Stable Nanoreporters
AssigneeNanoString Technologies, Inc.
InventorsPhilippa Jane Webster, Timothy Dahl
Life of this patent
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Abstract
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.
Claims63
A population of uniquely labeled nanoreporter probes, wherein the nanoreporter probe comprises:
- i) an unique target-specific region;
- and
- ii) a region comprising an unique, designed nanoreporter wherein said nanoreporter comprises a single-stranded nucleic acid backbone, said backbone comprising a plurality of label attachment regions covalently attached together in an unique linear combination, wherein the label attachment regions are selected from a population of designed polynucleotide sequences, wherein the 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;
- and wherein each nanoreporter has a detectable signal that distinguishes it from other nanoreporters in said population.
A method for determining the presence of at least one target molecule in a sample, comprising:
- (1) forming at least one molecular complex comprising:
- (a) at least one target molecule, and
- (b) at least one probe comprising an unique target-specific region and a region comprising an unique, designed nanoreporter, wherein said nanoreporter comprises a single-stranded nucleic acid backbone, said backbone comprising a plurality of label attachment regions covalently attached together in an unique linear combination, wherein each label attachment region 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;
- and
- (2) individually counting the presence of one or more molecular complex or at least part of said at least one molecular complex to determine the presence of said at least one target molecule in the sample.
An uniquely labeled nanoreporter probe comprising:
- i) an unique target-specific region;
- and
- ii) a region comprising a plurality of designed label attachment regions covalently attached together in a linear combination, wherein each label attachment region comprises about 800 to 1300 nucleotide bases and has a G/C content of about 50%, wherein each selected label attachment region is different from the other selected label attachment regions, and
- 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.
A method of preparing at least one uniquely labeled nanoreporter comprising:
- i) combining a plurality of label attachment region each comprising about 800 to 1300 nucleotide bases and a G/C content of about 50%, wherein each selected label attachment region is different from the other selected label attachment regions;
- ii) covalently attaching the plurality of label attachment regions to each other in linear combination;
- and
- iii) hybridizing a complementary polynucleotide sequence to said label attachment region, wherein said complementary polynucleotide sequence is having attached thereto one or more detectable molecules.
A kit for preparing at least one uniquely labeled nanoreporter comprising:
- a) at least three label attachment regions each comprising about 800 to 1300 nucleotide bases, a G/C content of about 50%;
- and
- b) at least three complementary polynucleotide sequences having attached thereto a detectable molecule, wherein the complementary polynucleotide sequences has a G/C ratio of at least 1/1.
Dependencies are inferred from the claim text.