MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression
Fig 4
Detection of DNA-DNA and RNA-DNA triplexes by EMSA and NMR, and molecular modeling of miRNA-duplex DNA triplex.
(A) EMSA; 5’ ROX-labeled hairpin duplex DNA (0.1 μM) was incubated for 3-hrs at 22°C in the presence (lanes 2–11) or absence (lane 1) of 2.5 μM 483-opti DNA oligo, and increasing concentration (30, 60, 150 μM) of Hoogsteen bond-optimized hsa-miR-483-5p (483-opti, lanes 3–5), hsa-miR-483-5 (483, lanes 6–8), or a scrambled RNA oligo (Scramble, lanes 9–11). Duplexes and triplexes were resolved on a 20% non-denaturing acrylamide gel, and the ROX-signal visualized. Triplex of 483-opti DNA oligo and duplex DNA is readily detected (lane 2). The 483-opti RNA oligo competes with 483-opti DNA oligo for binding to duplex DNA which is evident by increased amounts of duplex DNA and decreased amounts of triplex (compare lanes 3–5 with lane 2). Hsa-miR-483-5p (483) and scrambled RNA, because of the fewer number of favorable Hoogsteen bonds, did not compete with the 483-opti DNA oligo for binding to duplex DNA (lanes 6–7 and 9–10, respectively). (B-C) NMR; Two-Dimensional (2D) [1H, 1H] TOCSY spectra of free single stranded hairpin duplex DNA (blue contours), hairpin duplex DNA combined with hsa-miR-483-5p RNA oligo (green contours; 1:1.5 ratio), and hairpin duplex DNA with single stranded DNA oligo with the same sequence as hsa-miR-483-5p (red contours; 1:1ratio). (B) Thymidine cross-peaks between H6 and H7 (methyl), and (C) cytosine cross-peaks between H5 and H6. Single stranded RNA (hsa-miR-483-5p) or single stranded DNA with hairpin duplex DNA show similar improvement in peak the intensities, and similar chemical shift perturbations/appearance of new peaks highlighted in blue boxes, suggesting that single stranded DNA and single stranded RNA of the same sequence bind to DNA duplex in a similar manner; the major differences (peaks in red boxes) are one peak among thymidine cross-peaks, showing an intermediate change (peak disappearing) with singe stranded RNA while saturated with hairpin duplex DNA, and two new peaks among cytosine cross-peaks showing much higher intensities with single stranded DNA, indicating that the latter DNA binds to duplex DNA duplex with higher binding affinity than RNA, consistent with the results obtained by EMSA. (D) Molecular model of hsa-miR-483-5p-DNA triplex. (I): the model of predicted miRNA and corresponding DNA duplex sequences (16 favorable Hoogsteen pairings). All predicted Hoogsteen base pairs are well maintained after removal of positional and distance restraints(II): negative control (antisense hsa-miR-483-5p) of model with 9 favorable Hoogsteen pairings. Both RNA and DNA duplex are largely twisted and nearly all predicted Hoogsteen pairings cannot be stably maintained. Residues in favor of Hoogsteen hydrogen bond formation are shown in red while the others are shown in blue.