a plurality of designed label attachment regions covalently attached together in a synthetic linear combination comprises less than 85% homology to any sequence described in the REFSEQ public database.
19. The nanoreporter probe of claim 18 wherein the complementary polynucleotide sequence has a G/C ratio of about 3/2.
20. The nanoreporter probe of claim 18 further comprising a constant region, wherein the constant region comprises a plurality of repeat nucleotide sequences.
21. The nanoreporter probe of claim 18 wherein at least one of the designed label attachment regions comprises a regularly repeated pattern of adenine bases.
22. The nanoreporter probe of claim 21 wherein the adenine bases are spaced about an average of every 8 to 16 nucleotide bases.
23. The nanoreporter probe of claim 18 wherein the complementary polynucleotide sequence comprises a RNA polynucleotide sequence.
24. The nanoreporter probe of claim 23 wherein the RNA polynucleotide sequence comprises at least one amino-allyl modified uracil base.
25. The nanoreporter probe of claim 24 wherein the detectable molecule is attached to the amino-allyl modified uracil base.
26. The nanoreporter probe of claim 23 wherein the RNA polynucleotide sequence comprises a plurality of amino-allyl modified uracil bases that are spaced at about an average of every 8 to 16 bases in said RNA polynucleotide sequence.
27. The nanoreporter probe of claim 26 wherein the detectable molecule is attached to each of the allyl modified uracil bases.
28. The nanoreporter probe of claim 18 wherein the detectable molecules are fluorescent dyes.
29. The nanoreporter probe of claim 18 wherein the region comprising a plurality of designed label attachment regions covalently attached together in a synthetic linear combination contains less than 50% homology to any sequence described in the REFSEQ public database.
30. The nanoreporter probe of claim 18 wherein each label attachment region lacks a direct or inverted repeat that is greater than 12 nucleotides in length.
31. The nanoreporter probe of claim 30 wherein each label attachment region lacks a direct or inverted repeat that is greater than about 7 nucleotides in length.