Dual labeling methods for measuring cellular proliferation

Abstract

The present invention provides a method for measuring cellular nascent nucleic acid synthesis by dual pulse labeling of nucleic acid. The first pulse labeling of nucleic acid with a nucleoside analog allows establishment of a baseline nucleic acid synthesis rate. Pulse labeling of the nucleic acid with a second nucleoside analog then allows measurement of any changes to nucleic acid synthesis. The nucleic acid synthesis can be measured as cell proliferation, DNA, or gene expression, RNA. This method does not require a potentially artifact-inducing intermediary wash step between pulse labels. Additionally, this method may be used to screen compounds for their affect on cellular proliferation by treating cells or an organism with the test compound simultaneous to or before treatment with a competitive nucleoside analog.

1 . A method for measuring a change in cellular nucleic acid synthesis:
 a) incubating a sample with an effective amount of a first nucleoside or nucleotide analog to form a primary incubated sample;   b) incubating the primary incubated sample with at least one second nucleoside or nucleotide analog to form a secondary incubated sample;   c) incubating the secondary incubated sample with a first labeling reagent and at least one second labeling reagent to form a labeled sample;   d) detecting the labeled sample wherein a level of incorporation of the first and at least one second nucleoside or nucleotide analog is measured,
 wherein a difference in a level of incorporation of the at least one second nucleoside or nucleotide analog relative to the level of incorporation of the first nucleoside or nucleotide analog is measured as a change in cellular nucleic acid synthesis, 
 with the proviso that either the first nucleoside or nucleotide or the at least one second nucleoside or nucleotide contains a bioorthogonal functional moiety. 
   
     
     
         2 . A method for measuring a change in cellular DNA synthesis:
 a) incubating a sample with an effective amount of a first nucleoside or nucleotide analog to form a primary incubated sample;   b) incubating the primary incubated sample with at least one second nucleoside or nucleotide analog to form a secondary incubated sample;   c) incubating the secondary incubated sample with a first labeling reagent and at least one second labeling reagent to form a labeled sample;   d) detecting the labeled sample wherein a level of incorporation of the first and at least one second nucleoside or nucleotide analog is measured,
 wherein a difference in a level of incorporation of the at least one second nucleoside or nucleotide analog relative to the level of incorporation of the first nucleoside or nucleotide analog is measured as a change in cellular DNA synthesis, 
 with the proviso that either the first nucleoside or nucleotide or the at least one second nucleoside or nucleotide contains a bioorthogonal functional moiety. 
   
     
     
         3 . A method for measuring a change in cellular RNA synthesis:
 a) incubating a sample with an effective amount of a first nucleoside or nucleotide analog to form a primary incubated sample;   b) incubating the primary incubated sample with at least one second nucleoside or nucleotide analog to form a secondary incubated sample;   c) incubating the secondary incubated sample with a first labeling reagent and at least one second labeling reagent to form a labeled sample;   d) detecting the labeled sample wherein a level of incorporation of the first and at least one second nucleoside or nucleotide analog is measured,
 wherein a difference in a level of incorporation of the at least one second nucleoside or nucleotide analog relative to the level of incorporation of the first nucleoside or nucleotide analog is measured as a change in cellular RNA synthesis, 
 with the proviso that either the first nucleoside or nucleotide or the at least one second nucleoside or nucleotide contains a bioorthogonal functional moiety. 
   
     
     
         4 . The method according to  claim 1  wherein the sample is treated with a test compound simultaneous to or before treatment with the at least one second nucleoside or nucleotide analog. 
     
     
         5 . The method according to  claim 1 , wherein the first analog contains a bioorthogonal functional moiety. 
     
     
         6 . The method according to  claim 1 , wherein the at least one second analog contains a bioorthogonal functional moiety. 
     
     
         7 . The method according to  claim 1 , wherein the first analog or the at least one second analog contains a halogen moiety. 
     
     
         8 . The method according to  claim 1 , wherein the bioorthogonal functional moiety can undergo a [3+2] cycloaddition reaction. 
     
     
         9 . The method according to  claim 1 , wherein the bioorthogonal functional moiety can undergo a Staudinger ligation reaction. 
     
     
         10 . The method according to  claim 1 , wherein the bioorthogonal functional moiety contains an azido, alkyne or phosphine moiety. 
     
     
         11 . The method according to  claim 1 , wherein at least one, but not all, of the first and second analogs are ethynyl-deoxyuracil (EdU). 
     
     
         12 . The method according to  claim 1 , wherein least one, but not all, of the first and second analogs are 5-azido-2′-deoxyuracil (AzdU). 
     
     
         13 . The method according to  claim 7 , wherein the halogen moiety is bromo, chloro or iodo. 
     
     
         14 . The method according to  claim 1 , wherein at least one, but not all, of the first and second analogs are BrdU. 
     
     
         15 . The method according to  claim 1 , wherein the first labeling reagent and second labeling reagent is an antibody or a label that contains a bioorthogonal functional moiety. 
     
     
         16 . The method according to  claim 15 , wherein the bioorthogonal functional moiety can undergo a [3+2] cycloaddition reaction. 
     
     
         17 . The method according to  claim 15 , wherein the bioorthogonal functional moiety can undergo a Staudinger ligation reaction. 
     
     
         18 . The method according to  claim 15 , wherein the bioorthogonal functional moiety is an azido, alkyne or phosphine moiety. 
     
     
         19 . The method according to  claim 15 , wherein the label is a fluorescent dye. 
     
     
         20 . The method according to  claim 15 , wherein the antibody is an anti-BrdU antibody. 
     
     
         21 . The method according to  claim 15 , wherein the first labeling reagent or second labeling reagent is a dye-labeled azide. 
     
     
         22 . The method according to  claim 21 , wherein the dye-labeled azide is selected from the group consisting of:
 rhodamine-azide, Alexa Fluor® 350-azide, Alexa Fluor® 488-azide, Alexa Fluor® 555-azide, Alexa Fluor® 568-azide, Alexa Fluor® 568-azide, Alexa Fluor® 594-azide, Alexa Fluor® 633-azide, Alexa Fluor® 647-azide, Alexa Fluor® 680-azide, Pacific Blue™ azide, Cascade Blue® azide, fluorescein-azide, cyanine-azide, dapoxyl-azide and tetramethylrhodamine (TMR)-azide.   
     
     
         23 . The method according to  claim 1 , wherein incubating the first labeling reagent with the secondary incubated sample in a manner such that a covalent bond is formed between the first nucleoside analog and the labeling reagent. 
     
     
         24 . The method according to  claim 1 , wherein incubating the second labeling reagent with the secondary incubated sample in a manner such that a covalent bond is formed between the second nucleoside analog and the labeling reagent. 
     
     
         25 . The method according to  claim 1 , wherein incorporation of said first nucleoside analog and said at least one second nucleoside analog is detected by flow cytometry. 
     
     
         26 . The method according to  claim 1 , wherein incorporation of said first nucleoside analog and said at least one second nucleoside analog is detected by fluorescence microscopy. 
     
     
         27 . The method according to  claim 1 , wherein incorporation of said first nucleoside analog and said at least one second nucleoside analog is detected by multi-well plate assay. 
     
     
         28 . The method according to  claim 1 , wherein incorporation of said first nucleoside analog and said at least one second nucleoside analog is detected by imaging. 
     
     
         29 . The method according to  claim 1 , wherein incorporation of said first nucleoside analog and said at least one second nucleoside analog is detected by high content screening. 
     
     
         30 . The method according to  claim 1 , wherein the sample is an organism or cells in cell culture. 
     
     
         31 . A method for screening compounds for effects on cellular proliferation or gene expression comprising:
 a) incubating a sample with an effective amount of a first nucleoside or nucleotide analog to form a primary incubated sample;   b) treating the primary incubated sample with a test compound to form a treated sample;   c) incubating the treated sample with at least one second nucleoside or nucleotide analog simultaneous to or after treating the primary incubated sample with the test compound to form a secondary incubated sample;   d) incubating the secondary incubated sample with a first labeling reagent and at least one second labeling reagent to form a labeled sample;   e) detecting the labeled sample wherein a level of incorporation of the first and at least one second nucleoside or nucleotide analog is measured,
 wherein a difference in a level of incorporation of the at least one second nucleoside or nucleotide analog relative to the level of incorporation of the first nucleoside or nucleotide analog is measured as an effect of the screening compounds on cellular proliferation or gene expression, 
 with the proviso that either the first nucleoside or nucleotide or the at least one second nucleoside or nucleotide contains a bioorthogonal functional moiety. 
   
     
     
         32 . The method according to  claim 30 , wherein the sample is an organism or cells in cell culture. 
     
     
         33 . A kit for measuring a change in cellular nucleic acid synthesis, wherein the kit comprises:
 a) a first nucleoside or nucleotide analog;   b) at least one second nucleoside or nucleotide analog, wherein in at least the first analog or the at least one second nucleoside or nucleotide analog contains a bioorthogonal functional moiety;   c) a first labeling reagent; and   d) a second labeling reagent.