Methods and materials using signaling probes

Abstract

Methods of isolating cells or generating cell lines comprising the step of exposing the cells to signaling probes that produce a signal upon hybridization to a target sequence, as well as methods of quantifying the level of expression of an RNA of interest, methods for identifying genetic recombinational events in living cells and methods of generating a transgenic animal using the isolated cells. Methods for isolating a plurality of cells encoding a plurality of different RNAs associated with a same nucleic acid tag sequence, comprising the step of exposing the cells to a same signaling probe that produces a detectable signal upon hybridization to the same nucleic acid tag sequence, are also provided. Signaling probes and protease probes that form stem-loop structures, three-arm junction structures, and dumbbell structures may be used in the above methods.

What is claimed is: 
     
         1 . A signaling probe comprising a first nucleic acid strand and a second nucleic acid strand, wherein said first and second strands each comprise mutually complementary regions capable of hybridizing, wherein the first strand has a quencher moiety at one terminus and the second strand has a fluorophore at a terminus that is adjacent to the quencher moiety when the strands are hybridized, wherein one strand hybridizes to a target sequence in or associated with an RNA of interest, wherein binding of the target sequence to said strand produces a detectable fluorescent signal. 
     
     
         2 . The probe of  claim 1 , wherein said quencher moiety is a thallium or gold nanoparticle. 
     
     
         3 . The probe of  claim 2 , wherein said quencher moiety is a gold nanoparticle. 
     
     
         4 . The probe of  claim 1 , wherein the probe comprises more than one quencher moiety and fluorophore pair. 
     
     
         5 . The probe of  claim 1 , wherein the probe comprises multiple fluorophores. 
     
     
         6 . A method for detecting expression of an RNA of interest in cells, comprising the steps of:
 a) providing cells potentially expressing the RNA of interest;   a) exposing the cells to the probe of  claim 1 ; and   b) detecting a fluorescent signal produced upon binding of the probe to the target sequence in or associated with the RNA of interest.   
     
     
         7 . The method of  claim 6 , wherein said detection is carried out using fluorescence activated cell sorter technology, fluorescence microscopy, or fluorometer technology. 
     
     
         8 . The method of  claim 6 , further comprising the step of isolating the cells exhibiting the detectable signal. 
     
     
         9 . The method of  claim 8 , wherein the isolating step is performed using fluorescence activated cell sorter technology. 
     
     
         10 . A method of generating a cell line, comprising the step of culturing the cells isolated in the method of  claim 8 . 
     
     
         11 . The method of  claim 6 , wherein the RNA of interest comprises one or more of: a messenger RNA, an antisense RNA, a siRNA, a tRNA, a structural RNA, a ribosomal RNA, an hnRNA and an snRNA. 
     
     
         12 . The method of  claim 6 , wherein the cells are living cells. 
     
     
         13 . A method for quantifying the expression level of an RNA of interest in cells, comprising the steps of:
 a) exposing the cells to the signaling probe of  claim 1 ;   b) quantifying the level of the signal produced upon binding of the probe to the target sequence in or associated with the RNA of interest in the cells; and   c) correlating the level of the signal with the expression level of the RNA of interest.   
     
     
         14 . The method of  claim 13 , wherein said quantification is carried out using fluorescence activated cell sorter technology or fluorescence microscopy. 
     
     
         15 . The method of  claim 13 , further comprising the step of isolating the cells exhibiting the detectable signal. 
     
     
         16 . The method of  claim 15 , wherein the isolating step is performed using fluorescence activated cell sorter technology. 
     
     
         17 . A method of generating a cell line, comprising the step of culturing the cells isolated in the method of  claim 15 . 
     
     
         18 . The method of  claim 13 , wherein the RNA of interest comprises one or more of: a messenger RNA, an antisense RNA, a siRNA, a tRNA, a structural RNA, a ribosomal RNA, an hnRNA and an snRNA. 
     
     
         19 . The method of  claim 13 , wherein the cells are living cells.