Imaging individual mrna molecules using multiple singly labeled probes

Abstract

A method for probing a target sequence of messenger ribonucleic acid molecules (mRNA's) in a fixed, permeabilized cell, said target sequence including at least 30 non-overlapping probe binding regions of 15-100 nucleotides, comprising immersing said cell in an excess of at least 30 nucleic acid hybridization probes, each singly labeled with the same fluorescent label and each containing a nucleic acid sequence that is complementary to a different probe binding region of said target sequence; washing said fixed cell to remove unbound probes; and detecting fluorescence from said probes.

1 - 41 . (canceled) 
     
     
         42 . A probe set for in situ hybridization that enables detection of individual RNA molecules in cells, comprising at least 12 non-overlapping nucleic acid hybridization probes singly labelled with a same first fluorophore label of a first color, wherein the probes have sequences complementary to a target sequence. 
     
     
         43 . A kit comprising a first probe set of  claim 42 . 
     
     
         44 . The kit of  claim 43 , wherein the first probe set comprises at least 30 non-overlapping nucleic acid hybridization probes singly labelled with the same first fluorophore. 
     
     
         45 . The kit of  claim 44 , wherein the first probe set comprises 40 to 60 non-overlapping nucleic acid hybridization probes singly labelled with the same first fluorophore. 
     
     
         46 . The kit of  claim 43 , wherein the hybridization probes have target-complementary sequences 7-40 nucleotides in length. 
     
     
         47 . The kit of  claim 43 , wherein the hybridization probes have target-complementary sequences 15-30 nucleotides in length. 
     
     
         48 . The kit of  claim 43 , wherein the hybridization probes have target-complementary sequences 17-25 nucleotides in length. 
     
     
         49 . The kit of  claim 43 , wherein the hybridization probes have target-complementary sequences 17-22 nucleotides in length. 
     
     
         50 . The kit of  claim 43 , wherein all hybridization probes in the set are labelled with a fluorophore at their 3′ end. 
     
     
         51 . The kit of  claim 43 , wherein the hybridization probes comprise deoxyribonucleotides, ribonucleotides, or mixtures thereof. 
     
     
         52 . The kit of  claim 43 , wherein at least some of the hybridization probes include non-natural nucleotides. 
     
     
         53 . The kit of  claim 52 , wherein the non-natural nucleotides are 2′-O-methyl ribonucleotides. 
     
     
         54 . The kit of  claim 43 , further comprising a hybridization solution, a computer readable medium, or both. 
     
     
         55 . The kit of  claim 43 , further comprising a second probe set for in situ hybridization that enables detection of individual RNA molecules in cells, comprising at least 12 non-overlapping nucleic acid hybridization probes singly labelled with a same second fluorophore label of a second color, wherein the probes of the second probe set have sequences complementary to the target sequence or a different target sequence. 
     
     
         56 . The kit of  claim 43 , wherein the individual RNA molecules are mRNA molecules. 
     
     
         57 . The kit of  claim 43 , wherein the first probe set comprises DNA, RNA or a mixture of DNA and RNA that are free of non-natural nucleotides that increase binding affinity of probes. 
     
     
         58 . The kit of  claim 55 , wherein the probes of the second probe set are 7-40, 15-30, 17-25, or 15-22 nucleotides in length. 
     
     
         59 . The kit of  claim 55 , wherein the probes of the second probe set comprise deoxyribonucleotides, ribonucleotides, or mixtures thereof. 
     
     
         60 . The kit of  claim 55 , wherein at least some of the hybridization probes of the second probe include non-natural nucleotides. 
     
     
         61 . The kit of  claim 60 , wherein the non-natural nucleotides are 2′-O-methyl ribonucleotides.