Figure 4. The beneficial role for chimeric RDNA template in overcoming the template-product inhibition based on thermodynamic stability of duplexes.

(a) The expected difference between chimeric RDNA-RNA duplex and the homogeneous RNA-RNA duplex in being able to overcome the template-product inhibition. (b) Schematic representation of the proposal that hexadecameric (AU)-RDNA template C_T2 with RNA ligands R_L3+R_L4 produces R_P2, which in the presence of R_L5, R_L6 is expected to lead to R_P3, based on the greater thermodynamic stability of the R_P2:R_P3 duplex over the R_P2:C_T2 duplex, and release the C_T2 for another round of ligation reaction. (c) Time course of the EDC-mediated-ligation experiments documenting the effect of change in ratio of ligands, and the sequential-addition of ligands R_L5+R_L6 (0 h) followed by R_L5+R_L6 (at 20 h) versus all-in-one-pot reaction on the production of R_P2 and R_P3. (d) Comparison of the amount of R_P3 formed by the homogeneous RNA template R_T2 versus chimeric RDNA template C_T2 (at 48 h) demonstrating the higher efficiency of C_T2 in mediating the formation of R_P3 by overcoming the template-product inhibition. See supplementary figs. 63–78 for EDC-ligation conditions. A, U = RNA; A, T = DNA. Lines in graph (4c) are drawn as guide indicating the trend and are not mathematical curve fittings. % yields were calculated with respect to the template C_T2 or R_T2 respectively. Experiments were run in triplicate and the error range is less than ± 5%; error bars represent standard deviation.