WO2007002375A2 - Procédés et compositions pour l'analyse de microarn - Google Patents

Procédés et compositions pour l'analyse de microarn Download PDF

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Publication number
WO2007002375A2
WO2007002375A2 PCT/US2006/024448 US2006024448W WO2007002375A2 WO 2007002375 A2 WO2007002375 A2 WO 2007002375A2 US 2006024448 W US2006024448 W US 2006024448W WO 2007002375 A2 WO2007002375 A2 WO 2007002375A2
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mirna
nucleic acid
microrna
probes
probe
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PCT/US2006/024448
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WO2007002375A3 (fr
Inventor
Lori A. Neely
Maria Hackett
Sonal Patel
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U.S. Genomics, Inc.
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Publication of WO2007002375A2 publication Critical patent/WO2007002375A2/fr
Publication of WO2007002375A3 publication Critical patent/WO2007002375A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention provides methods and compositions for analysis of microRNA, including detection and quantitation.
  • miRNAs are short naturally occurring RNAs generally ranging in length from about 7 to about 27 nucleotides.
  • miRNAs Only a few hundred miRNAs have been identified. This number is far lower than the expected number of coding sequences in the human genome. However, it is not expected that each coding sequence has its own unique miRNA. This is because miRNAs generally hybridize to RNAs with one or more mismatches. The ability of the miRNA to bind to RNA targets in spite of these apparent mismatches provides the variability necessary to potentially modulate a number of transcripts with a single miRNA. miRNA therefore can act as regulators of cellular development, differentiation, proliferation and apoptosis. miRNAs can modulate gene expression by either impeding mRNA translation, degrading complementary mRNAs, or targeting genomic DNA for methylation.
  • miRNAs can modulate translation of mRNA transcripts by binding to and thereby making such transcripts susceptible to nucleases that recognize and cleave double stranded RNAs.
  • miRNAs have also been implicated as developmental regulators in mammals in two recent mouse studies characterizing specific miRNAs involved in stem cell differentiation (Houbaviy HB 2003; Chen CZ 2004). Numerous studies have demonstrated miRNAs are critical for cell fate commitment and cell proliferation (Brennecke J 2003) (Zhao Y 2005). Other studies have analyzed the role of miRNAs in cancer (Michael MZ 2003; Calin 2004; He 2005; Johnson SM 2005).
  • miRNAs may play a role in diabetes (Poy MN 2004) and neurodegeneration associated with Fragile X syndrome, spinal muscular atrophy, and early on-set Parkinson's disease (Caudy 2002; Hutvagner 2002; Mouelatos 2002; Dostie 2003).
  • Several miRNAs are virally encoded and expressed in infected cells (e.g., EBV, HPV and HCV).
  • the invention provides methods and systems (and corresponding reagents) for detecting and optionally quantitating microRNA (miRNA) in a sample.
  • the method may quantitate all known miRNAs within a complex total RNA sample. It is theoretically unlimited in its degree of multiplexing and offers increased specificity.
  • the method comprises contacting a template nucleic acid with a miRNA and allowing the template nucleic acid to bind to the miRNA thereby creating a double stranded hybrid with a 5' template overhang, polymerizing (i.e., synthesizing) a nucleic acid tail to the miRNA wherein the nucleic acid tail is complementary to the 5' template overhang (or a part thereof) and thereby creating a tailed miRNA, separating the template nucleic acid from the tailed miRNA, contacting a first and a second sequence-specific probe with the tailed miRNA and allowing the first and second sequence-specific probes to bind to the tailed miRNA wherein the first and second sequence-specific probes are complementary to the tailed miRNA, contacting the tailed miRNA to a nucleic acid complementary to the nucleic acid tail and conjugated to a solid support at a defined location (i.e., a capture nucleic acid or a capture probe) and allowing
  • the method involves contacting one sequence-specific probe with the tailed miRNA and allowing the sequence-specific probe to bind to the tailed miRNA wherein the sequence-specific probe is complementary to the tailed miRNA (preferably within the miRNA specific region), contacting the tailed miRNA to a nucleic acid complementary to the nucleic acid tail and conjugated to a solid support at a defined location (i.e., a capture nucleic acid or a capture probe) and allowing the tailed miRNA to bind to the solid support at the defined location (via binding to the capture nucleic acid), and detecting the level of binding of the tailed miRNA to the solid support based on the presence of the sequence-specific probe at the defined location.
  • the probe is conjugated to a detectable label.
  • the detectable label may be a fluorophore.
  • first and second sequence-specific probes are conjugated to first and second detectable labels, respectively.
  • the labels are preferably distinct from each other.
  • first and second detectable labels are first and second fluorophores.
  • the template nucleic acid is about 50% longer than the miRNA.
  • the miRNA is between 7 and 27 nucleotides in length, and preferably less than 25 nucleotides in length.
  • the 5' template overhang is at least 10 bases in length.
  • the tailed miRNA is contacted with the first and second sequence-specific probes prior to contact with and binding to the solid support (via the capture nucleic acid). In another embodiment, the tailed miRNA is contacted with the first and second sequence-specific probes after contact with and binding to the solid support (via the capture nucleic acid).
  • the template nucleic acid is a DNA. In other embodiments, it may comprise non-naturally occurring elements such as PNAs or LNAs or combinations thereof.
  • the first and second sequence-specific probes are LNA-DNA chimerae or co-polymers.
  • the solid support is a silica chip.
  • the method further comprises quantitating a plurality of miRNA.
  • the plurality of miRNA is greater than one and will be limited by the number of unique probe pairs (or unique detectable label pairs) and/or the capacity of the solid support.
  • the upper end of the plurality may be equal to or less than 10000, 3000, 1000, 500, 100, 50, 25, 10, or any integer in between as if explicitly recited herein.
  • the defined location on the solid support has a plurality of capture nucleic acids conjugated to it.
  • the plurality in this situation is dependent on the capacity and degree of derivatization of the solid support. Accordingly, the plurality of nucleic acids is at least two and equal to or less than 1000, 750, 500, 250, 100 or 50, in some embodiments.
  • the nucleic acid tail is polymerized by a primer extension reaction.
  • the primer extension reaction comprises a thermophilic exopolymerase.
  • the nucleic acid tail is fluorescent.
  • the nucleic acid complementary to the nucleic acid tail i.e., the capture nucleic acid
  • the nucleic acid complementary to the nucleic acid tail is a LNA.
  • the nucleic acid complementary to the nucleic acid tail i.e., the capture nucleic acid
  • the nucleic acid complementary to the nucleic acid tail is tethered to the solid support via a 3' ethylene glycol scaffold.
  • the invention provides a method for diagnosing a condition comprising determining a level of a miRNA in a test tissue sample, and comparing the level of the miRNA in the test tissue sample to a level of the miRNA in a control tissue sample.
  • a difference in the level of the miRNA in the test and the control tissue samples is indicative of the condition. In one embodiment, the difference in the level of the miRNA in the test and the control tissue samples is a greater level of miRNA in the test tissue sample. In one embodiment, the difference in the level of the miRNA in the test and the control tissue samples is a greater level of miRNA in the control tissue sample.
  • the level of the miRNA is determined by coincidence binding of one or more probes to the target miRNA. If more than one probe is used, preferably the probes are differentially and detectably labeled.
  • the coincidence binding is performed at a single molecule level.
  • coincidence binding comprises coincident detection of for example two signals from a first miRNA-specific probe labeled with a first detectable label and a second miRNA-specific probe labeled with a second detectable label distinguishable from the first detectable label. Such analysis may further comprise subtracting a random coincidence estimate from a raw coincidence count.
  • coincidence binding comprises use of a quencher probe.
  • the test tissue sample is a breast tissue sample, a cervical tissue sample, an ovarian tissue sample, or a prostate tissue sample.
  • the condition is cancer such as but not limited to breast cancer, cervical cancer, colon cancer, ovarian cancer, or prostate cancer. In another embodiment, the condition is cirrhosis.
  • the miRNA is mir-143 or mir-145.
  • the miRNA is a human miRNA and the samples are human samples.
  • the miRNA is present at a concentration of 1-1000 femtomolar, 1-100 femtomolar, or 1-10 femtomolar.
  • the invention provides a method for detecting microRNA in sample comprising contacting a sample with a first and a second nucleic acid probe under conditions and for a time sufficient to allow hybridization to a microRNA, wherein the first and second nucleic acid probes are conjugated to a first and second detectable label, respectively, that are distinct from each other, and detecting coincident binding of the first and second nucleic acid probes to a single microRNA as coincident signals from the first and second detectable labels.
  • Hybridization of the first and second nucleic acid probes to a microRNA results in a double stranded duplex (or hybrid) having at least a one or two base overhang at the 3 'and 5' end of the microRNA, and coincident signals are indicative of a microRNA.
  • the first and second nucleic acid probes have a sum total length that is at least 2, at least 3, at least 4, or at least 5 bases longer than the microRNA. In one embodiment, the first and second nucleic acid probes each is a DNA, PNA, LNA or a combination thereof.
  • the first nucleic acid probe is conjugated to a first fluorophore and the second nucleic acid probe is conjugated to a second fluorophore and the first and second fluorophores are a FRET pair.
  • the one or two base overhang at the 3 'and 5' end each comprises a cytosine or a guanosine. In one embodiment, the one or two base overhang at the 3'and 5' end each comprises an adenine or a thymidine. In one embodiment, the one or two base overhang at the 3' and 5' end each comprises an iso-guanosine or an iso-cytosine.
  • the first and second nucleic acid probes is each at least 12 or at least 13 bases long. In one embodiment, the first and second nucleic acid probes have a sum total length that is greater than the length of the microRNA. In one embodiment, the method further comprises isolating the double stranded duplex from the sample.
  • the double stranded duplex is isolated from the sample by size separation.
  • the sample comprises a plurality of RNA molecules.
  • the method further comprises column purification prior to detecting coincident binding.
  • the method further comprises addition of a quencher labeled nucleic acid probe to the sample prior to detecting coincident binding.
  • the method further comprises addition of a single stranded nuclease to the sample prior to detecting coincident binding.
  • the method further comprises ligating the first nucleic acid probe to the second nucleic acid probe prior to detecting coincident binding.
  • the invention provides a method for detecting microRNA in sample comprising contacting a sample with a dual labeled nucleic acid probe under conditions and for a time sufficient to allow hybridization to a microRNA, wherein the dual labeled nucleic acid probe comprises at least two distinct detectable labels, thereby allowing a substantially double stranded hybrid (or duplex) to form between the microRNA and the nucleic acid probe, contacting the sample with a single stranded nuclease under conditions and for a time sufficient to cleave single stranded regions within the hybrid, and detecting binding of the nucleic acid probe to a single microRNA as coincident signals from the distinct detectable labels. Coincident signals are indicative of a microRNA.
  • the nucleic acid probe has a length that is at least 2, at least 3, at least 4, or at least 5 bases longer than the microRNA.
  • the nucleic acid probe is a DNA, PNA, LNA or a combination thereof. In one embodiment, the nucleic acid probe is a molecular beacon. In one embodiment, the nucleic acid probe is 22-28 bases long.
  • the double stranded hybrid comprises a one or two base overhang at the 3 'and 5' end of the miRNA.
  • the one or two base overhang at the 3 'and 5' end each comprises a cytosine or a guanosine.
  • the one or two base overhang at the 3 'and 5' end each comprises an adenine or a thymidine.
  • the one or two base overhang at the 3' and 5' end each comprises an iso- guanosine or an iso-cytosine.
  • the method further comprises isolating the double stranded hybrid from the sample. In one embodiment, the method further comprises column purification prior to detecting coincident binding. In one embodiment, the method further comprises addition of a quencher labeled nucleic acid probe to the sample prior to detecting coincident binding.
  • the double stranded hybrid is isolated from the sample by size separation.
  • the sample comprises a plurality of RNA molecules.
  • the single stranded nuclease is RNase or Sl nuclease.
  • the invention provides a method for detecting microRNA in sample comprising contacting a sample with a dual labeled nucleic acid probe under conditions and for a time sufficient to allow hybridization to a microRNA, wherein the dual labeled nucleic acid probe comprises a FRET donor fluorophore and a FRET acceptor fluorophore, thereby allowing a substantially double stranded duplex to form between the microRNA and the nucleic acid probe, contacting the sample with a single stranded nuclease under conditions and for a time sufficient to cleave single stranded nucleic acids including single stranded nucleic acid regions within the hybrid, and detecting binding of the nucleic acid probe to a single microRNA as emission from the FRET acceptor fluorophore following excitation of the FRET donor fluorophore. Emission from the FRET acceptor fluorophore is indicative of a microRNA.
  • the invention provides a method for detecting microRNA in a sample comprising contacting a sample with a universal nucleic acid (or linker) having a first sequence specific for a microRNA conjugated to a second sequence that is a universal sequence, under conditions and for a time sufficient to allow hybridization of the universal nucleic acid to a microRNA, thereby forming a double stranded duplex with a 5' overhang comprising the universal sequence, synthesizing a nucleic acid tail from the miRNA wherein the tail is complementary to the 5' overhang, thereby creating a tailed miRNA, separating the tailed miRNA from the universal nucleic acid, contacting the tailed miRNA with a miRNA- specif ⁇ c probe labeled with a first detectable label and a universal sequence-specific probe labeled with a second detectable label, wherein the first and second detectable labels are distinct, and detecting coincident binding of the probes to a single microRNA as coincident signals from the
  • the invention provides a method for detecting microRNA in a sample comprising contacting a sample with a universal nucleic acid having a first sequence specific for a microRNA conjugated to a second sequence that is a universal sequence, under conditions and for a time sufficient to allow hybridization of the universal nucleic acid to a microENA, thereby forming a double stranded duplex with a 5' overhang comprising the universal sequence, synthesizing a nucleic acid tail from the miRNA wherein the tail is complementary to the 5' overhang, thereby creating a tailed miRNA, separating the tailed miRNA from the universal nucleic acid, contacting the tailed miRNA with a miRNA-specific probe labeled with a first fluorophore and a universal sequence-specific probe labeled with a second fluorophore, wherein the first and second fluorophores are a FRET pair comprised of a FRET donor fluorophore and a FRET acceptor fluor
  • first and second fluorophores are located at proximal ends of the probes when hybridized to the tailed miRNA.
  • the universal nucleic acid is at least 20 or at least 40 bases in length.
  • each of the probes is independently a DNA, PNA, LNA or a combination thereof.
  • the method further comprises isolating the tailed miRNA with coincidentally bound probes from the sample.
  • the tailed miRNA with coincidentally bound probes is isolated from the sample by size separation.
  • the sample comprises a plurality of RNA molecules.
  • the method further comprises column purification prior to detecting coincident binding or coincident signals.
  • the method further comprises addition of a quencher labeled nucleic acid probe to the sample prior to detecting coincident binding.
  • the method further comprises ligating the probes to each other prior to detecting coincident binding.
  • the detectable labels are located at distal ends of the probes when hybridized to the tailed miRNA.
  • the invention provides a method for detecting microRNA comprising contacting a sample with a microRNA-specif ⁇ c nucleic acid probe that is conjugated to a first detectable label under conditions and for a time sufficient for specific hybridization of the probe to a microRNA, thereby forming a double stranded duplex and a 5' overhang comprising microRNA sequence, synthesizing a nucleic acid tail from the microRNA-specific probe wherein the tail is complementary to a 5' region of the microRNA using nucleotides that are labeled with a second detectable label that is distinct from the first detectable label, thereby forming a dual labeled microRNA-specific probe hybridized to a microRNA, removing single stranded nucleic acids from the sample, and detecting coincident signals from the first and the second detectable labels. Coincident signals are indicative of a microRNA.
  • the invention provides a method for detecting microRNA comprising contacting a sample with a microRNA-specific nucleic acid probe that is conjugated to a first fiuorophore under conditions and for a time sufficient for specific hybridization of the probe to a microRNA, thereby forming a double stranded duplex and a 5' overhang comprising microRNA sequence, synthesizing a nucleic acid tail from the microRNA-specific probe wherein the tail is complementary to a 5' region of the microRNA using nucleotides that are labeled with a second fiuorophore, wherein the first and second fluorophores are a FRET pair comprised of a FRET donor and a FRET acceptor fiuorophore, thereby forming a dual labeled microRNA-specific probe hybridized to a microRNA, removing single stranded nucleic acids from the sample, and detecting emission from the FRET acceptor fiuorophore following excitation of the FRET donor fiu
  • the single stranded nucleic acids are removed from the sample by column purification prior to detecting coincident signals. In one embodiment, the single stranded nucleic acids are removed from the sample by addition of a single stranded nuclease to the sample prior to detecting coincident signals.
  • the microRNA-specific probe is at least 2, at least 3, at least 4, at least 5, at least 6, at least 6 or at least 7 bases shorter than the microRNA. In one embodiment, the microRNA-specific probes is at least 15 or at least 20 bases long. In one embodiment, wherein the microRNA-specific probe is a DNA, PNA, LNA or a combination thereof. In one embodiment, the method further comprises isolating the dual labeled microRNA-specific probe hybridized to a microRNA from the sample.
  • the dual labeled microRNA-specific probe hybridized to a microRNA is isolated from the sample by size separation.
  • the sample comprises a plurality of RNA molecules.
  • the method further comprises addition of a quencher labeled nucleic acid probe to the sample prior to detecting coincident signals.
  • FIG. 1 is a schematic of the method for quantitating miRNA as provided herein.
  • FIG. 2 shows the results of a hybridization reaction in which a DNA oligonucleotide was radiolabeled with P 32 and hybridized in solution to the lin-4 miRNA (SEQ ID NO:35) spiked into a complex total RNA sample (2 micrograms of E. coli total RNA).
  • FIG. 3 shows the results of a hybridization reaction in which a DNA oligonucleotide was radiolabeled with P 32 and hybridized in solution to the mutant lin-4 miRNA (SEQ ID NO:36) spiked into a complex total RNA sample (2 micrograms of E. coli total RNA).
  • FIG. 4 shows the specific extension of a fluorescently labeled DNA tail onto lin-4.
  • Extension reactions utilized Therminator (NEB) with sub-optimal concentrations of nucleotides (200 nM). The reactions were cycled 20 times (9O 0 C denaturation, 5O 0 C hybridization, 7O 0 C extension).
  • NEB Therminator
  • FIG. 5 is a schematic of a DirectTM miRNA assay.
  • FIG. 6 is a graph showing the sensitivity and linear dynamic range of the DirectTM miRNA assay.
  • the inset graph shows the linear range at the lower fM concentration range.
  • FIG. 7A is a table showing the results of a mir-16 analysis using the DirectTM miRNA assay.
  • FIG. 7B is a table showing the results from three separate determinations of mir-16 level in various tissues.
  • FIG. 7C is a graph showing a calibration curve of mir-16.
  • FIG. 7D is a bar graph showing the expression profile of mir-16 in human total RNA in various tissues.
  • FIG. 8 A is a table showing the results of two separate determinations of mir-126 levels in various human tissues. The assay is highly reproducible even when conducted by different operators on different instruments.
  • FIG. 8B is a table showing the results of two separate determinations of mir-191 levels in brain.
  • FIG. 8C is a table showing the results of two separate determinations of mir-136 levels in cervix.
  • FIG. 8D is a table showing the results of two separate determinations of mir-28 levels in cervix.
  • F ⁇ G. 8E is a table showing the results of two separate determinations of mir-195 levels in thymus and lymph.
  • FIG. 9A is a bar graph showing levels of mir-143 expression in human cancerous and normal tissues.
  • FIG. 9B is a bar graph showing levels of mir-145 expression in human cancerous and normal tissues.
  • FIG. 1 OA is a schematic of an miRNA assay using DNA or RNA probes.
  • FIG. 1OB is a schematic of an miRNA assay using DNA or RNA probes and a
  • FIG. 1OC is a schematic of an miRNA assay using DNA or RNA probes and a DNase/RNase protection step.
  • FIG. HA is a schematic of an miRNA assay using primer extension.
  • FIG. 1 IB is a schematic of an miRNA assay using primer extension and an enzyme clean-up step.
  • FIG. 12 is a schematic of an miRNA assay using DNA or RNA probes and a ligase.
  • FIG. 13 A is a schematic of an miRNA assay using DNA or RNA probes with a universal nucleic acid.
  • FIG. 13B is a schematic of an miRNA assay using miRNA assay using modified primer extension and a universal nucleic acid.
  • FIG. 13C is a schematic of an miRNA assay using a modified labeled primer extension and a universal nucleic acid.
  • FIG. 14 is a schematic of an miRNA assay using a universal nucleic acid and molecular beacons as probes.
  • FIG. 15 A is a schematic of an miRNA assay using RT extension followed by dual probe hybridization (coincident signal detection).
  • FIG. 15B is a schematic of an miRNA assay using RT extension followed by dual probe hybridization (FRET).
  • TABLE 2 shows mir-145 expression levels in tumor, normal adjacent tissues (NAT) and normal tissues.
  • TABLE 3 shows human miRNA expression levels in bladder and lung.
  • SEQ ID NOs: 1-34 are nucleotide sequences of a number of human miRNA, as shown herein.
  • SEQ ID NO:35 is the nucleotide sequence of a wild type lin-4 miRNA.
  • SEQ ID NO:36 is the nucleotide sequence of a point mutant lin-4 miRNA.
  • the methods of the invention can be used to generate information about miRNA.
  • the information obtained by analyzing a miRNA may include its detection in a sample, determination of the amount or level of the miRNA in a sample and how such amounts vary depending on one or more factors including conditions, timing or the presence of other molecules, determination of the relatedness of more than one miRNA, identification of the size of the miRNA, determination of the proximity or distance between two or more individual units within an miRNA, determination of the order of two or more individual units within an miRNA, and/or identification of the general composition of the miRNA.
  • the invention provides a method and system for detecting and quantitating one or more miRNAs simultaneously.
  • the ability to detect more than one miRNA simultaneously is referred to herein as multiplexing capacity.
  • the method of the invention is generally an assay involving the steps of i) hybridization of a template nucleic acid to a miRNA, ii) selective polymerization of a tail onto the end of the hybridized miRNA, iii) hybridization of two spectrally distinctly labeled probes to the tailed miRNA, iv) capture of the labeled tailed miRNAs to a solid surface, and v) measurement of the signal from the labeled tailed miRNA bound to the solid surface.
  • the schematic of the assay is presented in FIG. 1. Each of these steps is discussed in greater detail herein.
  • This method comprises contacting a template nucleic acid with a miRN A and allowing the template nucleic acid to bind to the miRNA thereby creating a 5' template overhang.
  • the amount of template used will depend upon the amount of miRNA target. Generally, a 10-50 fold is recommended although higher amounts can be used in some instances.
  • the template nucleic acid is a nucleic acid comprised of at least two nucleotide sequences.
  • the first sequence is miRNA specific (i.e., it binds to an miRNA target if that target is present in the sample being analyzed).
  • the second sequence is used to generate the tail off of the miRNA "primer” and thus controls the sequence of the tail and ultimately the capture nucleic acids used on the solid supports. This latter sequence may be random, although preferably it is known.
  • Templates that differ in their miRNA specific sequence may also differ in their tail specific sequence, particularly if miRNA identification relies on the location of binding onto the solid support. If miRNA identification relies on the specific probe or probe pairs (and more specifically the signal or coincident signals), then the tail specific sequences may be the same amongst different template nucleic acids.
  • the template nucleic acid may be comprised of naturally and/or non-naturally occurring elements.
  • it may be a DNA, RNA, PNA, LNA, or a combination thereof.
  • the template exhibits some degree of homology to one or more miRNA.
  • level of homology is at least 75%, and includes at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.
  • Binding of the template nucleic acid to the miRNA preferably occurs via Watson Crick binding due to the greater sequence specificity it provides.
  • Hybridization of the template to the miRNA is performed under conditions that provide the desired level of stringency and sequence specificity.
  • Those of ordinary skill in the art will be familiar with standard hybridization conditions and manipulation thereof. (See, for example, Maniatis' Handbook of Molecular Biology.)
  • binding and hybridizing are used interchangeably.
  • a 5' overhang is a single stranded region of the template lying 5' (along the length of the template) to the double stranded hybrid (or duplex) formed by hybridization of the template to the miRNA.
  • the length of the overhang is dependent on the length of the template and of the miRNA to which it hybridizes, as discussed below.
  • the template may be longer than the miRNA, but it is not so limited.
  • the template may not hybridize to the entire length of the miRNA, provided that it hybridizes to a sufficiently long region of the miRNA to provide specific hybridization and a stable hybrid (i.e., the template and miRNA hybrid should be sufficiently stable to allow synthesis of the miRNA tail).
  • the length of the template will contribute to this stability, with templates that hybridize to the entire miRNA being more suitable than those that bind to only a region of the miRNA.
  • the template is at least 5-10 nucleotides longer than the miRNA to which it is targeted, including at least 15, at least 20, at least 25, at least 50, or more nucleotides longer than the miRNA target.
  • the length of the template can be at least 25%, at least 50%, at least 75%, at least 100%, or at least 200% of the length of the target miRNA. Binding of the template to the miRNA may also create a 3' overhang, although no nucleic acid synthesis would be expected to occur from this end of the miRNA.
  • a plurality of template sequences may be added to a population of miRNA (or a population of RNA containing miRNA).
  • Each of the plurality may contain a random or quasi-random sequence in the region intended to bind to a miRNA. That is, the sequences of the target miRNA may not all be known a priori, and the invention can be used to determine those sequences.
  • the method further involves polymerizing a nucleic acid tail to the miRNA using the miRNA as a primer and the 5' overhang as the complementary strand (or template).
  • polymerization refers to the synthesis of new nucleic acid sequence attached to the miRNA (or in some instances to a probe that is functioning as a primer).
  • the nucleic acid tail is therefore complementary to all or part of the 5' overhang. Creation of the nucleic acid tail provides a way of localizing and potentially identifying the miRNA, as will be discussed below in greater detail.
  • Polymerization of the nucleic acid tail is accomplished enzymatically using a polymerase enzyme, the miRNA as a primer, the overhang as the template, and free nucleotides.
  • the polymerase enzyme is preferably a DNA polymerase such as DNA polymerase I or the Klenow fragment thereof.
  • the Klenow fragment from E. coli DNA polymerase I possesses polymerase activity and 3' -> 5' exonuclease activity but lacks 5' -> 3' exonuclease activity associated with DNA polymerase I.
  • the polymerase enzyme is a thermophilic exopolymerase. Use of a thermophilic exopolymerase allows for reaction cycling without significant loss of polymerase activity.
  • the nucleotides used to synthesize the tail are uniquely labeled and thus the synthesized tail is uniquely labeled.
  • Uniquely labeled means that the synthesized tail can be distinguished from the probes that later hybridize to the rm ' RNA (and optionally from all other probes used in a particular reaction mixture), based on different signal emissions. Examples of suitable detectable labels are provided herein.
  • the length of the nucleic acid tail is predominately controlled by the length of the 5' overhang.
  • the nucleic acid tail (and conversely the 5' overhang) must have a length sufficient to bind to a complementary capture nucleic acid (or capture probe) that is located on a solid support.
  • the length is preferably at least 6 nucleotides, but is more preferably longer (e.g., 10 nucleotides or longer).
  • the template nucleic acid tail is physically separated from the tailed miRNA. Physical separation can be accomplished by increasing temperature and/or reducing salt concentration to promote "melting" of the template/miRNA hybrid.
  • Identification of the miRNA is accomplished by binding one or more probes (e.g., first and second) sequence-specific probes to the tailed miRNA.
  • the probes may bind to the miRNA itself or to its tail region, or to a combination thereof. For example, if the tail is sufficiently long, one probe may bind to it (or to a region of it). More commonly, both sequence-specific probes will bind to the miRNA sequence itself.
  • the probes are bound to the tailed miRNA (regardless of the binding position) under stringent hybridization conditions, as discussed herein. If a combination of probes is used, then the combination must be capable of uniquely identifying the tailed miRNA.
  • the probes may be comprised of DNA, RNA, PNA, LNA or a combination thereof (e.g., a LNA-DNA chimerae). Sequence- specific probes are discussed in greater detail herein. A plurality of probes may be used and such a plurality may be synthesized using known or random sequences.
  • the tailed miRNA is positioned on a solid support by hybridizing it to a capture nucleic acid (or capture probe) that is complementary to the nucleic acid tail including a part thereof.
  • the capture nucleic acid is conjugated to the solid support using techniques known in the art.
  • the capture nucleic acid may be tethered to the solid support via a 3' ethylene glycol scaffold. (Matsuya et al. Anal Chem. 2003 Nov 15;75(22):6124-32.)
  • the capture nucleic acid is positioned on a solid support in a particular manner.
  • a solid support may be divided into a grid, each square of the grid having one or more capture nucleic acids of a particular sequence conjugated to it.
  • the number of capture nucleic acids that can be conjugated to a square in the grid will depend on a number of factors such as the size of the square, the conjugation technique used, the length of the capture nucleic acid, etc. In some instances, the number of capture nucleic acids may be in the tens, the hundreds, or even the thousands.
  • Each square may contain capture nucleic acids of a particular known sequence. Thus the location of the square can be representative of the particular miRNA being analyzed.
  • the solid support is then scanned using a detection system such as TrilogyTM for squares occupied by one or more sequence-specific probes. Presence of two probes (or two signals) at a given location is indicative of the presence of a miRNA.
  • the amount of dual signals in a defined location is representative of the amount of a particular miRNA captured (and thus the amount of that miRNA in the tested sample).
  • the method can be used to determine the presence of any number of miRNA, including but not limited to up to 5, 10, 25, 50, 100, 300, 100, 3000, or more.
  • the solid support is described herein as having a grid and therefore being divided into squares, the invention is not so limited. It is only necessary that the locations on the solid support be defined. The locations may be referred to, for example, by co-ordinates or by x-y distances relative to a reference spot on the support (e.g., a comer of the solid support).
  • binding of the sequence-specific probes to the tailed miRNA may occur before or after binding of the tailed miRNA to the capture nucleic acid on the solid support. Therefore, in some embodiments, the tailed miRNA is hybridized to the solid support following which detectably labeled probes are added to the solid support. In this way, smaller amounts of probes are necessary since the hybridization volume is small.
  • the methods of the invention can be used to determine amount or relative concentration of an miRNA species in a sample.
  • the data from a test sample i.e., a sample having unknown miRNA amount or concentration
  • control samples i.e., samples having known miRNA amount or concentration
  • a series of control samples are analyzed in order to generate a standard curve and the data from the test sample is plotted against the standard curve to arrive at an amount or concentration.
  • the invention provides a solution based hybridization assay referred to herein as "DirectTM miRNA” (see FIG. 5).
  • the DirectTM miRNA assay utilizes in some embodiments two spectrally distinguishable probes to label small RNAs of interest.
  • a first probe derivatized with Oyster 556 and a second LNA probe derivatized with Oyster 656 have been used.
  • the probes may be comprised of DNA, RNA, PNA, LNA, and the like, or some combination thereof.
  • both LNA probes are incubated in molar excess in a hybridization reaction with tissue total RNA.
  • probe complementary DNA quencher oligonucleotides are added and allowed to hybridize to the unbound fluorescent LNA probes.
  • the reactions are then diluted and subjected to single molecule analysis.
  • cross-correlation between the two red channels is used to monitor the flow velocity of the fluorescently labeled molecules to ensure even sampling rates across the entire 96-well plate. No enrichment, ligation, reverse transcription, amplification, or clean-up steps are required.
  • the number of coincident photon emissions above a pre-established threshold are counted.
  • This data analysis method provides an estimate of the number of random coincidences expected on the basis of the raw data. This estimate is subtracted from the raw coincidence count to give an estimate of the number of coincidences caused by dual-tagged molecules, and by inference, of the concentration of the analyte.
  • the expression of 47 different miRNAs within sixteen human tissues was analyzed.
  • the data shown in Table 1 are presented as ferntograms/microgram total RNA but could also be presented as femtomoles miRNA/microgram total RNA, or in terms of concentration.
  • the end result of the assay is a reproducible number that depends on the amount of dual-tagged molecules detected per two minute run.
  • the quantitative nature of these data makes the method suited for quantification of miRNA expression in disease and normal tissues.
  • the assay and platform has the ability to measure subtle fold changes in expression levels that may be missed using other approaches.
  • the assay has been further applied to examine the changes in mir-143 and mir-145 expression in adenocarcinoma tumors isolated from cervix, colon, prostate, breast and ovary (see FIG. 9 for detected molecules and Table 2 for femtogram amounts of miRNA) and mir- 122a expression in normal and cirrhotic liver total RNA.
  • the results suggest that miRNA expression is reduced in tumor tissue; however a reduction in miRNA expression in the
  • a direct detection method is one that minimally does not require pre- amplif ⁇ cation (e.g., via PCR) of the target miRNA prior to detection.
  • the method may also be performed using single miRNA specific probes in some embodiments.
  • the simplicity, sensitivity, rapidity and reproducibility of the assay and its detection platform represent a significant advance in the quantification of miRNA expression.
  • the instrument and assay are also completely automatable and as such a superior means for identifying and characterizing disease in a diagnostic setting.
  • the power of a quantifiable number e.g., femtograms miRNA
  • the method embraces the establishment of databases that contain miRNA expression level data for a variety of normal and abnormal tissue types, and comparison of miRNA expression data from test tissue samples to such databases.
  • the miRNA expression levels from a test tissue sample can be compared to a normal control from the same or a different subject prepared concurrently with (or prior to) the test tissue sample, or to expression levels previously determined for one or more abnormal (and optionally normal) tissue types.
  • the method further provides the ability to profile conditions or disease states based on expression (or lack thereof) of one or more miRNA. This allows a more accurate characterization of a disease state and its associated prognosis.
  • a method for detecting a miRNA in a sample that comprises contacting a sample with a first and a second probe wherein the first probe comprises a first detectable label and the second probe comprises a second detectable label, wherein the first and second detectable labels are distinct from each other, for a time and under conditions that allow binding of the first and second probes to their respective targets, and detecting a single miRNA that is bound to both the first and the second probes by coincident detection of the first and second detectable labels (FIGs. 1OA, 1OB and 12).
  • the probes may be of any length. Their combined length may be equal to or greater than the length of the miRNA target.
  • the probes may each be 11, 12, 13 or 14 bases in length. In some embodiments, the probes are of such a length that once bound to the target there exists a one or two or more base overhang at both ends of the duplex. Detection of the single miRNA may be preceded by a "clean-up" step. Such intervening steps are generally intended to separate unreacted reagents (such as unbound probes) from duplexes comprising the target and two probes bound thereto.
  • the clean up step may comprise use of a column that separates hybridization reaction components according to size (FIG. 10A).
  • Another clean up approach may comprise use of enzymes such as RNase and/or DNases to digest single stranded probes (which can be RNA or DNA in nature) as well as unbound targets (FIG. 10B).
  • the two bound probes may be ligated to each other through the action of a ligase, thereby resulting in a double stranded duplex at least 20 or 22 base pairs in length (FIG. 12).
  • miRNA is detected using a dual labeled probe.
  • a dual labeled probe is a probe comprising two distinct labels, preferably, one at each end of the probe.
  • the probes may be DNA or RNA in nature, and their lengths may range from 22- 28 bases in some embodiments.
  • the nuclease therefore would digest unbound probe as well as duplexes having single stranded mismatches.
  • the method would further comprise detection of a single miRNA target having a dual labeled probe bound thereto.
  • miRNA is detected using primer extension (FIGs. 1 IA, 1 IB,
  • the method comprises contacting miRNA with single stranded DNA probes that are labeled with a first detectable label, and then performing a reverse transcription (RT) reaction to extend the probe in the presence of nucleotides that are labeled with a second detectable label that is distinct from the first label.
  • RT reverse transcription
  • Single duplexes comprising the target miRNA and the dually labeled extended probe are then detected and are indicative of the presence of the miRNA in the sample.
  • the dually labeled extended probe may also be detected independent of its hybridization to the target miRNA.
  • the dually labeled extended probe is detected via coincident detection of signals from both labels.
  • the probes in the afore-mentioned method function as primers for the RT reaction.
  • the probes may be of any length that is less than the length of the target miRNA.
  • the probes may be 5-20 nucleotides in length.
  • a pool of labeled nucleotides may be used in the RT reaction, wherein the pool contains a first subset of nucleotides labeled with a second detectable label, a second subset of nucleotides labeled with a third detectable label, etc. provided that the each of the detectable labels is distinct from every other detectable label used in the reaction.
  • the RT reaction mixture may be manipulated in order to remove unreacted substrates such as unincorporated labeled nucleotides. This may be accomplished using a column purification step or a single stranded nuclease (e.g., RNase or Sl nuclease) digestion, or a combination of both, but it is not so limited.
  • Some detection methods may comprise the use of a universal nucleic acid having one sequence that is miRNA specific and a second region that comprises a universal sequence and therefore that acts as a universal linker (FIGs. 13 A, 13B, 13C 5 14, 15A and 15B).
  • a universal sequence is a sequence of known composition that may be common amongst a plurality of universal nucleic acids.
  • the invention provides a method in which single-stranded DNA or RNA probes are contacted with a sample, and allowed to hybridize to their respective target miRNA.
  • the probes are shorter than the target miRNA. They may range in size from 11-14 nucleotides, but they are not so limited.
  • the universal nucleic acid may be included in the initial hybridization reaction or it may be included in a subsequent hybridization reaction. The universal nucleic acid binds to the single stranded region of the target miRNA (i.e., the region not hybridized to the labeled probe). In doing so, a duplex is formed comprising the labeled probe, the target miRNA, and the universal nucleic acid.
  • This duplex further contains a single stranded region that is available as a template for an RT reaction that is primed from the miRNA.
  • the RT reaction is carried out in the presence of labeled nucleotides, as described above.
  • the method further comprises detecting a duplex comprising the labeled probe, the universal nucleic acid, and the labeled and extended target miRNA.
  • the labels are present on opposite strands of the duplex and thus coincident detection of distinct signals requires that the duplex remains intact.
  • a column purification step may be carried out prior to coincident detection analysis.
  • another method is provided for detecting miRNA that comprises a partially double stranded universal nucleic acid (FIG. 13C).
  • the linker may be pre- hybridized prior to further manipulation.
  • the universal nucleic acid comprises a first strand that is detectably labeled and a second strand that is not detectably labeled. The second strand is longer than the first strand, thereby creating a single stranded unlabeled region. This region is specific for a target miRNA.
  • the double stranded universal nucleic acid binds to its respective target miRNA resulting in a new single stranded overhang that then serves as a template for extending the second strand of the universal nucleic acid. This is accomplished by performing an RT reaction with labeled nucleotides.
  • a method for detecting miRNA uses both a universal nucleic acid and at least two probes that are molecular beacons (FIG. 14).
  • a first probe is specific for the target miRNA.
  • a second probe is specific for ' the universal nucleic acid.
  • Each probe further comprises a detectable label, the signal from which is quenched (by a quencher moiety) when the probe is not bound to its target.
  • the detectable labels on the first and second probes are distinct from each other.
  • the universal nucleic acid comprises an miRNA specific sequence (that may be for example 11-13 nucleotides in length) and a universal sequence (that may be for example 18-30 nucleotides in length).
  • the first probe When contacted and allowed to hybridize, the first probe will bind to the target miRNA, as will the universal nucleic acid.
  • the universal nucleic acid in turn will also bind to the second probe. This will result in a double stranded complex comprising both probes (with each emitting its own distinct signal), the target miRNA, and the universal nucleic acid. Single complexes will then be detected via coincident detection of signals from both probes.
  • a method for detecting miRNA by contacting a sample comprising miRNA with a universal nucleic acid (FIG. 15A).
  • the universal nucleic acid comprises an miRNA specific sequence and a universal sequence.
  • the universal nucleic acid is allowed to hybridize to its target miRNA, and the resulting complex comprises a double stranded region and a single stranded region.
  • the target miRNA acts as a primer that is extended using an RT reaction in the presence of nucleotides.
  • the resulting double stranded complex is then denatured and contacted with a first probe that is labeled with a first detectable label and a second probe that is labeled with a second detectable label that is distinct from the first label.
  • one label may be Tamra while the other may be Oyster 656.
  • One probe is therefore specific for the miRNA sequence while the other probe is specific for the universal sequence.
  • Single complexes comprising the extended miRNA strand hybridized to both probes are then detected via coincident detection of signals from both labels.
  • column separation, precipitation, and/or quencher conjugated probes may be used in order to remove unreacted substrates.
  • the universal nucleic acid may be any length that is greater than the target miRNA. For example, it may be 30, 40, 50, 60, or more nucleotides in length.
  • the universal sequence should be chosen so as not to interfere with hybridization to the target miRNA. For example, sequence from E. coli may be used.
  • the probes are each labeled at their opposite end such that, when hybridized to the extended miRNA, the labels are in sufficient proximity to undergo FRET (FIG. 15B).
  • FRET FRET
  • the coincident binding of the probes is detected via detection of signal from one of the labels upon excitation of the other label.
  • one probe may be labeled with Tamra and the other may be labeled with Oyster 656, and a green laser is used in order to detect a red signal.
  • the sample may be a sample that is harvested in accordance with RNA isolation methods.
  • miRNA may be enriched using a YM-100 column.
  • miRNA Targets are short non-coding RNA molecules, usually about 22 nucleotides in length. The sequences of numerous miRNA are known and publicly available. Accordingly, synthesis of miRNA-specific probes is within the ordinary skill in the art based on this information. miRNA sequences can be accessed at for example the website of the miRNA Registry of the Sanger Institute (Wellcome Trust), or the website of Ambion, Inc.
  • miRNA sequences are as follows:
  • RNA isolation protocols See, for example, Maniatis' Handbook of Molecular Biology.
  • the method does not require that miRNA be enriched from a standard RNA preparation.
  • miRNA can be enriched using, for example, a YM- 100 column.
  • the methods of the invention may be performed in the absence of prior nucleic acid amplification in vitro.
  • the miRNA is directly harvested and isolated from a biological sample (such as a tissue or a cell culture), without its amplification.
  • a biological sample such as a tissue or a cell culture
  • Such miRNA are referred to as "non in vitro amplified nucleic acids".
  • a “non in vitro amplified nucleic acid” refers to a nucleic acid that has not been amplified in vitro using techniques such as polymerase chain reaction or recombinant DNA methods.
  • a non in vitro amplified nucleic acid may, however, be a nucleic acid that is amplified in vivo (e.g., in the biological sample from which it was harvested) as a natural consequence of the development of the cells in the biological sample.
  • the non in vitro nucleic acid may be one which is amplified in vivo as part of gene amplification, which is commonly observed in some cell types as a result of mutation or cancer development.
  • miRNA to be detected and optionally quantitated are referred to as target miRNA or target nucleic acids.
  • miRNA may be harvested from a biological sample such as a tissue or a biological fluid.
  • tissue refers to both localized and disseminated cell populations including, but not limited, to brain, heart, breast, colon, bladder, uterus, prostate, stomach, testis, ovary, pancreas, pituitary gland, adrenal gland, thyroid gland, salivary gland, mammary gland, kidney, liver, intestine, spleen, thymus, bone marrow, trachea, and lung.
  • Biological fluids include saliva, sperm, serum, plasma, blood and urine, but are not so limited. Both invasive and non-invasive techniques can be used to obtain such samples and are well documented in the art.
  • the miRNA are harvested from one or few cells.
  • the biological sample can be normal or abnormal (e.g., malignant).
  • Malignant tissues and tumors include carcinomas, sarcomas, melanomas and leukemias generally and more specifically biliary tract cancer, bladder cell carcinoma, bone cancer, brain and CNS cancer, breast cancer, cervical cancer, choriocarcinoma, chronic myelogenous leukemia, colon cancer, connective tissue cancer, cutaneous T-cell leukemia, endometrial cancer, esophageal cancer, eye cancer, follicular lymphoma, gastric cancer, hairy cell leukemia, Hodgkin's lymphoma, intraepithelial neoplasms, larynx cancer, lymphomas, liver cancer, lung cancer (e.g.
  • melanoma multiple myeloma, neuroblastomas, oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cell carcinoma, sarcomas, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, and renal cancer.
  • the method may be used to distinguish between benign and malignant tumors.
  • Subjects from whom such tissue samples may be harvested include those at risk of developing a cancer.
  • a subject at risk of developing a cancer is one who has a high probability of developing cancer (e.g., a probability that is greater than the probability within the general public).
  • These subjects include, for instance, subjects having a genetic abnormality, the presence of which has been demonstrated to have a correlative relation to a likelihood of developing a cancer that is greater than the likelihood of the general public, and subjects exposed to cancer causing agents (i.e., carcinogens) such as tobacco, asbestos, or other chemical toxins, or a subject who has previously been treated for cancer and is in apparent remission.
  • cancer causing agents i.e., carcinogens
  • the method may be used to more finely characterize the cancer and optionally its stage of development, and thereby optionally provide a prognosis.
  • a subject having a cancer is a subject that has detectable cancerous cells.
  • the miRNA may be linearized or stretched prior to analysis, this is not necessary since the detection system is capable of analyzing both stretched and condensed forms. This is particularly the case with coincident events since these events simply require the presence of at least two labels, but are not necessarily dependent upon the relative positioning of the labels (provided however that if they are being detected using FRET, they are sufficiently proximal to each other to enable energy transfer).
  • stretching of the miRNA means that it is provided in a substantially linear, extended (e.g., denatured) form rather than a compacted, coiled and/or folded (e.g., secondary) form. Stretching the miRNA prior to analysis may be accomplished using particular configurations of, for example, a single molecule detection system, in order to maintain the linear form. These configurations are not required if the target can be analyzed in a compacted form.
  • the sample or reaction mixture may be cleaned prior to analysis, although the method provided herein does not require such a step.
  • cleaning refers to the process of removing unbound probes. This cleaning step can be accomplished in a number of ways including but not limited to column purification. Column purification generally involves capture of small molecules within a column with flow-through of larger molecules (such as the target miRNA and duplexes containing them). It is to be understood however that the method can be performed without removal of these reagents prior to analysis, particularly since coincident detection can distinguish between desired binding events and artifacts. Thus, in some embodiments, unreacted substrates including unbound detectable probes are not removed prior to analysis.
  • a quencher-conjugated probe is a probe that binds specifically to the detectable labeled probe used to analyze the target nucleic acid and comprises a quencher molecule.
  • Quencher molecules are molecules that absorb and thereby quench fluorescence from a sufficiently proximal fluorophore (approx. 10-100 A 0 ).
  • the quencher- fluorophore interaction is essentially a FRET phenomenon with the fluorophore being the donor and the quencher being the acceptor molecule.
  • quencher-conjugated probes can be designed such that the quencher will be proximal to the fluorophore on the complementary probe.
  • the sequence-specific probe has a fluorophore at its 3' end
  • the corresponding complementary quencher-conjugated probe may have the quencher located at its 5' end, and vice versa.
  • Quencher molecules do not re-emit fluorescence after interacting with a fluorophore.
  • interaction of unbound fluorescent probes with quencher-conjugated probes is effectively the same as physically removing the unbound probes from the reaction mixture, without the potential for any loss of sample or target nucleic acid.
  • Quenchers are usually multiple ring structures that dissipate the absorbed fluorescent energy via heat. Examples include Black Hole Quenchers (e.g., BHQ-I, BHQ-2, BHQ-3) from Molecular Probes and BioSearch Technologies (Novato, CA), and Iowa Black Quencher from IDT. A variety of quenchers are available such that fluorescence between 480-730 nm can be effectively quenched. The absorption spectra of quenchers can be quite broad and therefore a given quencher may be used to quench multiple fmorophore emissions.
  • BHQ-I has a maximum absorption wavelength of 534 nm but it can actually absorb emissions from 6-FAM (518 nm), TET (538 nm), HEX (553)/JOE (554) and Cy3 (565 nm), as well as others.
  • BHQ-2 has a maximum absorption wavelength of 579 nm but it can actually absorb emissions from TET, HEX/JOE, Cy3, TAMRA (583 nm) and ROX (607 nm), as well as others.
  • BHQ-3 has a maximum absorption wavelength of 672 nm but it can actually absorb emissions from LC Red (640 nm) and Cy5 (667 nm), as well as others.
  • quenchers generally conjugate the quencher to a nucleic acid probe of interest.
  • kits for performing such conjugation are also commercially available.
  • the quencher-conjugated probes are generally nucleic acid (e.g., DNA) in nature and are thus complementary to the miRNA-specif ⁇ c probes used. They must be sufficiently complementary to the sequence-specific probes used in order to bind to such probes specifically. Probes that bind specifically to the target of interest are probes that demonstrate preferential binding to the target than to any other compound. Specific probes have a higher binding affinity for their targets than for another compound. A higher binding affinity may be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 100-fold, or greater.
  • the quencher-conjugated probes are added to a reaction mixture at the same time as or following the sequence-specific probes.
  • the reaction conditions are manipulated in order to ensure that the sequence-specific probes preferably bind to the target nucleic acid (in a sequence-specific manner) and that once all probe target sites are saturated, the unbound probes will bind to the quencher-conjugated probes.
  • the invention contemplates modulation of factors such as temperature, buffer conditions (including pH and salt) and hybridization times in order to accomplish this result.
  • Coincident binding refers to the binding of two or more probes on a single molecule or complex. Coincident binding of two or more probes is used as an indicator of the molecule or complex of interest. It is also useful in discriminating against noise in the system and therefore increases the sensitivity and specificity of the system.
  • Coincident binding may take many forms including but not limited to a color coincident event, whereby two colors corresponding to a first and a second detectable label are detected.
  • Coincident binding may also be manifest as the proximal binding of a first detectable label that is a FRET donor fluorophore and a second detectable label that is a FRET acceptor fluorophore. In this latter embodiment, a positive signal is a signal from the FRET acceptor fluorophore upon laser excitation of the FRET donor fluorophore.
  • coincident detection refers to the detection of an emission signal from more than one detectable label in a given period of time.
  • the period of time is short, approximating the period of time necessary to analyze a single molecule. This time period may be on the order of a millisecond.
  • Coincident detection may be manifest as emission signals that overlap partially or completely as a function of time. The co-existence of the emission signals in a given time frame may indicate that two non-interacting molecules, each individually and distinguishably labeled, are present in the interrogation spot at the same time.
  • the coincident detection methods of the invention involve the simultaneous detection of more than one emission signal.
  • the number of emission signals that are coincident will depend on the number of distinguishable detectable labels available, the number of probes available, the number of components being detected, the nature of the detection system being used, etc. Generally, at least two emission signals are being detected. In some embodiments, three emission signals are being detected. However, the invention is not so limited. Thus, where multiple components are being detected in a single analysis, 4, 5, 6, 7, 8, 9, 10 or more emission signals can be detected simultaneously.
  • Coincident detection analysis is able to detect single molecules at very low concentrations. For example, as discussed herein, low femtomolar concentrations can be detected using a two or three emission signal approach. In addition, the analysis demonstrates a dynamic range of greater than four orders of magnitude.
  • a two emission signal approach is also able to detect single molecules such as single proteins at levels below 1 ng/ml.
  • Single miRNAs are detected using one or more probes that are specific to the miRNA (i.e., miRNA-specific probes, as discussed herein).
  • a sample may be tested for the presence of miRNA by contacting it with one or more miRNA-specific probes for a time and under conditions that allow for binding of the probe to the miRNA if it is present. Excess probe amounts may be used to ensure that all binding sites are occupied.
  • probes are preferably chosen so that they bind to different regions of the miRNA, and therefore cannot compete with each other for binding to the miRNA.
  • probes are labeled with distinguishable detectable labels (i.e., the detectable label on the first probe is distinct from that on the second probe).
  • the sample is analyzed for coincident emission signals (i.e., a distinct and detectable signal from each detectable label). For example, a miRNA bound by two probes is manifest as overlapping emission signals from the bound probes.
  • This detection is accomplished using a single molecule detection or analysis system.
  • a single molecule detection or analysis system is a system capable of detecting and analyzing individual, preferably intact, molecules.
  • the method is particularly suited to detecting miRNA in a rare or small sample (e.g., a nanoliter volume sample) or in a sample where miRNA concentration is low.
  • the invention allows more than one and preferably several different miRNA to be detected simultaneously, thereby conserving sample.
  • the method is capable of a high degree of multiplexing.
  • the degree of multiplexing may be 2 (i.e., 2 miRNA can be detected in a single analysis), 3, 4, 5, 6, 7, 8, 9, 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, or higher.
  • each miRNA is detected using a particular probe pair where preferably each member of the probe pair is specific to the miRNA (or at a minimum, one member of the pair is specific to the miRNA) and each probe used in an analysis is labeled with a distinguishable label.
  • a plurality of miRNA may be detected and analyzed. As used herein, a plurality is an amount greater than two but less than infinity. A plurality is sometimes less than a million, less than a thousand, less than a hundred, or less than ten.
  • a probe is a molecule that specifically binds to a target of interest.
  • the nature of the probe will depend upon the application and may also depend upon the nature of the target.
  • Specific binding means the probe demonstrates greater affinity for its target than for other molecules (e.g., based on the sequence or structure of the target).
  • the probe may bind to other molecules, but preferably such binding is at or near background levels. For example, it may have at least 2-fold, 5-fold, 10-fold or higher affinity for the desired target than for another molecule. Probes with the greatest differential affinity are preferred in most embodiments, although they may not be those with the greatest affinity for the target.
  • Probes can be virtually any compound that binds to a target with sufficient specificity. Examples include nucleic acids that bind to complementary nucleic acid targets via Watson- Crick and/or Hoogsteen binding (as discussed herein), aptamers that bind to nucleic acid targets due to structure rather than complementarity of sequence of the target, antibodies, etc. It is to be understood that although many of the exemplifications provided herein relate to nucleic acid probes, the invention is not so limited and other probes are envisioned. "Sequence-specific" when used in the context of a probe means that the probe recognizes a particular linear arrangement of nucleotides or derivatives thereof.
  • the sequence-specific probe is itself composed of nucleic acid elements such as DNA, RNA, PNA and LNA elements or combinations thereof (as discussed herein).
  • the linear arrangement includes contiguous nucleotides or derivatives thereof that each binds to a corresponding complementary nucleotide in the probe. In some embodiments, however, the sequence may not be contiguous as there may be one, two, or more nucleotides that do not have corresponding complementary residues on the probe, and vice versa.
  • any molecule that is capable of recognizing a nucleic acid with structural or sequence specificity can be used as a sequence-specific probe.
  • probes will be nucleic acids themselves and will form at least a Watson-Crick bond with the target.
  • the nucleic acid probe can form a Hoogsteen bond with the nucleic acid target, thereby forming a triplex.
  • a nucleic acid probe that binds by Hoogsteen binding enters the major groove of a nucleic acid target and hybridizes with the bases located there.
  • the nucleic acid probes can form both Watson-Crick and Hoogsteen bonds with the target.
  • BisPNA probes for instance, are capable of both Watson-Crick and Hoogsteen binding to a nucleic acid.
  • the length of the probe can also determine the specificity of binding.
  • the energetic cost of a single mismatch between the probe and its target is relatively higher for shorter sequences than for longer ones. Therefore, hybridization of smaller nucleic acid probes is more specific than is hybridization of longer nucleic acid probes to the same target because the longer probes can embrace mismatches and still continue to bind to the target.
  • One potential limitation to the use of shorter probes however is their inherently lower stability at a given temperature and salt concentration.
  • One way of avoiding this latter limitation involves the use of bisPNA probes which bind shorter sequences with sufficient hybrid stability.
  • the nucleic acid probes of the invention can be any length ranging from at least 4 nucleotides to in excess of 1000 nucleotides.
  • the probes are 5-100 nucleotides in length, more preferably between 5-25 nucleotides in length, and even more preferably 5-12 nucleotides in length.
  • the length of the probe can be any length of nucleotides between and including the ranges listed herein, as if each and every length was explicitly recited herein.
  • the length may be at least 5 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 20 nucleotides, or at least 25 nucleotides, or more, in length.
  • miRNA are detected using two or more probes. If two probes are used, each probe may be labeled at one of its ends such that when hybridized to the miRNA target, one probe is labeled at its 5' end while the other is labeled at its 3' end.
  • the combined length of the probes may be longer than the total length of the miRNA. For example, if the miRNA target is 22 bases long, then each of the probes may be 12, 13 or more bases in length. Hybridization of such probes is intended to yield a duplex with a one, two or more base overhang at both ends.
  • the bases to which the detectable labels are conjugated preferably are not themselves hybridized to complementary bases in the miRNA target. The use of longer probe pairs as described above has several advantages.
  • the overhangs may comprise an adenosine, thymine, guanine or cytosine, although modified bases such as LNA, iso-C or iso-G may also be used.
  • the probe may be 50 residues in length, yet only 45 of those residues hybridize to the nucleic acid target.
  • the residues that hybridize are contiguous with each other.
  • the length of the probe may also be represented as a proportion of the length of the miRNA to which it binds specifically.
  • the probe length may be at least 10%, at least 20%, at least 30%, at least 40 %, or at least 50% the length of its target miRNA, or longer.
  • the probes are preferably single-stranded, but they are not so limited.
  • the probe when it is a bisPNA it can adopt a secondary structure with the nucleic acid target (e.g., the miRNA) resulting in a triple helix conformation, with one region of the bisPNA clamp forming Hoogsteen bonds with the backbone of the miRNA and another region of the bisPNA clamp forming Watson-Crick bonds with the nucleotide bases of the miRNA.
  • the nucleic acid probe hybridizes to a complementary sequence within the miRNA.
  • the specificity of binding can be manipulated based on the hybridization conditions. For example, salt concentration and temperature can be modulated in order to vary the range of sequences recognized by the nucleic acid probes. Those of ordinary skill in the art will be able to determine optimum conditions for a desired specificity.
  • nucleic acid refers to multiple linked nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to an exchangeable organic base, which is either a pyrimidine (e.g., cytosine (C), thymidine (T) or uracil (U)) or a purine (e.g., adenine (A) or guanine (G)).
  • a pyrimidine e.g., cytosine (C), thymidine (T) or uracil (U)
  • purine e.g., adenine (A) or guanine (G)
  • Nucleic acid and “nucleic acid molecule” are used interchangeably and refer to oligoribonucleotides as well as oligodeoxyribonucleotides.
  • the terms shall also include polynucleosides (i.e., a polynucleotide minus a phosphate) and any other organic base containing nucleic acid.
  • the organic bases include adenine, uracil, guanine, thymine, cytosine and inosine.
  • the nucleic acids may be single- or double-stranded. Nucleic acids can be obtained from natural sources, or can be synthesized using a nucleic acid synthesizer. As used herein with respect to linked units of a nucleic acid, "linked” or “linkage” means two entities bound to one another by any physicocheniical means.
  • Natural linkages which are those ordinarily found in nature connecting for example the individual units of a particular nucleic acid, are most common. Natural linkages include, for instance, amide, ester and thioester linkages. The individual units of a nucleic acid may be linked, however, by synthetic or modified linkages. Nucleic acids where the units are linked by covalent bonds will be most common but those that include hydrogen bonded units are also embraced by the invention. It is to be understood that all possibilities regarding nucleic acids apply equally to nucleic acid tails, nucleic acid probes and capture nucleic acids.
  • the invention embraces nucleic acid derivatives in or as, for example, nucleic acid tails, nucleic acid probes and/or capture nucleic acids.
  • a "nucleic acid derivative” is a non-naturally occurring nucleic acid or a unit thereof. Nucleic acid derivatives may contain non-naturally occurring elements such as non-naturally occurring nucleotides and non-naturally occurring backbone linkages.
  • substituted purines and pyrimidines such as C-5 propyne modified bases, 5-methylcytosine, 2-aminopurine, 2 ⁇ amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, 2-thiouracil and pseudoisocytosine.
  • substituted purines and pyrimidines such as C-5 propyne modified bases, 5-methylcytosine, 2-aminopurine, 2 ⁇ amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, 2-thiouracil and pseudoisocytosine.
  • substituted purines and pyrimidines such as C-5 propyne modified bases, 5-methylcytosine, 2-aminopurine, 2 ⁇ amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, 2-thiouracil and pseudoisocytosine.
  • Other such modifications are well known to those of skill in the art.
  • the nucleic acid derivatives may also encompass substitutions or modifications, such as in the bases and/or sugars.
  • they include nucleic acids having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.
  • modified nucleic acids may include a 2'-O-alkylated ribose group.
  • modified nucleic acids may include sugars such as arabinose instead of ribose.
  • the nucleic acids may be heterogeneous in backbone composition thereby containing any possible combination of nucleic acid units linked together such as peptide nucleic acids (which have amino acid linkages with nucleic acid bases, and which are discussed in greater detail herein).
  • the nucleic acids are homogeneous in backbone composition. Nucleic acid probes and capture nucleic acids can be stabilized in part by the use of backbone modifications.
  • the invention intends to embrace, in addition to the peptide and locked nucleic acids discussed herein, the use of the other backbone modifications such as but not limited to phosphorothioate linkages, phosphodiester modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acid, methylphosphonate, alkylphosphonates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof.
  • backbone modifications such as but not limited to phosphorothioate linkages, phosphodiester modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acid, methylphosphonate, alkylphosphonates, phosphate esters, alkylphosphonothioates, phosphoramidates,
  • nucleic acid probes and/or capture nucleic acids may include a peptide nucleic acid (PNA), a bisPNA clamp, a pseudocomplementary PNA, a locked nucleic acid (LNA), DNA, RNA, or co-nucleic acids of the above such as DNA-LNA co-nucleic acids (as described in co-pending U.S. Patent Application having serial number 10/421,644 and publication number US 2003-0215864 Al and published November 20, 2003, and PCT application having serial number PCT/XJS03/12480 and publication number WO 03/091455 Al and published November 6, 2003, filed on April 23, 2003), or co-polymers thereof (e.g., a DNA-LNA co-polymer).
  • PNA peptide nucleic acid
  • bisPNA clamp a pseudocomplementary PNA
  • LNA locked nucleic acid
  • DNA RNA
  • co-nucleic acids of the above such as DNA-LNA co-nucleic acids (as
  • the nucleic acid probe is a LNA/DNA chimeric probe.
  • LNA content may vary from more than 0% to less than 100%, and may include at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.
  • 10- or 11- mer probes may contain on average about 3-4 LNAs, for example.
  • PNAs are DNA analogs having their phosphate backbone replaced with 2-aminoethyl glycine residues linked to nucleotide bases through glycine amino nitrogen and methylenecarbonyl linkers. PNAs can bind to both DNA and RNA targets by Watson-Crick base pairing, and in so doing form stronger hybrids than would be possible with DNA- or RNA-based probes.
  • PNAs are synthesized from monomers connected by a peptide bond (Nielsen, P.E. et al. Peptide Nucleic Acids, Protocols and Applications, Norfolk: Horizon Scientific Press, p. 1-19 (1999)). They can be built with standard solid phase peptide synthesis technology.
  • PNA chemistry and synthesis allows for inclusion of amino acids and polypeptide sequences in the PNA design.
  • lysine residues can be used to introduce positive charges in the PNA backbone.
  • AU chemical approaches available for the modifications of amino acid side chains are directly applicable to PNAs.
  • PNA has a charge-neutral backbone, and this attribute leads to fast hybridization rates of PNA to DNA (Nielsen, P.E. et al. Peptide Nucleic Acids, Protocols and Applications, Norfolk: Horizon Scientific Press, p. 1-19 (1999)).
  • the hybridization rate can be further increased by introducing positive charges in the PNA structure, such as in the PNA backbone or by addition of amino acids with positively charged side chains (e.g., lysines).
  • PNA can form a stable hybrid with DNA molecule.
  • the stability of such a hybrid is essentially independent of the ionic strength of its environment (Orum, H. et al., BioTechniques 19(3):472-480 (1995)), most probably due to the uncharged nature of PNAs.
  • This provides PNAs with the versatility of being used in vivo or in vitro.
  • the rate of hybridization of PNAs that include positive charges is dependent on ionic strength, and thus is lower in the presence of salt.
  • PNA single strand PNA
  • pcPNA pseudocomplementary PNA
  • Single stranded PNA binds to single-stranded DNA (ssDNA) preferably in anti- parallel orientation (i.e., with the N-terminus of the ssPNA aligned with the 3' terminus of the ssDNA) and with a Watson-Crick pairing.
  • PNA also can bind to DNA with a Hoogsteen base pairing, and thereby forms triplexes with double stranded DNA (dsDNA) (Wittung, P. et al., Biochemistry 36:7973 (1997)).
  • Single strand PNA is the simplest of the PNA molecules. This PNA form interacts with nucleic acids to form a hybrid duplex via Watson-Crick base pairing.
  • the duplex has different spatial structure and higher stability than dsDNA (Nielsen, P.E. et al. Peptide Nucleic Acids, Protocols and Applications, Norfolk: Horizon Scientific Press, p. 1-19 (1999)).
  • PNA/DNA/PNA or PNA/DNA/DNA triplexes can also be formed (Wittung, P. et al., Biochemistry 36:1913> (1997)).
  • the formation of duplexes or triplexes additionally depends upon the sequence of the PNA.
  • Thymine-rich homopyrimidine ssPNA forms PNA/DNA/PNA triplexes with dsDNA targets where one PNA strand is involved in Watson-Crick antiparallel pairing and the other is involved in parallel Hoogsteen pairing.
  • Cytosine-rich homopyrimidine ssPNA preferably binds through Hoogsteen pairing to dsDNA forming a PNA/DNA/DNA triplex. If the ssPNA sequence is mixed, it invades the dsDNA target, displaces the DNA strand, and forms a Watson-Crick duplex. Polypurine ssPNA also forms triplex PNA/DNA/PNA with reversed Hoogsteen pairing.
  • BisPNA includes two strands connected with a flexible linker. One strand is designed to hybridize with DNA by a classic Watson-Crick pairing, and the second is designed to hybridize with a Hoogsteen pairing.
  • the target sequence can be short (e.g., 8 bp), but the bisPNA/DNA complex is still stable as it forms a hybrid with twice as many (e.g., a 16 bp) base pairings overall.
  • the bisPNA structure further increases specificity of their binding. As an example, binding to an 8 bp site with a probe having a single base mismatch results in a total of 14 bp rather than 16 bp.
  • Pseudocomplementary PNA (pcPNA) (Izvolsky, K.I. et al., Biochemistry 10908- 10913 (2000)) involves two single-stranded PNAs added to dsDNA.
  • One pcPNA strand is complementary to the target sequence, while the other is complementary to the displaced DNA strand.
  • the displaced DNA generally does not restore the dsDNA structure.
  • the PNA/PNA duplex is more stable than the DNA/PNA duplex and the PNA components are self-complementary because they are designed against complementary DNA sequences. Hence, the added PNAs would rather hybridize to each other.
  • modified bases are used for their synthesis including 2,6-diamiopurine (D) instead of adenine and 2-thiouracil ( S U) instead of thymine. While D and S U are still capable of hybridization with T and A respectively, their self-hybridization is sterically prohibited.
  • Locked nucleic acids are modified RNA nucleotides.
  • LNAs form hybrids with DNA which are at least as stable as PNA/DNA hybrids. Therefore, LNA can be used just as PNA molecules would be. LNA binding efficiency can be increased in some embodiments by adding positive charges to it.
  • Commercial nucleic acid synthesizers and standard phosphoramidite chemistry are used to make LNAs. Therefore, production of mixed LNA/DNA sequences is as simple as that of mixed PNA/peptide sequences. Naturally, most of biochemical approaches for nucleic acid conjugations are applicable to LNA/DNA constructs.
  • backbone modifications particularly those relating to PNAs, include peptide and amino acid variations and modifications.
  • the backbone constituents of PNAs may be peptide linkages, or alternatively, they may be non-peptide linkages. Examples include acetyl caps, amino spacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein as O- linkers), amino acids such as lysine (particularly useful if positive charges are desired in the PNA), and the like.
  • O- linkers amino spacers
  • amino acids such as lysine (particularly useful if positive charges are desired in the PNA)
  • Various PNA modifications are known and probes incorporating such modifications are commercially available from sources such as Boston Probes, Inc. Labeling of Sequence-Specific Probes
  • the probes, and in some instances the miRNA tails are detectably labeled (i.e., they comprise a detectable label).
  • a detectable label is a moiety, the presence of which can be ascertained directly or indirectly.
  • detection of the label involves the creation of a detectable signal such as for example an emission of energy.
  • the label may be of a chemical, lipid, peptide or nucleic acid nature although it is not so limited. The nature of label used will depend on a variety of factors, including the nature of the analysis being conducted, the type of the energy source and detector used.
  • the label should be sterically and chemically compatible with the constituents to which it is bound.
  • the label can be detected directly for example by its ability to emit and/or absorb electromagnetic radiation of a particular wavelength.
  • a label can be detected indirectly for example by its ability to bind, recruit and, in some cases, cleave another moiety which itself may emit or absorb light of a particular wavelength (e.g., an epitope tag such as the FLAG epitope, an enzyme tag such as horseradish peroxidase, etc.).
  • a particular wavelength e.g., an epitope tag such as the FLAG epitope, an enzyme tag such as horseradish peroxidase, etc.
  • nucleic acids de novo (e.g., using automated nucleic acid synthesizers) using fluorescently labeled nucleotides.
  • nucleotides are commercially available from suppliers such as Amersham Pharmacia Biotech, Molecular Probes, and New England Nuclear/Perkin Elmer.
  • the detectable label can be selected from the group consisting of directly detectable labels such as a fluorescent molecule (e.g., fluorescein, rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), fluorescein amine, eosin, dansyl, umbelliferone, 5- carboxyfluorescein (FAM) 5 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfiuorescein (JOE) 5 6 carboxyrhodamine (R6G), N 5 N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6- carboxy-X-rhodamine (ROX), 4-(4'-dimethylamino ⁇ henylazo) benzoic acid (DABCYL), 5- (2'-aminoethyl
  • RTM. Brilliant Red 3B- A lissamine rhodamine B sulfonyl chloride, rhodamine B 5 rhodamine 123, rhodamine X 5 sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101, tetramethyl rhodamine, riboflavin, rosolic acid, and terbium chelate derivatives), a chemiluminescent molecule, a bioluminescent molecule, a chromogenic molecule, a radioisotope (e.g., P 32 or H 3 , 14 C, 125 I and 131 I), an electron spin resonance molecule (such as for example nitroxyl radicals), an optical or electron density molecule, an electrical charge transducing or transferring molecule, an electromagnetic molecule such as a magnetic or paramagnetic bead or particle, a
  • the detectable label can also be selected from the group consisting of indirectly detectable labels such as an enzyme (e.g., alkaline phosphatase, horseradish peroxidase, ⁇ - galactosidase, glucoamylase, lysozyme, luciferases such as firefly luciferase and bacterial luciferase (U.S. Patent No.
  • an enzyme e.g., alkaline phosphatase, horseradish peroxidase, ⁇ - galactosidase, glucoamylase, lysozyme
  • luciferases such as firefly luciferase and bacterial luciferase
  • saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase
  • heterocyclic oxidases such as uricase and xanthine oxidase coupled to an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase
  • an enzyme substrate an affinity molecule, a ligand, a receptor, a biotin molecule, an avidin molecule, a streptavidin molecule, an antigen (e.g., epitope tags such as the FLAG or HA epitope), a hapten (e.g., biotin, pyridoxal, digoxigenin fluorescein and dinitrophenol), an antibody, an antibody fragment, a microbead, and the like.
  • Antibody fragments include Fab, F(ab)2, Fd and antibody fragments which include a CDR3 region
  • the first and second probes may be labeled with fluorophores that form a fluorescence resonance energy transfer (FRET) pair.
  • FRET fluorescence resonance energy transfer
  • one excitation wavelength is used to excite fluorescence of donor fluorophores.
  • a portion of the energy absorbed by the donors can be transferred to acceptor fluorophores if they are close enough spatially to the donor molecules (i.e., the distance between them must approximate or be less than the Forster radius or the energy transfer radius).
  • acceptor fluorophore absorbs the energy, it in turn fluoresces in its characteristic emission wavelength. Since energy transfer is possible only when the acceptor and donor are located in close proximity, acceptor fluorescence is unlikely if both probes are not bound to the same miRNA. Acceptor fluorescence therefore can be used to determine presence of miRNA.
  • a FRET fluorophore pair is two fluorophores that are capable of undergoing FRET to produce or eliminate a detectable signal when positioned in proximity to one another.
  • donors include Alexa 488, Alexa 546, BODIPY 493, Oyster 556, Fluor (FAM), Cy3 and TMR (Tamra).
  • acceptors include Cy5, Alexa 594, Alexa 647 and Oyster 656. Cy5 can work as a donor with Cy3, TMR or Alexa 546, as an example.
  • FRET should be possible with any fluorophore pair having fluorescence maxima spaced at 50-100 nm from each other.
  • the FRET embodiment can be coupled with another label on the target miRNA such as a backbone label, as discussed below.
  • the miRNA target may be additionally labeled with a backbone label. These labels generally label nucleic acids in a sequence non-specific manner.
  • the miRNA may be detected by the coincident signals from the backbone label and one or more of the bound probes.
  • backbone labels include intercalating dyes such as phenanthridines and acridines (e.g., ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide, and ACMA); minor grove binders such as indoles and imidazoles (e.g., Hoechst 33258, Hoechst 33342, Hoechst 34580 and DAPI); and miscellaneous nucleic acid stains such as acridine orange (also capable of intercalating), 7-AAD, actinomycin D, LDS751 , and hydroxystilbamidine. All of the aforementioned nucleic acid stains are commercially available from suppliers such as Molecular Probes, Inc.
  • nucleic acid stains include the following dyes from Molecular Probes: cyanine dyes such as SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-I, POPO-3, YOYO-I, Y0Y0-3, TOTO-I, TOTO-3, JOJO-I, LOLO-I, BOBO-I, BOBO-3, PO-PRO-I, PO-PRO-3, BO-PRO-I, BO-PRO-3, TO-PRO-I, TO-PRO-3, TO-PRO-5, JO- PRO-I, LO-PRO-I, YO-PRO-I, Y0-PR0-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO- 13, -16, -24, -21, -23, -12, -11, -20,
  • some embodiments of the invention embrace three color coincidence, hi these embodiments, single or multiple lasers may be used.
  • three different lasers may be used for excitation at the following wavelengths: 488 nm (blue), 532 nm (green), and 633 nm (red).
  • These lasers excite fluorescence of Alexa 488, TMR (tetramethylrhodamine), and TOTO-3 fluorophores, respectively. Fluorescence from all these fluorophores can be detected independently.
  • one sequence-specific probe may be labeled with Alexa 488 fluorophore
  • a second sequence-specific probe may be labeled with TMR
  • the miRNA backbone may be labeled with TOTO-3.
  • TOTO-3 is an intercalating dye that non-specifically stains nucleic acids in a length-proportional manner.
  • Another suitable set of fluorophores that can be used is the combination of POPO- 1 , TMR and Alexa 647 (or Cy5) which are excited by 442, 532 and 633 nm lasers respectively.
  • conjugation means two entities stably bound to one another by any physicochemical means. It is important that the nature of the attachment is such that it does not substantially impair the effectiveness of either entity. Keeping these parameters in mind, any covalent or non-covalent linkage known to those of ordinary skill in the art is contemplated unless explicitly stated otherwise herein.
  • Non-covalent conjugation includes hydrophobic interactions, ionic interactions, high affinity interactions such as biotin-avidin and biotin-streptavidin complexation and other affinity interactions. Such means and methods of attachment are known to those of ordinary skill in the art. Conjugation can be performed using standard techniques common to those of ordinary skill in the art.
  • functional groups which are reactive with various labels include, but are not limited to, (functional group: reactive group of light emissive compound) activated este ⁇ amines or anilines; acyl azide:amines or anilines; acyl halide:amines, anilines, alcohols or phenols; acyl nitrile:alcohols or phenols; aldehyde:amines or anilines; alkyl halide:amines, anilines, alcohols, phenols or thiols; alkyl sulfonate:thiols, alcohols or phenols; anhydride:alcohols, phenols, amines or anilines; aryl halide:thiols; aziridine:thiols or thioethers; carboxylic acid:amines, anilines, alcohols or alkyl halides; diazoalkane:carboxylic acids; epoxide
  • Linkers and/or spacers may be used in some instances.
  • Linkers can be any of a variety of molecules, preferably nonactive, such as nucleotides or multiple nucleotides, straight or even branched saturated or unsaturated carbon chains of C 1 -C 30 , phospholipids, amino acids, and in particular glycine, and the like, whether naturally occurring or synthetic.
  • Additional linkers include alkyl and alkenyl carbonates, carbamates, and carbamides. These are all related and may add polar functionality to the linkers such as the C1-C3 0 previously mentioned.
  • linker and spacer are used interchangeably.
  • spacers can be used, many of which are commercially available, for example, from sources such as Boston Probes, Inc. (now Applied Biosystems). Spacers are not limited to organic spacers, and rather can be inorganic also (e.g., -0-Si-O-, or O-P-O-). Additionally, they can be heterogeneous in nature (e.g., composed of organic and inorganic elements). Essentially, any molecule having the appropriate size restrictions and capable of being linked to the various components such as fluorophore and probe can be used as a linker.
  • Examples include the E linker (which also functions as a solubility enhancer), the X linker which is similar to the E linker, the O linker which is a glycol linker, and the P linker which includes a primary aromatic amino group (all supplied by Boston Probes, Inc., now Applied Biosystems).
  • Other suitable linkers are acetyl linkers, 4-aminobenzoic acid containing linkers, Fmoc linkers, 4-aminobenzoic acid linkers, 8-amino-3, 6-dioxactanoic acid linkers, succinimidyl nialeimidyl methyl cyclohexane carboxylate linkers, succinyl linkers, and the like.
  • linker is that described by Haralambidis et al. in U.S. Patent 5,525,465, issued on June 11, 1996.
  • the length of the spacer can vary depending upon the application and the nature of the components being conjugated.
  • the linker molecules may be homo-bifunctional or hetero-bifunctional cross-linkers, depending upon the nature of the molecules to be conjugated.
  • Homo-bifunctional cross-linkers have two identical reactive groups.
  • Hetero-bifunctional cross-linkers are defined as having two different reactive groups that allow for sequential conjugation reaction.
  • Various types of commercially available cross-linkers are reactive with one or more of the following groups: primary amines, secondary amines, sulphydryls, carboxyls, carbonyls and carbohydrates.
  • amine-specif ⁇ c cross-linkers are bis(sulfosuccmimidyl) suberate, bis[2-(succinimidooxycarbonyloxy)ethyl] sulfone, disuccinimidyl suberate, disuccinimidyl tartarate, dimethyl adipimate-2 HCl, dimethyl pimelimidate-2 HCl, dimethyl suberimidate-2 HCl, and ethylene glycolbis-fsuccinimidyl- [succinate]].
  • Cross-linkers reactive with sulfhydryl groups include bismaleimidohexane, l,4-di-[3'-(2'-pyridyldithio)-propionamido)] butane, l-[p-azidosalicylamido]-4- [iodoacetamido] butane, and N-[4-(p-azidosalicylamido) butyl]-3'-[2'-pyridyldithio] propionamide.
  • Cross-linkers preferentially reactive with carbohydrates include azidobenzoyl hydrazine.
  • Cross-linkers preferentially reactive with carboxyl groups include 4-[p-azidosalicylamido] butylamine.
  • Heterobifunctional cross-linkers that react with amines and sulfhydryls include
  • N-succinimidyl-S-p-pyridyldithio] propionate succinimidyl [4-iodoacetyl]aminobenzoate
  • succinimidyl 4-[N-maleimidomethyl] cyclohexane- 1- carboxylate m-maleimidobenzoyl-N-hydroxysuccinimide ester
  • sulfosuccinimidyl 6-[3-[2-pyridyldithio]propionamido]hexanoate and sulfosuccinimidyl 4-[N- maleimidomethyl] cyclohexane-l-carboxylate.
  • Heterobifunctional cross-linkers that react with carboxyl and amine groups include l-ethyl-3-[3-diniethylaminopropyl]- carbodiimide hydrochloride.
  • Heterobifunctional cross-linkers that react with carbohydrates and sulfhydryls include 4-[N-maleimidomethyl]-cyclohexane-l-carboxylhydrazide-2 HCl, 4-(4-N-maleimidophenyl)-butyric acid hydrazide-2 HCl, and 3-[2-pyridyldithio] propionyl hydrazide.
  • the cross-linkers are bis-[ ⁇ -4-azidosalicylamido)ethyl]disulfide and glutaraldehyde.
  • Amine or thiol groups may be added at any nucleotide of a synthetic nucleic acid so as to provide a point of attachment for a bifunctional cross-linker molecule.
  • the nucleic acid may be synthesized incorporating conjugation-competent reagents such as Uni-Link AminoModifier, 3'-DMT-C6-Amine-ON CPG, AminoModifier II, N-TFA-C6-AminoModifier, C6-ThiolModif ⁇ er, C6-Disulfide Phosphoramidite and C6-Disulfide CPG (C ⁇ ontech, Palo Alto, CA).
  • conjugation-competent reagents such as Uni-Link AminoModifier, 3'-DMT-C6-Amine-ON CPG, AminoModifier II, N-TFA-C6-AminoModifier, C6-ThiolModif ⁇ er, C6-Disulfide Phosphoramidite and
  • a linker or spacer comprising a bond that is cleavable under certain conditions.
  • the bond can be one that cleaves under normal physiological conditions or that can be caused to cleave specifically upon application of a stimulus such as light, whereby the conjugated entity is released leaving its conjugation partner intact.
  • cleavable bonds include readily hydrolyzable bonds, for example, ester bonds, amide bonds and Schiff s base-type bonds. Bonds which are cleavable by light are known in the art.
  • a "substrate” can be any substrate on which one or more capture nucleic acids can be immobilized.
  • substrates that can be used in the compositions and methods provided herein include, for example, include glass, silicon oxides, plastics or metals.
  • Plastic substrates include, for example, acrylonitrile butadiene styrene, polyamide (Nylon), polyamide, polybutadiene, Polybutylene terephthalate, Polycarbonates, poly(ether sulphone) (PES, PES/PEES), poly(ether ether ketone)s, polyethylene (or polyethene), polyethylene glycol, polyethylene oxide, polyethylene terephthalate (PET, PETE, PETP), polyimide, polypropylene, polytetrafluoroethylene (Teflon) perfluoroalkoxy polymer resin (PFA), polystyrene, styrene acrylonitrile, poly(trimethylene terephthalate) (PTT), polyurethane (PU), polyvinylchloride (PVC), polyvmyldifluorine (PVDF), polyvinyl pyrrolidone) (PVP), Kynar, Mylar, Rilsan, (e.g.
  • Substrates further include but are not limited to membranes, e.g., natural and modified celluloses such as nitrocellulose or nylon, Sepharose, Agarose, polystyrene, polypropylene, polyethylene, dextran, amylases, polyacrylamides, polyvinylidene difluoride, PEGylated or calcium alginate spheres, other agaroses, and magnetite, including magnetic beads.
  • membranes e.g., natural and modified celluloses such as nitrocellulose or nylon, Sepharose, Agarose, polystyrene, polypropylene, polyethylene, dextran, amylases, polyacrylamides, polyvinylidene difluoride, PEGylated or calcium alginate spheres, other agaroses, and magnetite, including magnetic beads.
  • Substrates also include coblock polymers, which have both hydrophilic and hydrophobic components.
  • Nucleic acid microarray technology which is also known by other names including DNA chip technology, gene chip technology, and solid-phase nucleic acid array technology, is well known to those of ordinary skill in the art. Many components and techniques utilized in nucleic acid microarray technology are presented in The Chipping Forecast, Nature Genetics, Vol.21, Jan 1999, the entire contents of which is incorporated by reference herein.
  • Nucleic acid microarray substrates may include but are not limited to glass, silica, aluminosilicates, borosilicates, metal oxides such as alumina and nickel oxide, various clays, nitrocellulose, or nylon.
  • Capture nucleic acids may range in length from 5 to 25 nucleotides, although other lengths may be used. Appropriate capture nucleic acid length may be determined by one of ordinary skill in the art by following art-known procedures.
  • the microarray substrate may be coated with a compound to enhance synthesis of the capture nucleic acid on the substrate.
  • a compound to enhance synthesis of the capture nucleic acid on the substrate include, but are not limited to, oligoethylene glycols.
  • coupling agents or groups on the substrate can be used to covalently link the first nucleotide or oligonucleotide to the substrate. These agents or groups may include, for example, amino, hydroxy, bromo, and carboxy groups.
  • These reactive groups are preferably attached to the substrate through for example an alkylene or phenylene divalent radical, one valence position occupied by the chain bonding and the remaining attached to the reactive groups.
  • These groups may contain up to about ten carbon atoms, preferably up to about six carbon atoms.
  • Alkylene radicals are usually preferred containing two to four carbon atoms in the principal chain. These and additional details of the process are disclosed, for example, in U.S. Patent 4,458,066, which is incorporated by reference in its entirety.
  • capture nucleic acids are synthesized directly on the substrate in a predetermined grid pattern using methods such as light-directed chemical synthesis, photochemical deprotection, or delivery of nucleotide precursors to the substrate and subsequent capture probe synthesis.
  • the substrate may be coated with a compound to enhance binding of the capture probe to the substrate.
  • a compound to enhance binding of the capture probe to the substrate include, but are not limited to, polylysine, amino silanes, amino-reactive silanes or chromium.
  • presynthesized capture probes are applied to the substrate in a precise, predetermined volume and grid pattern, utilizing a computer-controlled robot to apply probe to the substrate in a contact-printing manner or in a non-contact manner such as ink jet or piezo-electric delivery.
  • Probes may be covalently linked to the substrate with methods that include, but are not limited to, UV irradiation.
  • probes are linked to the substrate with heat.
  • one or more control capture probes are attached to the substrate.
  • control probes allow determination of factors such as miRNA quality and binding characteristics, reagent quality and effectiveness, hybridization success, and analysis thresholds and success.
  • the nucleic acids may be analyzed using a single molecule analysis system.
  • a single molecule analysis system is capable of analyzing single, preferably intact, molecules separately from other molecules. Such a system is sufficiently sensitive to detect signals emitting from a single molecule and its bound probes.
  • the system may be a linear molecule analysis system in which single molecules are analyzed in a linear manner (i.e., starting at a point along the polymer length and then moving progressively in one direction or another). Many of the methods provided herein do not require linear analysis of miRNA.
  • the system is preferably not an electrophoretic method and thus is sometimes referred to as a non-electrophoretic single molecule detection (or analysis) system.
  • a non-electrophoretic single molecule detection (or analysis) system Such systems do not rely on gel electrophoresis or capillary electrophoresis to separate molecules from each other.
  • TrilogyTM An example of a single molecule detection/analysis system is the TrilogyTM instrument which is based on the Gene EngineTM technology described in PCT patent applications WO98/35012 and WO00/09757, published on August 13, 1998, and February 24, 2000, respectively, and in issued U.S. Patent 6,355,420 Bl, issued March 12, 2002.
  • the Gene EngineTM system allows single polymers to be passed through an interaction station, whereby the units of the polymer or labels of the compound are interrogated individually in order to determine whether there is a detectable label conjugated to the target. Interrogation involves exposing the label to an energy source such as optical radiation of a set wavelength. In response to the energy source exposure, the detectable label emits a detectable signal. The mechanism for signal emission and detection will depend on the type of label sought to be detected.
  • the TrilogyTM system is a single molecule confocal fluorescence detection platform.
  • the platform enables four-color fluorescent detection in a microfluidic flow stream with engineering modifications to automate sample handling and delivery.
  • photons emitted by the fluorescently tagged molecules pass through the dichroic mirror and are band-pass filtered to remove stray laser light and any Rayleigh or Raman scattered light.
  • the emission is focused and filtered through 100 micrometer pinholes of multi-mode fiber optic cables coupled to single photon counting modules.
  • a high-speed data acquisition card is used to store photon counts from each channel using a 10 kHz sampling rate. It should be noted that this system has single fluorophore detection sensitivity of four spectrally distinct fluorophores.
  • the TrilogyTM provides real-time counting of individually labeled molecules in a nanoliter interrogation zone.
  • the system detects labeled molecules at low femtomolar concentrations and displays a dynamic range over 4+ logs.
  • the system can accommodate standard sample carriers such as but not limited to 96 well plates or microcentrifuge (e.g., Eppendorf) tubes.
  • the sample volumes may be on the order of microliters (e.g., 1 ul volume).
  • the systems described herein will encompass at least one detection system.
  • the nature of such detection systems will depend upon the nature of the detectable label.
  • the detection system can be selected from any number of detection systems known in the art. These include an electron spin resonance (ESR) detection system, a charge coupled device (CCD) detection system, a fluorescent detection system, an electrical detection system, a photographic film detection system, a chemiluminescent detection system, an enzyme detection system, an atomic force microscopy (AFM) detection system, a scanning tunneling microscopy (STM) detection system, an optical detection system, a nuclear magnetic resonance (NMR) detection system, a near field detection system, and a total internal reflection (TIR) detection system, many of which are electromagnetic detection systems.
  • ESR electron spin resonance
  • CCD charge coupled device
  • fluorescent detection system an electrical detection system
  • photographic film detection system a chemiluminescent detection system
  • an enzyme detection system an atomic force microscopy (AFM) detection system
  • STM scanning tunneling microscopy
  • RNA oligonucleotide identical in sequence to the lin-4 miRNA was titrated in increasing concentrations into 2 micrograms of E. coli total RNA.
  • a radiolabeled DNA oligonucleotide complementary in sequence to lin-4 but containing 10 extra nucleotide bases at its 5' end was hybridized in solution to the lin-4 spiked NR solutions. When hybridized, this DNA oligomer will generate a 10 base 5' overhang on the DNA/RNA duplex.
  • the left gel shows the resulting autoradiograph. Specific hybridization of lin-4 to the radiolabeled DNA oligonucleotide probe was observed.
  • Lane 7 is a positive control in which a small amount of radiolabeled DNA oligomer was hybridized to several fold molar excess of lin-4 to ensure complete hybridization of radiolabeled oligomer to target miRNA.
  • Sybr Gold staining of the same gel shows the degree of background RNA present in the hybridization reactions. The process was repeated using a lin-4 point mutant as the target miRNA. There was no measurable hybridization of the radiolabeled oligonucleotide to the point mutant miRNA. Similarly, there was no non-specific hybridization to total RNA.
  • radiolabeled bands on the gel are the results of radiolabeled branch products that were generated during the synthesis of the DNA oligonucleotide, as is apparent when a high concentration of the radiolabeled DNA oligonucleotide is loaded alone. (FIG. 3, lane 8.)
  • the long overhang generated when the DNA oligonucleotide hybridizes to the miRNA is used as a template for the primer extension reaction.
  • This reaction uses the miRNA as a primer.
  • a nucleic acid tail of known sequence can be added to each miRNA. It is will be clear that the system can be designed such that every miRNA has its own specific tail.
  • RNA primer The ability of a DNA polymerase to extend off an RNA primer is a vital biological process.
  • the replication of lagging strand requires DNA pol I extension off of short RNA primers.
  • the invention takes advantage of this fundamental process to add capture tails to miRNAs.
  • Several commercially available polymerases are able to extend off the miRNA primers, however they vary in their extension efficiencies.
  • the experiments reported herein used a commercially available thermophilic exopolymerase (i.e., Therminator, New England BioLabs).
  • the miRNA targets are not being amplified. Therefore, it is possible to drive the extension reactions to completion with only a limited number of templates. To ensure that miRNA were being specifically extended, extension reactions were conducted using fluorescently labeled nucleotides.
  • Extension reactions used Therminator (New England Biolabs) with sub-optimal concentrations of nucleotides (200 nM). The reactions were cycled (90 0 C denaturation, 5O 0 C hybridization, 7O 0 C extension) twenty times.
  • the gel in FIG. 4 shows the results of extension reactions conducted on both wild-type lin-4 and the point mutant lin-4.
  • a fluorescently labeled product indicates nucleic acid tail synthesis or polymerization at the 3' end of the miRNA.
  • Lane 1 represents the reaction without added enzyme.
  • Lane 2 represents the reaction with added enzyme and wild-type lin-4.
  • Lane 3 represents the reaction with added enzyme and point mutant lin-4. Only lane 2 contains the extended lin-4 product.
  • the reaction can be adapted to generate a non-fluorescently labeled unique nucleic acid tail for each miRNA.
  • the process also involves hybridizing two distinctly labeled probes to the miRNA. This may be done either before or after the tailed miRNA is captured onto a solid support or surface.
  • the probes may be distinct fluorescently labeled probes, 10 nucleotides in length and composed of LNA/DNA elements (i.e., LNA/DNA chimeric probes).
  • the LNA/DNA chimeric probes offer some advantage over standard DNA oligonucleotide probes. For example, they can off-compete hybridized DNA or RNA probes and they form thermally stable duplexes. This thermal stability ensures complete hybridization will be retained at room temperature and enables hybridization reactions to be carried out at higher temperatures thereby improving hybridization specificity.
  • the process further involves capture of the tailed miRNA to a solid support or surface.
  • the unique sequence of the nucleic acid tails on the miRNA are hybridized to complementary capture nucleic acids positioned on pre-determined 2-dimensional locations on a surface or support such as a silica chip.
  • LNA capture probes are immobilized on for example silica chips.
  • Linkers or spacers can be used to position the capture probes away from the solid surfaces in order to minimize steric hindrance the might interfere with hybridization of the capture probe to the tailed miRNA.
  • An example of a linker or spacer is a 3 '-ethylene glycol scaffold.
  • the capture hybridization is carried out under conditions that do not cause denaturation of the probes from the miRNA, Moreover, shorter capture probes are possible by incorporating LNA elements into the probes.
  • the final step in the process involves measuring signal from the captured miRNAs.
  • a single molecule detection platform can be used to scan the surface of the solid support (e.g., the silica chip surface) and thereby quantitate the amount of signal from each pre-determined region. It is possible that up to 5,000 different miRNA may be analyzed per day using automated detection systems.
  • the TrilogyTM analysis system may be used and/or adapted for this purpose.
  • the instrument's confocal microscopy arrangement may be replaced with a linear array, single electron multiplying CCD (EM- CCD).
  • EM-CCDs are an emerging technology with on-cbip signal amplification that essentially eliminates the largest source of noise in conventional CCDs (i.e., the read-out noise).

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Abstract

La présente invention a trait à des procédés et des systèmes pour la détection et la mesure de microARN.
PCT/US2006/024448 2005-06-23 2006-06-23 Procédés et compositions pour l'analyse de microarn WO2007002375A2 (fr)

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EP2280078A1 (fr) * 2008-03-27 2011-02-02 Kuroda, Masahiko Marqueur pour la détermination du cancer du sein, méthode d'essai, et kit d'essai
CN101633925B (zh) * 2009-08-25 2011-08-31 南京医科大学 一种与精子生成障碍相关的精浆微小核糖核酸标志物及其应用
WO2011157617A1 (fr) * 2010-06-17 2011-12-22 Febit Holding Gmbh Ensemble complexe de banques de miarn
CN102296112A (zh) * 2011-08-09 2011-12-28 南京医科大学 人类非梗阻性无精子症相关的精浆微小核糖核酸标志物及其应用
US8202848B2 (en) 2008-03-17 2012-06-19 Board Of Regents, The University Of Texas System Identification of micro-RNAS involved in neuromuscular synapse maintenance and regeneration
US8592384B2 (en) 2005-04-04 2013-11-26 The Board Of Regents Of The University Of Texas System Micro-RNA's that regulate muscle cells
EP3049539A4 (fr) * 2013-09-25 2017-08-23 Bio-Id Diagnostic Inc. Procédés de détection de fragments d'acide nucléique

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9023823B2 (en) 2005-04-04 2015-05-05 The Board Of Regents Of The University Of Texas System Micro-RNA's that regulate muscle cells
US8592384B2 (en) 2005-04-04 2013-11-26 The Board Of Regents Of The University Of Texas System Micro-RNA's that regulate muscle cells
WO2008040355A3 (fr) * 2006-10-06 2008-05-29 Exiqon As Nouveaux procédés de quantification de micro-arn et de petits arn interférants
WO2008040355A2 (fr) * 2006-10-06 2008-04-10 Exiqon A/S Nouveaux procédés de quantification de micro-arn et de petits arn interférants
US8202848B2 (en) 2008-03-17 2012-06-19 Board Of Regents, The University Of Texas System Identification of micro-RNAS involved in neuromuscular synapse maintenance and regeneration
US8728724B2 (en) 2008-03-17 2014-05-20 Board Of Regents, The University Of Texas System Identification of micro-RNAs involved in neuromuscular synapse maintenance and regeneration
EP2280078A4 (fr) * 2008-03-27 2011-08-17 Kuroda Masahiko Marqueur pour la détermination du cancer du sein, méthode d'essai, et kit d'essai
EP2280078A1 (fr) * 2008-03-27 2011-02-02 Kuroda, Masahiko Marqueur pour la détermination du cancer du sein, méthode d'essai, et kit d'essai
JP5737935B2 (ja) * 2008-03-27 2015-06-17 雅彦 黒田 乳癌判定用のマーカーおよび検査方法
CN101633925B (zh) * 2009-08-25 2011-08-31 南京医科大学 一种与精子生成障碍相关的精浆微小核糖核酸标志物及其应用
WO2011157617A1 (fr) * 2010-06-17 2011-12-22 Febit Holding Gmbh Ensemble complexe de banques de miarn
CN102296112A (zh) * 2011-08-09 2011-12-28 南京医科大学 人类非梗阻性无精子症相关的精浆微小核糖核酸标志物及其应用
CN102296112B (zh) * 2011-08-09 2013-06-05 南京医科大学 人类非梗阻性无精子症相关的精浆微小核糖核酸标志物及其应用
EP3049539A4 (fr) * 2013-09-25 2017-08-23 Bio-Id Diagnostic Inc. Procédés de détection de fragments d'acide nucléique

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