Figure 1.

(A) Cy-Dye molecular toolkit; Cy3BdT phosphoramidite A; 5-(hexyn-1-ol)-6-Cy3B phosphoramidite for 5′-addition B; β-Cy3BdR phosphoramidite C; α-Cy3BdR oligonucleotide synthesis resin for 3′-addition D; 5-ethynyl-Cy3B E; Cy3dT phosphoramidite F; Cy5dT phosphoramidite G. The CydT and β-Cy3BdR phosphoramidite monomers can be added either internally or at the 5′-end of oligonucleotides but the latter will not form a base pair. 5-Ethynyl-Cy3B can be used for modification of oligonucleotides by click chemistry. (B) Flexible dye linker (left) and short rigid dye linker (right) in the context of a DNA helix cross-section (only labelled strand shown). Blue shading (Cy5) and pink shading (Cy3) suggests the volume of space that can be sampled by the dyes when the bases are paired. A short rigid linker restricts the dye and prevents dye–DNA interactions.