Nanoreporters and methods of manufacturing and use thereof

Abstract

The present invention relates to compositions and methods for detection and quantification of individual target molecules in biomolecular samples. In particular, the invention relates to coded, labeled probes that are capable of binding to and identifying target molecules based on the probes' label codes. Methods of making and using such probes are also provided. The probes can be used in diagnostic, prognostic, quality control and screening applications.

1 . A probe pair comprising a first probe and a second probe,
 said first probe being a complex comprising:
 (a) a first molecule, comprising: (i) a first label attachment region to which are attached one or more label monomers that emit light constituting a first signal; (ii) a second label attachment region, which is non-overlapping with the first label attachment region, to which are attached one or more label monomers that emit light constituting a second signal; and 
 (b) a first target-specific sequence attached to the first molecule, 
   said second probe being a second molecule, comprising (i) a second target-specific sequence; (ii) optionally, a third label attachment region to which are attached one or more label monomers that emit light constituting a third signal; and (iii) optionally, an affinity tag attached to said second molecule;   wherein the first target-specific sequence and the second target-specific sequence bind to different regions of the same target molecule,   wherein the target molecule is a naturally occurring molecule or a cDNA of a naturally occurring molecule or the complement of said cDNA,   and wherein when said probe pair is bound to its target molecule, the identity of the first and second signals and their locations relative to each other constitute at least part of a code that identifies the target molecule.   
     
     
         2 . A probe pair comprising a first probe and a second probe,
 said first probe being a complex comprising:
 (a) a first molecule, comprising: (i) a first label attachment region to which are attached one or more label monomers that emit light constituting a first signal; (ii) a second label attachment region to which are attached one or more label monomers that emit light constituting a second signal; and 
 (b) a first target-specific sequence attached to the first molecule, 
   said second probe being a second molecule, comprising (i) a second target-specific sequence; (ii) optionally, a third label attachment region to which are attached one or more label monomers that emit light constituting a third signal; and (iii) optionally, an affinity tag attached to said second molecule;   wherein the first target-specific sequence and the second target-specific sequence bind to different regions of the same target molecule,   wherein the first signal and the second signal are spatially or spectrally distinguishable;   wherein the target molecule is a naturally occurring molecule or a cDNA of a naturally occurring molecule or the complement of said cDNA,   and wherein when said probe pair is bound to its target molecule, the identity of the first and second signals and their locations relative to each other constitute at least part of a code that identifies the target molecule.   
     
     
         3 . The probe pair of  claim 1  or  2 , wherein said first and second label attachment regions are predetermined nucleotide sequences. 
     
     
         4 . The probe pair of  claim 3 , wherein a first DNA molecule is hybridized to the first label attachment region, to which first DNA molecule are bound said one or more label monomers that emit light constituting said first signal; and wherein a second DNA molecule is hybridized to the second label attachment region, to which second DNA molecule are bound said one or more label monomers that emit light constituting said second signal. 
     
     
         5 . The probe pair of  claim 3 , wherein a first RNA molecule is hybridized to the first label attachment region, to which first RNA molecule are bound said one or more label monomers that emit light constituting said first signal; and wherein a second RNA molecule is hybridized to the second label attachment region, to which second RNA molecule are bound said one or more label monomers that emit light constituting said second signal. 
     
     
         6 . The probe pair of  claim 3 , wherein a plurality of first DNA molecules are hybridized to the first label attachment region, to which DNA molecules are bound said one or more label monomers that emit light constituting said first signal; and wherein a plurality of second DNA molecules are hybridized to the second label attachment region, to which second DNA molecules are bound said one or more label monomers that emit light constituting said second signal. 
     
     
         7 . The probe pair of  claim 3 , wherein a plurality of first RNA molecules are hybridized to the first label attachment region, to which RNA molecules are bound said one or more label monomers that emit light constituting said first signal; and wherein a plurality of second RNA molecules are hybridized to the second label attachment region, to which second RNA molecules are bound said one or more label monomers that emit light constituting said second signal. 
     
     
         8 . The probe pair of any one of  claims 1 - 7 , wherein the first probe further comprises an affinity tag. 
     
     
         9 . The probe pair of  claim 1 - 7 , wherein the second probe comprises an affinity tag. 
     
     
         10 . The probe pair of  claim 1 - 7  wherein the second probe comprises a third label attachment region to which are attached one or more label monomers that emit light constituting a third signal. 
     
     
         11 . The probe pair of  claim 10 , wherein said third label attachment region is a predetermined nucleotide sequence, and wherein a third RNA molecule is hybridized to the third label attachment region, to which third RNA molecule are attached said one or more label monomers that emit light constituting said third signal. 
     
     
         12 . The probe pair of  claim 11 , wherein when the probe pair is bound to its target molecule, the code comprises the identity of the first signal, second signal and third signal and their locations relative to each other. 
     
     
         13 . The probe pair of  claim 1  or  2 , wherein the code comprises the identity of the first and second signals, their locations relative to each other, and the size of the spot resulting from at least one of said signals. 
     
     
         14 . The probe pair of  claim 9 , wherein the first and second molecules are nucleic acid molecules. 
     
     
         15 . The probe pair of  claim 14 , wherein the label attachment regions and target-specific sequences are predetermined nucleotide sequences. 
     
     
         16 . The probe pair of  claim 15  in which said one or more label monomers are covalently bound to nucleic acids hybridized to their respective attachment regions. 
     
     
         17 . The probe pair of  claim 16 , wherein said nucleic acids are hybridized to the respective attachment regions by way of one or more non-covalently bound bridging nucleic acids. 
     
     
         18 . The probe pair of  claim 1  or  2  in which the first and second target-specific sequences are unlabeled with any one or more of said label monomers. 
     
     
         19 . The probe pair of  claim 9 , wherein the second molecule further comprises a fourth label attachment region, which is non-overlapping with the third label attachment region, to which is attached one or more label monomers that emit light constituting a fourth signal. 
     
     
         20 . The probe pair of  claim 19 , wherein when the probe pair is bound to its target molecule, the code comprises the identity of the first signal, second signal, third signal and fourth signal and their locations relative to each other. 
     
     
         21 . The probe pair of  claim 8 , wherein the second probe further comprises an affinity tag. 
     
     
         22 . The probe pair of  claim 8 , wherein a first RNA molecule is hybridized to the first label attachment region, to which first RNA molecule are bound said one or more label monomers that emit light constituting said first signal; and wherein a second RNA molecule is hybridized to the second label attachment region, to which second RNA molecule are bound said one or more label monomers that emit light constituting said second signal and wherein the first probe and/or the second probe further comprises an affinity tag. 
     
     
         23 . The probe pair of  claim 8 , wherein the affinity tag is covalently attached to the first molecule or to the second molecule. 
     
     
         24 . The probe pair of  claim 1 , wherein the label monomers attached to the first label attachment region emit light at the same wavelength, which light constitutes said first signal, and wherein the label monomers attached to the second label attachment region emit light at the same wavelength, which light constitutes the second signal. 
     
     
         25 . The probe pair of  claim 1 , wherein at least one of the first signal and the second signal comprises light at a plurality of different wavelengths. 
     
     
         26 . The probe pair of  claim 1 , wherein first and second signals are spectrally distinguishable. 
     
     
         27 . The probe pair of  claim 5 , wherein the first and second RNA molecules are each 100 to 3,000 nucleotides in length. 
     
     
         28 . The probe pair of  claim 5 , wherein the first and second RNA molecules are each 500 to 1,500 nucleotides in length. 
     
     
         29 . The probe pair of  claim 5 , wherein the second probe is a nucleic acid complex comprising:
 (a) the second molecule, wherein the second molecule comprises a third label attachment region to which is hybridized a third RNA molecule, to which third RNA molecule are bound one or more label monomers that emit light constituting a third signal; and   (b) the second target-specific sequence attached to the second molecule,   wherein the code comprises the identity of the first, second and third signals and their locations relative to each other.   
     
     
         30 . The probe pair of  claim 29 , wherein the label monomers attached to the first label attachment regions emit light at the same wavelength, which light constitutes said first signal; wherein the label monomers attached to the second label attachment region emit light at the same wavelength, which light constitutes the second signal; and wherein the label monomers attached to the third label attachment region emit light at the same wavelength, which light constitutes the third signal. 
     
     
         31 . The probe pair of  claim 29 , wherein at least one of the first signal, the second signal and the third signal comprises light at a plurality of different wavelengths. 
     
     
         32 . The probe pair of  claim 29 , in which the first target-specific sequence and the second target-specific sequence are unlabeled with any one or more of said label monomers. 
     
     
         33 . The probe pair of  claim 29 , wherein first, second and third signals are spectrally distinguishable. 
     
     
         34 . The probe pair of  claim 29 , wherein first and third signals emit at the same wavelength or wavelengths. 
     
     
         35 . The probe pair of  claim 29 , wherein the first probe further comprises an affinity tag. 
     
     
         36 . The probe pair of  claim 29 , wherein the second probe further comprises an affinity tag. 
     
     
         37 . The probe pair of  claim 35 , wherein the second probe further comprises an affinity tag. 
     
     
         38 . The probe pair of  claim 35 , wherein the affinity tag is covalently attached to the first molecule. 
     
     
         39 . The probe pair of  claim 36 , wherein the affinity tag is covalently attached to the second molecule. 
     
     
         40 . The probe pair of  claim 29 , wherein the first, second and third RNA molecules are each 100 to 3,000 nucleotides. 
     
     
         41 . The probe pair of  claim 29 , wherein the first, second and third RNA molecules are each 500 to 1,500 nucleotides. 
     
     
         42 . A method of detecting a target molecule in a biomolecular sample comprising: (i) contacting said sample with a probe pair according to any one of  claims 1 - 41  under conditions that allow binding of the first target-specific sequence and the second target-specific sequence to the target molecule and (ii) detecting the code that identifies the target molecule. 
     
     
         43 . The method of  claim 42 , further comprising quantitating the amount of said target molecule in said biomolecular sample. 
     
     
         44 . The method of  claim 42  or  43 , wherein the concentration of each of said first and second target-specific sequences is in excess of the concentration of said target molecule. 
     
     
         45 . A method of detecting a plurality of target molecules in a biomolecular sample comprising: (i) contacting said sample with a population of probe pairs according to any one of  claims 1 - 41 , under conditions that allow binding of the first target-specific sequence and the second target-specific sequence of each probe pair to their respective target molecule, wherein each probe pair in said population when bound to its respective target molecule is associated with a distinguishable code, and (ii) detecting the codes that identify the plurality of target molecule6. 
     
     
         46 . The method of  claim 45 , further comprising quantitating the amount of each of said plurality of target molecules in said biomolecular sample. 
     
     
         47 . The method of  claim 45  or  46 , wherein the concentration of each of said first and second target-specific sequences of each probe pair of said population is in excess of the concentration of its respective target molecule.