Multivalent RNA aptamers and their expression in multicellular organisms

Abstract

The present invention relates to a monovalent RNA aptamer that binds to Drosophila splicing factor B52 and a multivalent RNA aptamer that includes at least two RNA aptamer sequences linked together. Also disclosed are isolated or constructed DNA molecules which encode either a monovalent RNA aptamer or a multivalent RNA aptamer of the present invention, an engineered gene encoding a multivalent RNA aptamer of the present invention, and host cells and expression systems which contain either a heterologous DNA molecule or a heterologous gene of the present invention. Further aspects of the present invention relate to a method of expressing a multivalent RNA aptamer in a cell, a method of increasing activity of a splicing factor protein in a cell, and a method of inhibiting activity of a target molecule in a cell. A transgenic non-human organism whose somatic and germ cell lines contain an engineered gene encoding a multivalent RNA aptamer is also disclosed.

What is claimed:  
     
         1 . A constructed DNA molecule comprising: 
 a plurality of monomeric DNA sequences linked together to form a single DNA chain, each monomeric DNA sequence encoding an independent, functional RNA molecule.    
     
     
         2 . The constructed DNA molecule of  claim 1 , wherein each of the plurality of monomeric sequences also encodes a cis-acting ribozyme.  
     
     
         3 . A method of expressing a functional RNA molecule in a cell comprising: 
 introducing a constructed DNA molecule of  claim 1  into a cell under conditions effective to express the functional RNA molecule.    
     
     
         4 . An engineered gene comprising: 
 the constructed DNA molecule of  claim 1  and    a regulatory sequence coupled to the constructed DNA molecule to control expression thereof.    
     
     
         5 . A method of expressing a functional RNA molecule in a cell comprising: 
 introducing an engineered gene of  claim 4  into a cell under conditions effective to express the functional RNA molecule.    
     
     
         6 . A method of inhibiting activity of a target molecule in a cell comprising: 
 expressing a multivalent RNA aptamer in a cell, the multivalent RNA aptamer having an affinity for a target molecule sufficient to inhibit activity of the target molecule.    
     
     
         7 . The method of  claim 6  further comprising: 
 introducing into the cell, prior to said expressing, a DNA molecule encoding the multivalent RNA aptamer.  
 
     
     
         8 . The method of  claim 7 , wherein the DNA molecule includes a promoter sequence which regulates transcription of the DNA molecule.  
     
     
         9 . The method of  claim 8 , wherein said expressing includes exposing the cell to conditions effective to induce the promoter sequence to initiate transcription of the DNA molecule.  
     
     
         10 . The method of  claim 6 , wherein the multivalent RNA aptamer has at least two RNA aptamer sequences linked together.  
     
     
         11 . The method of  claim 10 , wherein the multivalent RNA aptamer has five aptamer sequences linked together.  
     
     
         12 . The method of  claim 6 , wherein the target molecule is Drosophila splicing factor B52.  
     
     
         13 . A method of increasing activity of a splicing factor protein comprising: 
 inserting a multivalent RNA aptamer, which binds to a splicing factor protein, into an RNA transcript, which contains exons and introns, under conditions effective to enable splicing of the RNA transcript.    
     
     
         14 . The method of  claim 13 , wherein said inserting comprises: 
 inserting a heterologous DNA molecule which encodes the multivalent RNA aptamer into the genome of a host cell under conditions effective to cause the multivalent RNA aptamer to be transcribed in cis with the RNA transcript.    
     
     
         15 . The method of  claim 13 , wherein the splicing factor protein is Drosophila splicing factor B52.  
     
     
         16 . A transgenic non-human organism whose somatic and germ cell lines contain an engineered gene encoding a multivalent RNA aptamer which inhibits activity of a target molecule to treat a condition associated with an expression level of the target molecule.  
     
     
         17 . The transgenic non-human organism of  claim 16 , wherein the non-human organism is an insect.  
     
     
         18 . The transgenic non-human organism of  claim 17 , wherein the insect is a species of Drosophila.  
     
     
         19 . The transgenic non-human organism of  claim 18 , wherein the target molecule is Drosophila splicing factor B52.  
     
     
         20 . The transgenic non-human organism of  claim 16 , wherein the engineered gene encoding a multivalent RNA aptamer comprises: 
 a DNA sequence encoding the multivalent RNA aptamer and    a regulatory sequence which controls expression of the DNA sequence encoding a multivalent RNA aptamer.    
     
     
         21 . The transgenic non-human organism of  claim 20 , wherein the DNA sequence comprises: 
 a plurality of monomeric DNA sequences each encoding a multivalent RNA aptamer.    
     
     
         22 . The transgenic non-human organism of  claim 21 , wherein each of the plurality of monomeric DNA sequences is substantially identical.  
     
     
         23 . The transgenic non-human organism of  claim 21 , wherein each of the plurality of monomeric sequences also encodes a cis-acting ribozyme.  
     
     
         24 . The transgenic non-human organism of  claim 23 , wherein the cis-acting ribozyme is a hammerhead-type ribozyme.  
     
     
         25 . A host cell in a non-human living organism, the host cell comprising: 
 a DNA molecule encoding a multivalent RNA aptamer comprising at least two RNA aptamer sequences linked together.    
     
     
         26 . A host cell in a non-human living organism, the host cell comprising: 
 a constructed DNA molecule comprising a plurality of monomeric DNA sequences linked together to form a single DNA chain, each monomeric DNA sequence encoding a multivalent RNA aptamer comprising at least two RNA aptamer sequences linked together, each of the at least two RNA aptamer sequences being capable of binding a target molecule.    
     
     
         27 . A host cell in a non-human living organism, the host cell comprising: 
 a heterologous gene comprising (i) a DNA sequence encoding a multivalent RNA aptamer comprising at least two RNA aptamer sequences linked together and (ii) a regulatory sequence which controls expression of the DNA sequence encoding the multivalent RNA aptamer.    
     
     
         28 . A host cell in a non-human living organism, the host cell comprising: 
 an engineered gene comprising (i) a constructed DNA molecule comprising a plurality of monomeric DNA sequences linked together to form a single DNA chain, each monomeric DNA sequence encoding a multivalent RNA aptamer comprising at least two RNA aptamer sequences linked together, each of the at least two RNA aptamer sequences being capable of binding a target molecule and (ii) a regulatory sequence which controls expression of each monomeric DNA sequence encoding a multivalent RNA aptamer.