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. 2015 Feb 23;5:8534. doi: 10.1038/srep08534

Figure 2. Free energy directly affected the interaction between hsa-miR-128-3p and its counterparts.

Figure 2

(a) RNA EMSA with the cy5.5TM-labeled hsa-miR-128-3p or hsa-miR-143-3p oligonucleotides, and 2′ O-Methyl modified and IRDye®800-labeled CYP2C9 mRNA oligonucleotides. Lanes 1, 2, 4, and 5 show mobility of the labeled oligonucleotides; Lanes 3 and 6 show mobility of the labeled miRNA oligonucleotides with corresponding CYP2C9 mRNA oligonucleotides; Lanes 7 and 8 show mobility of the labeled miRNA oligonucleotides with unmatched CYP2C9 mRNA oligonucleotides (negative controls). Arrow indicates an oligonucleotide complex (yellow) in Lane 3. * or † indicates the CYP2C9 mRNA oligonucleotides retaining the hsa-miR-128-3p or hsa-miR-143-3p recognition sites, repectively. (b) RNA EMSA with the cy5.5TM-labeled hsa-miR-128-3p probe, and 2′ O-Methyl modified and IRDye®800-labeled CYP2C9, PAIP2, or PFKFB4 mRNA oligonucleotides. Lanes 1, 2, 3, 4, and 5 show mobility of the labeled oligonucleotides; Lanes 6, 7, 8 and 9 show mobility of the labeled hsa-miR-128-3p oligonucleotide with corresponding CYP2C9, PAIP2, or PFKFB4 mRNA probes with different free energy. Arrows indicate the oligonucleotide complex (yellow) in Lane 6 and 8. *, †, and ‡ indicate two PAIP2 mRNA oligonucleotides, and one PFKFB4 mRNA oligonucleotide with the binding free energy of −18.0 kcal/mol, −20.5 kcal/mol, and −15.8 kcal/mol, respectively. (c) RNA EMSA with the cy5.5TM-labeled hsa-miR-128-3p probe, and 2′ O-Methyl modified and IRDye®800-labeled CYP2C9 or PAIP2 mRNA oligonucleotides. Lanes 1 and 4 show mobility of the labeled hsa-miR-128-3p oligonucleotide with corresponding CYP2C9, and PAIP2 mRNA probes with the free energy of −23.9 kcal/mol and −20.5 kcal/mol, respectively. Lanes 2, 3, 5 or 6 show mobility of the labeled hsa-miR-128-3p oligonucleotide with corresponding CYP2C9 and PAIP2 mRNA probes, in the presence of unlabeled excess nonspecific competitor (Cold-NC) and excess specific competitor (Cold hsa-miR-128-3p), respectively. NC, nonspecific competitor. Arrows indicate the oligonucleotide complex (yellow) in Lane 1, 2, 3, 4 and 5.