To compare preferences to the two ends, we made perfect WC complementarity the second-stage filtering criterion, and sorted the results into one set of potential targets with perfect complementarity to the 5' ends of miRNAs, and another set with perfect complementarity to the 3' ends.
Based on our results and those studies, we designed a target sequence with six tandem repeats with perfect complementarity to the mature strand of the miR122 duplex.
Unexpectedly, we were unable to identify by BLAST a single target transcript with perfect complementarity to any of our recovered shRNA sequences.
The 10 nt motif returned by MEME included a 7 nt stretch with perfect complementarity to positions 2 8 in miR-124.
The next three most highly overrepresented 6mers were overlapping sequences with perfect complementarity to positions 1 8 in miR-1 (Figure 4Ai).
The ratio of the calculated free energy of miRNA: target to the free energy if the miRNA had perfect complementarity to the target gene (percent minimum free energy) was the final feature.
Similarly, the multiple expectation maximization for motif elicitation (MEME) motif discovery algorithm identified a 10 nt motif sequence from the high confidence miR-1 targets with perfect complementarity to positions 1 8 in miR-1 (Figure 4Aiii) [66].
The miR-145 pMIR-REPORT luciferase reporter vector was cloned by inserting a oligo with perfect complementarity to mature miR-145 into the HindII/SpeI site of pMIR-REPORT Luciferase vector (AM5795, Applied Biosystems).
Computational analysis revealed eleven additional 7 8mer binding sites for miR-29 in the coding sequence (CDS) of elastin (Fig. 4A, dashed lines), as well as eight MREs with perfect complementarity to the seed sequence of the miR-15 family members miR-195/miR-497 (Fig. 4A, arrows).
Using the microRNA Registry release 2.0 prediction of the mir-250 sequence [17], we identified potential target genes computationally, using the same criteria that we used for mir-61 targets: we required that 3' UTRs have at least 7 bases of perfect complementarity to the 5' end of mir-250, with this putative "seed match" conserved in the C. briggsae orthologs of the C. elegans genes [11].
