RNA MOLECULES AND USES THEREOF
The invention relates to a method of designing a short RNA molecule to increase the expression of a target gene in a cell through the down-regulation of a non-coding RNA transcript, said method comprising the steps of: a) obtaining the nucleotide sequence of the coding strand of the target gene, at least between 200 nucleotides upstream of the gene's transcription start site and 200 nucleotides downstream of the gene's transcription start site; b) determining the reverse complementary RNA sequence to the nucleotide sequence determined in step a); and c) designing a short RNA molecule which is the reverse complement or has at least 80% sequence identity with the reverse complement of a region of the sequence determined in step b); wherein said method does not include a step in which the existence of said non-coding RNA transcript is determined; and to such short RNA molecules and uses thereof.
1 . A method of designing a short RNA molecule to increase the expression of a target gene in a cell through the down-regulation of a non-coding RNA transcript, said method comprising the steps of:
a) obtaining a nucleotide sequence of the coding strand of the target gene, said nucleotide sequence being obtained by selecting the sequence between 200 nucleotides upstream of the gene's transcription start site and the gene's transcription start site or the sequence of an intron between the gene's transcription start site and 200 nucleotides downstream of the gene's start site;
b) determining the reverse complementary RNA sequence to the nucleotide sequence selected in step a);
c) designing a short RNA molecule which is the reverse complement or has at least 80% sequence identity with the reverse complement of a region of the nucleotide sequence determined in step b), wherein the short RNA molecule increases the expression of the target gene; and
d) synthesizing the short RNA molecule designed in step c); wherein said method does not include a step in which the existence of said non-coding RNA transcript is determined prior to step a).
2 . The method of claim 1 , wherein the target gene is a pluripotency-inducing gene.
3 . The method of claim 1 , wherein the region defined in c) includes the reverse complement of the gene's transcription start site.
4 . The method of claim 1 , wherein the short RNA molecule is from 16 nucleotides to 30 nucleotides in length.
5 . The method of claim 1 , which further comprises the step of generating a double-stranded siRNA molecule which incorporates said short RNA molecule.
6 . The method of claim 5 , wherein each strand of said double-stranded siRNA molecule is 16 to 30 nucleotides in length and wherein said molecule is hybridised over a length of at least 12 nucleotides.
7 . The method of claim 1 , wherein the short RNA molecule is 21 nucleotides in length.
8 . The method of claim 1 , wherein the short RNA molecule is the reverse complement or has at least 95% sequence identity with the reverse complement of a region of the nucleotide sequence determined in step b).
9 . A method of designing a short RNA molecule to increase the expression of a target gene in a cell through the down-regulation of a non-coding RNA transcript, said method comprising the steps of:
a) obtaining a nucleotide sequence of the coding strand of the target gene, said nucleotide sequence being obtained by selecting the sequence between 500 nucleotides upstream of the gene's transcription start site and the gene's transcription start site or the sequence of an intron between the gene's transcription start site and 500 nucleotides downstream of the gene's start site;
b) determining the reverse complementary RNA sequence to the nucleotide sequence selected in step a);
c) designing a short RNA molecule which is the reverse complement or has at least 80% sequence identity with the reverse complement of a region of the nucleotide sequence determined in step b), wherein the short RNA molecule increases the expression of the target gene; and
d) synthesizing the short RNA molecule designed in step c); wherein said method does not include a step in which the existence of said non-coding RNA transcript is determined prior to step a).