Figure 1.
Enzymatic probing (A, B, E, F) and NMR analysis (C, D, G, H, I) of miR-21 (A-D), miR-93 (E-H) and miR-296 (I).
A, B, E, F. Cleavage patterns obtained for limited hydrolysis of 5'-end labeled miR-21 (A, B) and miR-93 (E, F) with RNase V1, nuclease S1 and RNase T1 in native conditions. Lanes: C - reaction control; L – OH ladder; T1 - limited hydrolysis by RNase T1 (0.025u/µl) in denaturing condition. A. Lines V1 - limited hydrolysis with RNase V1 (0, 0.03125, 0.0625 or 0.125 u/µl). B. Lanes T1(N) - limited hydrolysis with RNase T1 (0, 0.04, 0.02 or 0.01 u/µl) in native conditions. E. Line V1 - limited hydrolysis with RNase V1 (0.125 u/µl). F. Lines S1 - limited hydrolysis with nuclease S1 (0.00475 u/µl). The increasing concentrations of RNase V1 and RNase T1 are indicated by arrows. Cleavage sites are indicated in autoradiogram. C, D, G, H, I. NMR analysis of miR-21 (C, D) miR-93 (G, H) and miR-296 (I) structures. C. The imino region of 1H NMR spectrum of miR-21 (0.7 mM) recorded at 7°C in H2O:D2O (90%:10%) with 150 mM sodium chloride, 10 mM phosphate buffer and 0.1 mM EDTA. Resonances arising from the hairpin form are indicated with *. D. Imino region of the 1H-1H 2D NOESY spectrum of miR-21 at 15°C in H2O:D2O (90%:10%) with 150 mM sodium chloride, 10 mM phosphate buffer and 0.1 mM EDTA. The lines indicate the imino proton connectivity. G. The imino region of 1H NMR spectrum of miR-93 (0.75 mM) recorded at 25°C in H2O:D2O (90%:10%) with 150 mM sodium chloride (top) or 50 mM sodium chloride (bottom), 10 mM phosphate buffer and 0.1 mM EDTA. Resonances arising from the hairpin form are indicated with *. H. Imino region of the 1H-1H 2D NOESY spectrum of miR-93 at 15°C in H2O:D2O (90%:10%) with 150 mM sodium chloride, 10 mM phosphate buffer and 0.1 mM EDTA. The lines indicate the imino proton connectivity. I. The imino region of 1H NMR spectrum of miR-296 (0.3 mM) recorded at 25°C in H2O:D2O (90%:10%) with 150 mM sodium chloride, 10 mM phosphate buffer and 0.1 mM EDTA.
Figure 2.
Distribution of linear and nonlinear human miRNAs and of hairpin stem location in predicted human secondary structure (A); and folding distribution of human mature miRNAs strands (B).
Figure 3.
Concentration dependency plots of miR-21, miR-93, miR-296 monomer and dimer (A) and the scheme illustrating concentration dependent equilibriums of these miRNAs (B).
Table 1.
Some parameters of miRNAs and the partition of monomer and dimer formation in concentration dependence.
Figure 4.
miR-21, miR-93 and miR-296 stability in GBM lysate.
A, B. Hydrolysis of 5′-end labeled miR-21, miR-93 and miR-296 in 0.01 mg/ml GBM lysate. C. Half-lives of miR-21, miR-93 and miR-296.
Figure 5.
Similarity of structures of miR-21, miR-93, miR-296 and anti-Tn-C aptamer TN-9.6.
A. RNA structure cluster of miR-21, miR-93, miR-296 and TN-9.6. B. ASCII representation of a consensus sequence and structure of miR-21, miR-93, miR-296 and TN-9.6. Multiple structure alignments of anti-Tn-C aptamer TN-9, its derivatives (TN-9.4, TN-9.6), miR-21 and miR-93 based. C. Graphic representation of miR-21, miR-93, miR-296 and TN-9.6 structures similarity. Regions of structures identical within compared group are marked in black. Calculations are made by RNAforester algorithms. The obtained score is 80.25.