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. 2012 Nov 5;109(49):E3340–E3349. doi: 10.1073/pnas.1208618109

Fig. 5.

Fig. 5.

MDA5 binds to dsRNA via multiple, rate-limiting nucleation steps to assemble discontinuous filaments on a single dsRNA. (A) Schematic of the single-molecule fluorescence assay. The filament assembly reaction was monitored using TIRF single-molecule microscopy upon the addition of fluorescently labeled MDA5 or MDA5h to surface-immobilized dsRNA (t = 0) (Methods and Materials). (B) Representative traces of filament assembly reactions using Alexa 647-labeled MDA5hSNAP (50 nM) and 2,012-bp dsRNA. The immediate increase in fluorescence upon MDA5hSNAP injection (t = 0) indicates the background noise, which also was observed without dsRNA. (C) A model of stepwise assembly reaction. The alternation between elongation and pause in assembly traces indicates the involvement of multiple nucleation events. Elongation ends abruptly upon encounter of dsRNA ends or neighboring filament termini. The insufficiency of a single nucleus to propagate and saturate the entire length of dsRNA suggests unidirectional elongation of MDA5 filaments. The direction of elongation likely is determined by asymmetric binding of a nucleus to symmetric dsRNA with an equal probability of facing either direction. (D) A representative electron micrograph of full-length MDA5 (0.3 μM) in complex with 2,012-bp dsRNA (1.2 μg/mL; i.e., 0.34 μM MDA5 binding site). Gaps (arrows) indicate the presence of multiple filaments propagated from independent nuclei on a single dsRNA. (E) Histogram of the elongation rates of MDA5hSNAP filaments calculated from the slope of the first linear elongation phase of ∼350 assembly traces. Fluorescence intensity was converted to base pair numbers by assuming 100% coverage of 512 bp at saturation. (F) Averaged traces of filament propagation of MDA5, MDA5h, and MDA5hSNAP synchronized by the initial nucleation event (n = 331, 257, and 542, respectively). (G) Histogram of the nucleation times of MDA5hSNAP (50 nM) on 512-bp dsRNA and the single-exponential fit. (H) Dependence of the nucleation time (mean value) on dsRNA length (n = 250–500).