Methods and systems for identifying micro-rna targets and synthesizing novel micro-rnas and uses of the same

Abstract

Method of identifying a microRNA-recognition element and of generating microRNAs are disclosed. System and computer programs for performing such methods are disclosed. Recombinant nucleic acid molecule comprising a heterologous coding sequences and one or more MREs are also disclosed as are isolated nucleic acid molecule comprising one or more MRE sequences and being free of a coding sequence operably linked to regulatory elements. MicroRNA generated by a methods of the invention and the use of the microRNAs to downregulate gene expression are disclosed.

1 . (canceled) 
     
     
         2 . The method of  claim 27  further comprising determining free energy of microRNA bound to said mRNA sequence wherein a free energy determination of −10 kcal/mole or less indicates that said mRNA sequence is a microRNA-recognition element for the microRNA. 
     
     
         3 . The method of  claim 2  wherein a free energy determination of −20 kcal/mole or less indicates that said mRNA sequence is a microRNA-recognition element for the microRNA. 
     
     
         4 . The method of  claim 2  wherein a free energy determination of −30 kcal/mole or less indicates that said mRNA sequence is a microRNA-recognition element for the microRNA. 
     
     
         5 . The method of any of  claims 1 - 4  wherein mRNA sequences in a database of mRNA sequences are compared to the miRNA sequence. 
     
     
         6 . The method of  claim 5  wherein repetitive Alu sequences are removed from mRNA sequences in a database of mRNA sequences and the remaining mRNA sequences are compared to the miRNA sequence. 
     
     
         7 . The method of  claim 27  wherein the miRNA sequence is compared to the mRNA 3′ untranslated region. 
     
     
         8 - 22 . (canceled) 
     
     
         23 . An recombinant nucleic acid molecule comprising a heterologous coding sequences and one or more MREs identified by the method of  claim 27 . 
     
     
         24 . An isolated nucleic acid molecule comprising one or more MRE sequences identified by the method of  claim 27 , said nucleic acid molecule being free of a coding sequence operably linked to regulatory elements. 
     
     
         25 . A microRNA generated by a method comprising the steps of:
 step i) identifying a selected mRNA sequence to be the target of the microRNA;   step ii) generating an oligonucleotide sequence of a microRNA that is 17-25 nucleotides and has a degree of complementarity to the selected mRNA sequence that is indicative of a microRNA-recognition element for the microRNA wherein the microRNA includes a proximal region that is 7-9 nucleotides, has a 5′ end and a 3′ end and includes a nucleotide at the 5′ end which is the microRNA's 5′ terminus nucleotide, a distal region that is 7-15 nucleotides, has a 5′ end and a 3′ end and includes a nucleotide at the 3′ end which is the microRNA's 3′ terminus nucleotide, and a loop region that is 6-9 nucleotides, wherein the 3′ end of the proximal region is contiguous to the 5′ end of the loop region, and the 3′ end of the loop region is contiguous to the 5′ end of the distal region, wherein complementarity of the selected mRNA sequence to the microRNA sequence that is indicative of a microRNA-response element for the microRNA refers to: the selected mRNA sequence having a sequence that:
 a) includes a-region corresponding to the proximal region of the microRNA that is either:
 (i) completely complementary to the proximal region, or 
 (ii) has a single mismatch
 (A) to the 5′ end of the proximal region, or 
 (B) symmetrically placed between the 5′ end of the proximal region and the 3′ end of the proximal region; and 
 
 
 b) includes a region corresponding to the loop region of the microRNA, wherein said region of the selected mRNA has 0 nucleotides; and 
 c) includes a region corresponding to the distal region of the microRNA that is either:
 (i) completely complementary to at least 7 contiguous nucleotides of the distal region of the microRNA, including the 5′ end of the distal region, or 
 (ii) contains
 (A) mismatches of 1-4 contiguous nucleotides and 
 (3) matches of at least 5 nucleotides to a contiguous nucleotide sequence of the distal region of the microRNA, including the 5′ end of the distal region; wherein the oligonucleotide sequence has a degree of complementarity to the selected mRNA sequence that is indicative of a microRNA for a microRNA-recognition element; 
 
 
   step iii) synthesizing an oligonucleotide having the microRNA sequence.   
     
     
         26 . A method of down regulating expression of an mRNA comprising the steps of selecting a nucleotide sequence of the mRNA, preparing a microRNA by a method comprising the steps of:
 step i) identifying a selected mRNA sequence to be the target of the microRNA;   step ii) generating an oligonucleotide sequence of a microRNA that is 17-25 nucleotides and has a degree of complementarity to the selected mRNA sequence that is indicative of a microRNA-recognition element for the microRNA wherein the microRNA includes a proximal region that is 7-9 nucleotides, has a 5′ end and a 3′ end and includes a nucleotide at the 5′ end which is the microRNA's 5′ terminus nucleotide, a distal region that is 7-15 nucleotides, has a 5′ end and a 3′ end and includes a nucleotide at the 3′ end which is the microRNA's 3′ terminus nucleotide, and a loop region that is 6-9 nucleotides, wherein the 3′ end of the proximal region is contiguous to the 5′ end of the loop region, and the 3′ end of the loop region is contiguous to the 5′ end of the distal region, wherein complementarity of the selected mRNA sequence to the microRNA sequence that is indicative of a microRNA-response element for the microRNA refers to: the selected mRNA sequence having a sequence that:
 a) includes a-region corresponding to the proximal region of the microRNA that is either:
 (i) completely complementary to the proximal region, or 
 (ii) has a single mismatch
 (A) to the 5′ end of the proximal region, or 
 (B) symmetrically placed between the 5′ end of the proximal region and the 3′ end of the proximal region; and 
 
 
 b) includes a region corresponding to the loop region of the microRNA, wherein said region of the selected mRNA has 0 nucleotides; and 
 c) includes a region corresponding to the distal region of the microRNA that is either:
 (i) completely complementary to at least 7 contiguous nucleotides of the distal region of the microRNA, including the 5′ end of the distal region, or 
 (ii) contains
 (A) mismatches of 1-4 contiguous nucleotides and 
 (B) matches of at least 5 nucleotides to a contiguous nucleotide sequence of the distal region of the microRNA, including the 5′ end of the distal region; wherein the oligonucleotide sequence has a degree of complementarity to the selected mRNA sequence that is indicative of a microRNA for a microRNA-recognition element; 
 
 
   step iii) synthesizing an oligonucleotide having the microRNA sequence and   step iv) contacting the microRNA with a cell expressing the mRNA.   
     
     
         27 . A method of identifying a microRNA-recognition element comprising the steps of:
 comparing degree of complementarity of a microRNA nucleotide sequence to an mRNA sequence to identify the presence of an mRNA sequence that exhibits a degree of complementarity to the microRNA sequence that is indicative of a microRNA recognition element for the microRNA,   wherein the microRNA is 17-25 nucleotides and includes   a proximal region that is 7-9 nucleotides, has a 5′ end and a 3′ end and includes a nucleotide at the 5′ end which is the microRNA's 5′ terminus nucleotide,   a distal region that is 7-15 nucleotides, has a 5′ end and a 3′ end and includes a nucleotide at the 3′ end which is the microRNA's 3′ terminus nucleotide, and   a loop region that is 0 nucleotide, 2-3 nucleotides or 6-3 nucleotide,   wherein when the loop region is 0 nucleotides the 3′ end of the proximal region is contiguous to the 5′ end of the distal region and when the loop region is 2-3 or 6-9 nucleotides the 3′ end of the proximal region is contiguous to the 5′ end of the loop region and the 3″ end of the loop region is contiguous to the 5′ end of the distal region,   wherein complementarity of the mRNA sequence to the microRNA sequence that is indicative of a microRNA-response element for the microRNA is characterized by:   an mRNA sequence having a sequence that:   a) includes a region corresponding to the proximal region of the microRNA that is either completely complementary to the proximal region, or has a single mismatch to the 5′ end of the proximal region, or symmetrically placed between the 5′ end of the proximal region and the 3′ end of the proximal region;   b) includes a region corresponding to the loop region of the microRNA that either forms a loop of 2-5 non-paired nucleotides when the loop region is 0, or has 0 nucleotides when the loop region is 6-9 nucleotides, or has 2-3 nucleotides which forms a bulge of 2-3 non-complementary nucleotides of the loop region when the loop region is 2-3 nucleotides; and   c) includes a region corresponding to the distal region that is either completely complementary to at least 7 contiguous nucleotides of the distal region including the 5′ end of the distal region, or contains mismatches of 1-4 contiguous nucleotides and matches of at least 5 nucleotides to a contiguous nucleotide sequence of the distal region including the 5′ end of the distal region;   wherein an mRNA sequence that has complementarity to the microRNA sequence that is indicative of a microRNA-recognition element for the microRNA indicates that said mRNA sequence is a microRNA-recognition element for the microRNA.