Figure 1.
Conformational Dynamics of Cas12a Ternary Complexes on Fully Matched dsDNA
(A) Representative smFRET trajectories and cartoons illustrating corresponding states. Cy3 (green dots) is labeled at 3′ end of crRNA, Cy5 (red dots) is labeled at the TS 28 nucleotides away from the PAM. Black lines are apparent FRET efficiencies, and red lines are hidden Markov modeling of FRET. Immobilized dsDNA contains 23 matched bases toward the crRNA. Under 532 nm excitation, spontaneous appearance of Cy3 and FRET signals represents the formation of Cas12a ternary complexes on immobilized dsDNAs (black arrows), and disappearance of FRET corresponds to dissociation of the cleaved PAM-distal DNA fragments (blue arrows). Four distinctive FRET states are indicated and assigned as S1–S4 from low to high values.
(B) Time-dependent FRET probability density plots synchronized at the appearance of FRET (defined as t = 0 in the left panel) or at the disappearance of FRET (defined as t = 0 in the right panel). From them, evolution of FRET after complex formation or before release of cleaved DNA can be carefully examined. Overall, FRET efficiencies increase over time after complex formation.
(C) A transition density plot reflecting transition frequencies among FRET states, in which initial and final FRET values for each transition event are accumulated into a two-dimensional histogram.
(D) A kinetic scheme displaying transition pathways and rates (s−1) among different FRET states. All experiments were repeated three times, and SEMs were used as error bars.