1. Human Argonaute 2 Has Diverse Reaction Pathways on Target RNAs
Myung Hyun Jo, Soochul Shin, Seung-Ryoung Jung, Eunji Kim, Ji-Joon Song, Sungchul Hohng 
Molecular Cell 
Volume 59, Issue 1, Pages (July 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 59, 117-124DOI: (10.1016/j.molcel.2015.04.027)
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3. Figure 1 Experimental Scheme
(A) The standard design of guide and target RNAs for single molecule FRET experiments.
(B) Experimental procedure. After target RNA immobilization, pre-assembled core-RISC was added to a reaction channel while single-molecule fluorescence signals were being monitored.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Acceleration of Target Binding by Argonaute
(A) Single-molecule images of donor channel (green) and acceptor channel (red) before (left) and 60 s after the addition of 2 nM core-RISC (top) and 2 nM free guide (bottom).
(B) Binding time distributions of core-RISC (black squares) and free guide (red circles), and their single-exponential fits (black and red lines). Data are mean ± SD for three independent experiments.
(C) Guide RNA sequences to study dinucleotide mismatch effects (red, mismatch sites; underline, dye labeling position).
(D) Comparison of the binding rate constant between dinucleotide mismatched guide RNAs. Data are mean ± SD for three independent experiments.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 RISC Recycling after Target Cleavage
(A and B) Representative fluorescence intensity time traces reporting the recycling of core-RISC after target cleavage (Cy3 [green] and Cy5 [red] at Cy3 excitation [top], and at Cy5 excitation [bottom]).
(C) Dinucleotide mismatch effects on the cleavage probability per core-RISC binding. Data are mean ± SD for three independent experiments.
(D and E) Distributions of T1 and T2 (dissociation times of cleaved targets as defined in A and B) and their single exponential fits (red lines).
(F) Intrinsic binding lifetime of cleaved guide-target duplexes (the RNA duplex made from a guide RNA and a target RNA truncated to the 10th nucleotide). The guide and truncated target RNAs were preannealed and immobilized on a glass surface, and the number of remaining FRET spots was counted at different times. Data are mean ± SD for three independent experiments, and the red lines are a single exponential fit of the data.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 Diverse Reaction Pathways of RISC
(A) Representative fluorescence intensity (top) and corresponding FRET (bottom) time traces showing core-RISC binding without target cleavage. (Cy3 [green], Cy5 [red], FRET [black]). Existence of two distinct binding modes (transient and long) is clear.
(B) Binding lifetime of the transient binding and the long binding modes. The number of FRET spots was counted at different times after flushing out free core-RISC for long binding mode. Data are mean ± SD for three independent experiments
(C) Comparison of single-molecule Cy5 images at Cy3 excitation before and 10 min after RNase III treatment for the free guide-target duplex (top), and the long binding mode (bottom).
(D) RNA degradation rates of the free guide-target duplex and the long binding mode after RNase III treatment.
(E) Comparison of single-molecule Cy5 images at Cy3 excitation before and 10 min after RNase A treatment for the free guide-target duplex (top) and the long binding mode (bottom). Not to hinder the activity of RNase A, target RNA with longer linker (Linker39U) was used.
(F) The survival probabilities of the free guide-target duplex and the long binding mode for RNase III and RNase A treatments. Data are mean ± SD for three independent experiments.
(G) Binding lifetimes of the stable binding.
(H) Binding lifetimes of the free-guide duplexes.
See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 Mg2+ and Sequence Effects on RISC Activities
(A) Probabilities of target cleavage (black) and transient core-RISC binding (red) at varying Mg2+ concentrations. Data are mean ± SD for three independent experiments.
(B) Core-RISC recycling rates at varying Mg2+ concentrations. Data are mean ± SD for three independent experiments.
(C) Guide RNA sequences (left) and their probabilities for target cleavage, transient core-RISC binding, stable core-RISC binding, and Argonaute unloading (right). Data are mean ± SD for three independent experiments.
See also Figures S5 and S6.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 Reaction Pathways of Core-RISC
Reaction pathways of core-RISC are summarized with their probabilities and kinetic parameters. let7a miRNA and its fully complementary target are used as an example. The binding rate of core-RISC was obtained from Figure 2D. The Argonaute unloading rate was obtained from Figure 4D by assuming that the cleavage of unloaded guide-target duplexes by RNase III was fast. The dissociation rates of cleaved targets were obtained from Figures 3D and 3E by assuming two branched dissociation pathways with k1 = 1/τ1 and k2 = 1/τ2, respectively. In this model, the histograms of T1 and T2 provide only the sum of k1 and k2. However, k1 and k2 can be uniquely determined using the fact that the ratio of k1 and k2 determines the relative probability of the two dissociation events. The dissociation rate of the transient binding mode was obtained from Figure 4B. The cleavage rate and the dissociation rate of the stable binding mode could not be directly measured, so only their boundaries are provided.
Molecular Cell  , DOI: ( /j.molcel )
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