Methods and Compositions for Detection and Enrichment of Target Small RNAs

Abstract

Methods and compositions are provided for detecting small target RNAs where the target RNA may be single-stranded or double-stranded and may be contained in a mixture of RNAs of different types and sizes. The methods and compositions utilize a p19 fusion protein that is capable of binding double-stranded RNA in a size-specific but sequence-independent manner and is further capable of binding to a matrix such as beads or plastic microwell plates. By labeling the p19 fusion protein or the target RNA in a polynucleotide duplex either directly or indirectly, low levels of target RNA including microRNAs can be detected from cells. This can be applied to diagnosis of pathological conditions.

1 . A recombinant protein having at least 90% sequence homology to SEQ ID NO:33 and capable of binding a 21 nucleotide double-stranded RNA (dsRNA). 
     
     
         2 . The recombinant protein according to  claim 1 , further comprising: a detectable label selected from a fluorescent label, a radioactive label, a chemiluminescent label, a protein label and a small molecule label. 
     
     
         3 . A DNA encoding the protein of  claim 1 . 
     
     
         4 . A vector, comprising: the DNA of  claim 3  for expressing the recombinant protein in a host cell. 
     
     
         5 . A method; comprising:
 mixing a target RNA with a p19 fusion protein, wherein either the p19 fusion protein or the target RNA is labeled, the label being   (i) directly linked to the protein or target RNA, or   (ii) indirectly linked by means of a molecule capable of binding to the p19 fusion protein or target RNA; and   immobilizing on a matrix the p19 fusion protein bound to the target RNA for detecting the target RNA.   
     
     
         6 . A method according to  claim 5 , wherein the target RNA is a single-stranded RNA (ssRNA), the ssRNA hybridizing to a complementary polynucleotide probe to form a double-stranded hybrid polynucleotide for binding to the p19 fusion protein. 
     
     
         7 . A method according to  claim 6 , wherein the complementary polynucleotide probe is an RNA, a DNA molecule or a locked nucleic acid. 
     
     
         8 . A method according to  claim 6 , wherein the complementary polynucleotide probe extends at the 3′ end beyond the target RNA. 
     
     
         9 . A method according to  claim 6 , wherein the single-stranded molecule is an miRNA. 
     
     
         10 . A method according to  claim 5 , wherein the target RNA is a dsRNA. 
     
     
         11 . A method according to  claim 10 , wherein the dsRNA is an siRNA. 
     
     
         12 . A method according to  claim 5 , wherein the p19 fusion protein is immobilized prior to binding the target RNA. 
     
     
         13 . A method according to  claim 5 , wherein the target RNA is immobilized prior to binding the p19 fusion protein. 
     
     
         14 . A method according to  claim 5 , wherein the detectable label is selected from the group consisting of a fluorescent label, a radioactive label, a chemiluminescent label, a protein label and a small molecule label. 
     
     
         15 . A method according to  claim 5 , wherein the p19 fusion protein is bound to the target RNA to form a complex which is then immobilized on a matrix. 
     
     
         16 . A method according to  claim 5 , wherein the matrix has a colored or fluorescent label that differs from the label on the p19 protein or polynucleotide probe. 
     
     
         17 . A method according to  claim 5  or  16 , wherein the matrix is a bead. 
     
     
         18 . A method according to  claim 17 , wherein the bead is magnetic. 
     
     
         19 . A method according to  claim 17 , wherein the bead is coated with a carbohydrate to which the p19 fusion protein can bind. 
     
     
         20 . A method according to  claim 5 , wherein the immobilized target RNA is diagnostic for an abnormal condition of a cell. 
     
     
         21 . A method according to  claim 5 , wherein the target RNA is diagnostic of an abnormal host cell and is present in a mixture comprising a plurality of types and sizes of RNA. 
     
     
         22 . A method for detecting a target RNA in a mixture of RNAs, such that if the target RNA is
 (i) single-stranded, adding a complementary polynucleotide probe for forming a dsRNA and allowing the dsRNA to bind to a p19 fusion protein; or   (ii) double-stranded, binding the dsRNA directly to the p19 fusion protein,   wherein either the complementary polynucleotide probe or the p19 fusion protein is linked to a label selected from a fluorescent label, a radioactive label, a chemiluminescent label, a protein label and a small molecule label.   
     
     
         23 . A method according to  claim 20 , further comprising:
 immobilizing the p19 fusion protein on a matrix for binding labeled dsRNA and removing unbound RNA.   
     
     
         24 . A method according to  claim 20 , further comprising:
 immobilizing the dsRNA on a matrix and binding a labeled p19 fusion protein to the dsRNA.   
     
     
         25 . A kit, comprising: the recombinant protein in  claim 1 , instructions for detecting a small RNA, and optionally a matrix for binding p19 fusion protein or a polynucleotide probe. 
     
     
         26 . A kit according to  claim 25 , wherein the kit contains a matrix and the p19 fusion protein is bound to the matrix. 
     
     
         27 . A kit according to  claim 25 , wherein the kit contains a matrix and the matrix further comprises one or more polynucleotide probes; and
 the recombinant p19 fusion protein is labeled.   
     
     
         28 . A kit according to  claim 25 , wherein the matrix is a bead. 
     
     
         29 . A kit according to  claim 25 , wherein the bead is colored or fluorescent. 
     
     
         30 . A kit according to  claim 27 , wherein the bead is magnetic. 
     
     
         31 . A small RNA, comprising a 21 nucleotide dsRNA.