Figure 5. Analysis of Ternary Complex Formation by Native Gel Electrophoresis.

(A) Titration of labeled tDNA with increasing amounts of the preformed gRNA-RsAgo complex (1:2 ratio, RsAgo concentrations are shown at the top) for either fully complementary (“Comp,” left) or 3+2A′ (middle) targets. Titration of preformed gRNA-tDNA duplex with RsAgo (right).

(B) Formation of binary and ternary complexes containing labeled gRNA₁*. The components were mixed as described in the text; gRNA¹ and gRNA⁴ are indicated as “1” and “4.” The arrowheads indicate the order of addition of the labeled and unlabeled competitor gRNAs; the competitor gRNAs were added either before or 5 min after mixing of RsAgo and gRNA₁*, followed by the addition of tDNA (when indicated) and incubation for 20 min at 30°C. The concentrations of gRNA₁*, competitor gRNAs, and RsAgo were 20 nM, 1 μM, and 40 nM; tDNA was added to 40 nM. Positions of free nucleic acids and binary and ternary complexes are shown on the sides of the gels. The labeled components in each experiment are indicated with asterisks.