Fig. 6: Base-stacking in biotechnology applications.
From: High-throughput single-molecule quantification of individual base stacking energies in nucleic acids
a A DNA tetrahedron is assembled from four 3-point-star DNA motifs connected on each edge with two pairs of sticky ends. b Non-denaturing PAGE analysis shows DNA tetrahedron assembly from oligonucleotide components. (n = 1). c Designs of base stacking interactions tested in DNA tetrahedra with conserved sticky end sequences. d Assembly of DNA tetrahedra with different base stacks (full gels in Supplementary Fig. 14). e Thermal stability of DNA tetrahedra with various base-stacks. f Ligation of two DNA duplexes with 3 or 4 base pair sticky ends. g Non-denaturing PAGE confirms the ligation of the two DNA duplexes (full gel in Supplemental Fig. 15) (n = 1). h Designs of base stacking interactions of sticky ends tested for ligation. i Gel images show the increase in band intensity of ligated fragments (full gels in Supplementary Figs. 16–17). j Quantified ligation product over time. k Ligated product for different base stacks at 8 min. l Molecular dynamics simulations of base stacking interactions with different force fields. m Simulation scheme showing two 3 bp duplexes with A|A in red and T|T in blue, in the initial (stacked) and the final (unstacked) confirmation. The pulling force is applied on the C1’ atoms of the T|T stacked pair, orthogonal to the base-pairs. n Potential of mean force (PMF) of the A|A-T|T construct as a function of the distance between the pull groups (ξ), for the Amber99-Chen-Garcia52 and parmbsc154 force-fields. o Designs of base stacking interactions tested in MD simulation. p Free energy of stacking (ΔGstack) as calculated from the simulations compared to experimentally determined values (values added from Table 1). Data in (d, e, j, k) is presented as mean values +/− standard deviation from triplicate experiments. Data in (n, p) presented as calculated values +/− standard deviations of the energies sampled at each distance.
